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Nievergelt AP. Genome editing in the green alga Chlamydomonas: past, present practice and future prospects. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2025; 122:e70140. [PMID: 40186543 PMCID: PMC11971955 DOI: 10.1111/tpj.70140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/30/2024] [Revised: 03/20/2025] [Accepted: 03/26/2025] [Indexed: 04/07/2025]
Abstract
The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.
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Affiliation(s)
- Adrian P. Nievergelt
- Max Planck Institute of Molecular Cell Biology and GeneticsPfotenhauerstraße 108Dresden01307Germany
- Max Planck Institute of Molecular Plant PhysiologyAm Mühlenberg 1Potsdam14476Germany
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2
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Niraula A, Danesh A, Merindol N, Meddeb-Mouelhi F, Desgagné-Penix I. Aromatic Amino Acids: Exploring Microalgae as a Potential Biofactory. BIOTECH 2025; 14:6. [PMID: 39982273 PMCID: PMC11843938 DOI: 10.3390/biotech14010006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2024] [Revised: 01/24/2025] [Accepted: 01/27/2025] [Indexed: 02/22/2025] Open
Abstract
In recent times, microalgae have emerged as powerful hosts for biotechnological applications, ranging from the production of lipids and specialized metabolites (SMs) of pharmaceutical interest to biofuels, nutraceutical supplements, and more. SM synthesis through bioengineered pathways relies on the availability of aromatic amino acids (AAAs) as an essential precursor. AAAs, phenylalanine, tyrosine, and tryptophan are also the building blocks of proteins, maintaining the structural and functional integrity of cells. Hence, they are crucial intermediates linking the primary and specialized metabolism. The biosynthesis pathway of AAAs in microbes and plants has been studied for decades, but not much is known about microalgae. The allosteric control present in this pathway has been targeted for metabolic engineering in microbes. This review focuses on the biosynthesis of AAAs in eukaryotic microalgae and engineering techniques for enhanced production. All the putative genes involved in AAA pathways in the model microalgae Chlamydomonas reinhardtii and Phaeodactylum tricornutum are listed in this review.
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Affiliation(s)
| | | | | | | | - Isabel Desgagné-Penix
- Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, Trois-Rivières, QC G8Z 4M3, Canada; (A.N.); (A.D.); (N.M.); (F.M.-M.)
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3
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Sookhoo JRV, Schiffman Z, Ambagala A, Kobasa D, Pardee K, Babiuk S. Protein Expression Platforms and the Challenges of Viral Antigen Production. Vaccines (Basel) 2024; 12:1344. [PMID: 39772006 PMCID: PMC11680109 DOI: 10.3390/vaccines12121344] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2024] [Revised: 11/19/2024] [Accepted: 11/19/2024] [Indexed: 01/11/2025] Open
Abstract
Several protein expression platforms exist for a wide variety of biopharmaceutical needs. A substantial proportion of research and development into protein expression platforms and their optimization since the mid-1900s is a result of the production of viral antigens for use in subunit vaccine research. This review discusses the seven most popular forms of expression systems used in the past decade-bacterial, insect, mammalian, yeast, algal, plant and cell-free systems-in terms of advantages, uses and limitations for viral antigen production in the context of subunit vaccine research. Post-translational modifications, immunogenicity, efficacy, complexity, scalability and the cost of production are major points discussed. Examples of licenced and experimental vaccines are included along with images which summarize the processes involved.
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Affiliation(s)
- Jamie R. V. Sookhoo
- Canadian Food Inspection Agency, National Centre for Foreign Animal Disease, Winnipeg, MB R3E 3R2, Canada; (J.R.V.S.); (A.A.)
- Department of Immunology, University of Manitoba, Winnipeg, MB R3E 0T5, Canada
| | - Zachary Schiffman
- Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada; (Z.S.); (D.K.)
- Department of Medical Microbiology, University of Manitoba, Winnipeg, MB R3E 0W2, Canada
| | - Aruna Ambagala
- Canadian Food Inspection Agency, National Centre for Foreign Animal Disease, Winnipeg, MB R3E 3R2, Canada; (J.R.V.S.); (A.A.)
- Department of Medical Microbiology, University of Manitoba, Winnipeg, MB R3E 0W2, Canada
| | - Darwyn Kobasa
- Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada; (Z.S.); (D.K.)
- Department of Medical Microbiology, University of Manitoba, Winnipeg, MB R3E 0W2, Canada
| | - Keith Pardee
- Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON M5S 3M2, Canada;
- Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ON M5S 3G8, Canada
| | - Shawn Babiuk
- Canadian Food Inspection Agency, National Centre for Foreign Animal Disease, Winnipeg, MB R3E 3R2, Canada; (J.R.V.S.); (A.A.)
- Department of Immunology, University of Manitoba, Winnipeg, MB R3E 0T5, Canada
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4
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Rajput BK, Ikram SF, Tripathi BN. Harnessing the potential of microalgae for the production of monoclonal antibodies and other recombinant proteins. PROTOPLASMA 2024; 261:1105-1125. [PMID: 38970700 DOI: 10.1007/s00709-024-01967-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/17/2024] [Accepted: 06/25/2024] [Indexed: 07/08/2024]
Abstract
Monoclonal antibodies (mAbs) have become indispensable tools in various fields, from research to therapeutics, diagnostics, and industries. However, their production, primarily in mammalian cell culture systems, is cost-intensive and resource-demanding. Microalgae, diverse photosynthetic microorganisms, are gaining attention as a favorable option for manufacturing mAbs and various other recombinant proteins. This review explores the potential of microalgae as a robust expression system for biomanufacturing high-value proteins. It also highlights the diversity of microalgae species suitable for recombinant protein. Nuclear and chloroplast genomes of some microalgae have been engineered to express mAbs and other valuable proteins. Codon optimization, vector construction, and other genetic engineering techniques have significantly improved recombinant protein expression in microalgae. These accomplishments demonstrate the potential of microalgae for biopharmaceutical manufacturing. Microalgal biotechnology holds promise for revolutionizing the production of mAbs and other therapeutic proteins, offering a sustainable and cost-effective solution to address critical healthcare needs.
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Affiliation(s)
- Balwinder Kaur Rajput
- Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, 484887, India
| | - Sana Fatima Ikram
- Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, 484887, India
| | - Bhumi Nath Tripathi
- Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, 484887, India.
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Hammel A, Cucos LM, Caras I, Ionescu I, Tucureanu C, Tofan V, Costache A, Onu A, Hoepfner L, Hippler M, Neupert J, Popescu CI, Stavaru C, Branza-Nichita N, Bock R. The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein. Proc Natl Acad Sci U S A 2024; 121:e2400145121. [PMID: 38833465 PMCID: PMC11181018 DOI: 10.1073/pnas.2400145121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Accepted: 05/03/2024] [Indexed: 06/06/2024] Open
Abstract
Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.
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Affiliation(s)
- Alexander Hammel
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
| | - Lia-Maria Cucos
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Iuliana Caras
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Irina Ionescu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Catalin Tucureanu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Vlad Tofan
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Adriana Costache
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Adrian Onu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Lara Hoepfner
- Institute of Plant Biology and Biotechnology, University of Münster, D-48143Münster, Germany
| | - Michael Hippler
- Institute of Plant Biology and Biotechnology, University of Münster, D-48143Münster, Germany
- Institute of Plant Science and Resources, Okayama University, Kurashiki710-0046, Japan
| | - Juliane Neupert
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
| | - Costin-Ioan Popescu
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Crina Stavaru
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Norica Branza-Nichita
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Ralph Bock
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
- NIBIO, Norwegian Institute of Bioeconomy Research, NO-1431 Ås, Norway
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6
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Kumar V, Barwal A, Sharma N, Mir DS, Kumar P, Kumar V. Therapeutic proteins: developments, progress, challenges, and future perspectives. 3 Biotech 2024; 14:112. [PMID: 38510462 PMCID: PMC10948735 DOI: 10.1007/s13205-024-03958-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2023] [Accepted: 02/13/2024] [Indexed: 03/22/2024] Open
Abstract
Proteins are considered magic molecules due to their enormous applications in the health sector. Over the past few decades, therapeutic proteins have emerged as a promising treatment option for various diseases, particularly cancer, cardiovascular disease, diabetes, and others. The formulation of protein-based therapies is a major area of research, however, a few factors still hinder the large-scale production of these therapeutic products, such as stability, heterogenicity, immunogenicity, high cost of production, etc. This review provides comprehensive information on various sources and production of therapeutic proteins. The review also summarizes the challenges currently faced by scientists while developing protein-based therapeutics, along with possible solutions. It can be concluded that these proteins can be used in combination with small molecular drugs to give synergistic benefits in the future.
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Affiliation(s)
- Vimal Kumar
- University Institute of Biotechnology, Chandigarh University, Gharuan, Mohali, Punjab 140413 India
| | - Arti Barwal
- Department of Microbial Biotechnology, Panjab University, South Campus, Sector-25, Chandigarh, 160014 India
| | - Nitin Sharma
- Department of Biotechnology, Chandigarh Group of Colleges, Mohali, Punjab 140307 India
| | - Danish Shafi Mir
- University Institute of Biotechnology, Chandigarh University, Gharuan, Mohali, Punjab 140413 India
| | - Pradeep Kumar
- Faculty of Applied Sciences and Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, 173229 India
| | - Vikas Kumar
- University Institute of Biotechnology, Chandigarh University, Gharuan, Mohali, Punjab 140413 India
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7
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Liu Y, Aimutis WR, Drake M. Dairy, Plant, and Novel Proteins: Scientific and Technological Aspects. Foods 2024; 13:1010. [PMID: 38611316 PMCID: PMC11011482 DOI: 10.3390/foods13071010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Revised: 03/19/2024] [Accepted: 03/20/2024] [Indexed: 04/14/2024] Open
Abstract
Alternative proteins have gained popularity as consumers look for foods that are healthy, nutritious, and sustainable. Plant proteins, precision fermentation-derived proteins, cell-cultured proteins, algal proteins, and mycoproteins are the major types of alternative proteins that have emerged in recent years. This review addresses the major alternative-protein categories and reviews their definitions, current market statuses, production methods, and regulations in different countries, safety assessments, nutrition statuses, functionalities and applications, and, finally, sensory properties and consumer perception. Knowledge relative to traditional dairy proteins is also addressed. Opportunities and challenges associated with these proteins are also discussed. Future research directions are proposed to better understand these technologies and to develop consumer-acceptable final products.
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Affiliation(s)
- Yaozheng Liu
- Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA; (Y.L.); (W.R.A.)
| | - William R. Aimutis
- Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA; (Y.L.); (W.R.A.)
- North Carolina Food Innovation Lab, North Carolina State University, Kannapolis, NC 28081, USA
| | - MaryAnne Drake
- Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA; (Y.L.); (W.R.A.)
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8
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Goold HD, Moseley JL, Lauersen KJ. The synthetic future of algal genomes. CELL GENOMICS 2024; 4:100505. [PMID: 38395701 PMCID: PMC10943592 DOI: 10.1016/j.xgen.2024.100505] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Revised: 12/18/2023] [Accepted: 01/24/2024] [Indexed: 02/25/2024]
Abstract
Algae are diverse organisms with significant biotechnological potential for resource circularity. Taking inspiration from fermentative microbes, engineering algal genomes holds promise to broadly expand their application ranges. Advances in genome sequencing with improvements in DNA synthesis and delivery techniques are enabling customized molecular tool development to confer advanced traits to algae. Efforts to redesign and rebuild entire genomes to create fit-for-purpose organisms currently being explored in heterotrophic prokaryotes and eukaryotic microbes could also be applied to photosynthetic algae. Future algal genome engineering will enhance yields of native products and permit the expression of complex biochemical pathways to produce novel metabolites from sustainable inputs. We present a historical perspective on advances in engineering algae, discuss the requisite genetic traits to enable algal genome optimization, take inspiration from whole-genome engineering efforts in other microbes for algal systems, and present candidate algal species in the context of these engineering goals.
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Affiliation(s)
- Hugh D Goold
- New South Wales Department of Primary Industries, Orange, NSW 2800, Australia; ARC Center of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW 2109, Australia; School of Natural Sciences, Macquarie University, Sydney, NSW 2109, Australia
| | - Jeffrey L Moseley
- California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Division of Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Phycoil Biotechnology International, Inc., Fremont, CA 94538, USA
| | - Kyle J Lauersen
- Bioengineering Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia.
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Jin C, Kang YJ, Park SR, Oh YJ, Ko K. Production, expression, and function of dual-specific monoclonal antibodies in a single plant. PLANTA 2023; 259:15. [PMID: 38071691 DOI: 10.1007/s00425-023-04284-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/13/2023] [Accepted: 11/07/2023] [Indexed: 12/18/2023]
Abstract
MAIN CONCLUSION LSC CO17-1AK and anti-HER2 VHH-FcK can be produced in a single plant and exhibit anti-tumor activities comparable to those of their respective parent antibodies. Recombinant monoclonal antibodies (mAbs) which can be applied to treat various cancers, are primarily produced using mammalian, insect, and bacteria cell culture systems. Plant expression systems have also been developed to produce antibodies. Plant expression systems present several advantages, including a lack of human pathogenic agents, efficient production costs, and easy large-scale production. In this study, we generated a transgenic plant expressing anti-colorectal cancer large single chain (LSC) CO17-1AK and anti-human epidermal growth factor receptor 2 (HER2) VHH-FcK mAbs by cross-pollinating plants expressing LSC CO17-1AK and anti-HER2 VHH-FcK, respectively. F1 siblings expressing both LSC CO17-1AK and anti-HER2 VHH-FcK were screened using polymerase chain reaction and Western-blot analyses. The cell enzyme-linked immunosorbent assay (Cell ELISA) confirmed the binding of LSC CO17-1AK and anti-HER2 VHH-FcK to target proteins in the SW620 human colorectal cancer and the SKBR-3 human breast cancer cell lines, respectively. The wound healing assay confirmed the inhibitory activity of both antibodies against SW620 and SKBR-3 cell migration, respectively. In conclusion, both LSC CO17-1AK mAb and anti-HER2 VHH-FcK can be produced in a single plant, achieve binding activities to SW620 and SKBR-3 cancer cells, and inhibitory activity against SW620 and SKBR-3 cell migration similar to their parental antibodies, respectively.
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Affiliation(s)
- Caiquan Jin
- Department of Medicine, College of Medicine, Chung-Ang University, Seoul, 06974, South Korea
| | - Yang Joo Kang
- Department of Medicine, College of Medicine, Chung-Ang University, Seoul, 06974, South Korea
| | - Se Ra Park
- Department of Medicine, College of Medicine, Chung-Ang University, Seoul, 06974, South Korea
- Department of Pathology, University of Michigan, Ann Arbor, 48109, USA
| | - Yoo Jin Oh
- Department of Applied Experimental Biophysics, Johannes Kepler University Linz, Linz, 4040, Austria
| | - Kisung Ko
- Department of Medicine, College of Medicine, Chung-Ang University, Seoul, 06974, South Korea.
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Dubey KK, Kumar A, Baldia A, Rajput D, Kateriya S, Singh R, Nikita, Tandon R, Mishra YK. Biomanufacturing of glycosylated antibodies: Challenges, solutions, and future prospects. Biotechnol Adv 2023; 69:108267. [PMID: 37813174 DOI: 10.1016/j.biotechadv.2023.108267] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 09/03/2023] [Accepted: 09/28/2023] [Indexed: 10/11/2023]
Abstract
Traditionally, recombinant protein production has been done in several expression hosts of bacteria, fungi, and majorly CHO (Chinese Hamster Ovary) cells; few have high production costs and are susceptible to harmful toxin contamination. Green algae have the potential to produce recombinant proteins in a more sustainable manner. Microalgal diversity leads to offer excellent opportunities to produce glycosylated antibodies. An antibody with humanized glycans plays a crucial role in cellular communication that works to regulate cells and molecules, to control disease, and to stimulate immunity. Therefore, it becomes necessary to understand the role of abiotic factors (light, temperature, pH, etc.) in the production of bioactive molecules and molecular mechanisms of product synthesis from microalgae which would lead to harnessing the potential of algal bio-refinery. However, the potential of microalgae as the source of bio-refinery has been less explored. In the present review, omics approaches for microalgal engineering, methods of humanized glycoproteins production focusing majorly on N-glycosylation pathways, light-based regulation of glycosylation machinery, and production of antibodies with humanized glycans in microalgae with a major emphasis on modulation of post-translation machinery of microalgae which might play a role in better understanding of microalgal potential as a source for antibody production along with future perspectives.
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Affiliation(s)
- Kashyap Kumar Dubey
- Biomanufacturing and Process Development Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
| | - Akshay Kumar
- Biomanufacturing and Process Development Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
| | - Anshu Baldia
- Biomanufacturing and Process Development Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
| | - Deepanshi Rajput
- Biomanufacturing and Process Development Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
| | - Suneel Kateriya
- Laboratory of Optobiotechnology, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
| | - Rajani Singh
- Laboratory of Optobiotechnology, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
| | - Nikita
- Laboratory of AIDS Research and Immunology, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
| | - Ravi Tandon
- Laboratory of AIDS Research and Immunology, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
| | - Yogendra Kumar Mishra
- Mads Clausen Institute, NanoSYD, University of Southern Denmark, Alison 2, 6400 Sønderborg, Denmark.
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11
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Pessoa JDS, de Oliveira CFM, Mena-Chalco JP, de Carvalho JCM, Ferreira-Camargo LS. Trends on Chlamydomonas reinhardtii growth regimes and bioproducts. Biotechnol Appl Biochem 2023; 70:1830-1842. [PMID: 37337370 DOI: 10.1002/bab.2486] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Accepted: 05/31/2023] [Indexed: 06/21/2023]
Abstract
The green microalga Chlamydomonas reinhardtii is a model microorganism for several areas of study. Among the different microalgae species, it presents advantageous characteristics, such as genomes completely sequenced and well-established techniques for genetic transformation. Despite that, C. reinhardtii production is still not easily commercially viable, especially due to the low biomass yield. So far there are no reports of scientometric study focusing only on C. reinhardtii biomass production process. Considering the need for culture optimization, a scientometric research was conducted to analyze the papers that investigated the growth regimes effects in C. reinhardtii cultivation. The search resulted in 130 papers indexed on Web of Science and Scopus platforms from 1969 to December 2022. The quantitative analysis indicated that the photoautotrophic regime was the most employed in the papers. However, when comparing the three growth regimes, the mixotrophic one led to the highest production of biomass, lipids, and heterologous protein. The production of bioproducts was considered the main objective of most of the papers and, among them, biomass was the most frequently investigated. The highest biomass production reported among the papers was 40 g L-1 in the heterotrophic growth of a transgenic strain. Other culture conditions were also crucial for C. reinhardtii growth, for instance, temperature and cultivation process.
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12
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Jiji MG, Ninan MA, Thomas VP, Thomas BT. Edible microalgae: potential candidate for developing edible vaccines. VEGETOS (BAREILLY, INDIA) 2023:1-6. [PMID: 37359124 PMCID: PMC10136395 DOI: 10.1007/s42535-023-00636-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/14/2023] [Revised: 04/05/2023] [Accepted: 04/12/2023] [Indexed: 06/28/2023]
Abstract
Infectious diseases are always a threat to all living beings. Today, in this world pathogens have no difficulty reaching anywhere. Every year new and deadly diseases are born and most of them are caused by viruses. Vaccines can provide lifelong immunity against infectious diseases, but the production cost of vaccines is unaffordable for a layman and traditional vaccines have certain limitations with storage and delivery. However, edible vaccines have shifted this paradigm and have received acceptance all over the world, especially in developing countries. Microalgae are one of the potential candidates for developing edible vaccines. Modifying microalgae as edible vaccines are gaining worldwide attention, especially in the world of science. Microalgae can augment the immune system as they are a promising source for antigen carriers and many of them are regarded as safe to eat. Moreover, they are a pantry of proteins, vitamins, minerals, and other secondary metabolites like alkaloids, phenols, and terpenes. In addition, being resistant to animal pathogens they are less sophisticated for genetic modification. This review analyses the potential scope of microalgae as an edible vaccine source.
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Affiliation(s)
- Merin Grace Jiji
- Phycotechnology laboratory, Post Graduate and Research Department of Botany, Catholicate college, Pathanamthitta, Kerala 689645 India
| | - Merin Ann Ninan
- Phycotechnology laboratory, Post Graduate and Research Department of Botany, Catholicate college, Pathanamthitta, Kerala 689645 India
| | - V. P. Thomas
- Phycotechnology laboratory, Post Graduate and Research Department of Botany, Catholicate college, Pathanamthitta, Kerala 689645 India
| | - Binoy T. Thomas
- Phycotechnology laboratory, Post Graduate and Research Department of Botany, Catholicate college, Pathanamthitta, Kerala 689645 India
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13
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Barbosa MJ, Janssen M, Südfeld C, D'Adamo S, Wijffels RH. Hypes, hopes, and the way forward for microalgal biotechnology. Trends Biotechnol 2023; 41:452-471. [PMID: 36707271 DOI: 10.1016/j.tibtech.2022.12.017] [Citation(s) in RCA: 44] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Revised: 12/22/2022] [Accepted: 12/27/2022] [Indexed: 01/26/2023]
Abstract
The urge for food security and sustainability has advanced the field of microalgal biotechnology. Microalgae are microorganisms able to grow using (sun)light, fertilizers, sugars, CO2, and seawater. They have high potential as a feedstock for food, feed, energy, and chemicals. Microalgae grow faster and have higher areal productivity than plant crops, without competing for agricultural land and with 100% efficiency uptake of fertilizers. In comparison with bacterial, fungal, and yeast single-cell protein production, based on hydrogen or sugar, microalgae show higher land-use efficiency. New insights are provided regarding the potential of microalgae replacing soy protein, fish oil, and palm oil and being used as cell factories in modern industrial biotechnology to produce designer feed, recombinant proteins, biopharmaceuticals, and vaccines.
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Affiliation(s)
- Maria J Barbosa
- Bioprocess Engineering & AlgaePARC, Wageningen University and Research, PO Box 16, Wageningen, 6700, AA, The Netherlands.
| | - Marcel Janssen
- Bioprocess Engineering & AlgaePARC, Wageningen University and Research, PO Box 16, Wageningen, 6700, AA, The Netherlands
| | - Christian Südfeld
- Bioprocess Engineering & AlgaePARC, Wageningen University and Research, PO Box 16, Wageningen, 6700, AA, The Netherlands
| | - Sarah D'Adamo
- Bioprocess Engineering & AlgaePARC, Wageningen University and Research, PO Box 16, Wageningen, 6700, AA, The Netherlands
| | - Rene H Wijffels
- Bioprocess Engineering & AlgaePARC, Wageningen University and Research, PO Box 16, Wageningen, 6700, AA, The Netherlands; Biosciences and Aquaculture, Nord University, Bodø, N-8049,Norway
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14
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Vilatte A, Spencer-Milnes X, Jackson HO, Purton S, Parker B. Spray Drying Is a Viable Technology for the Preservation of Recombinant Proteins in Microalgae. Microorganisms 2023; 11:microorganisms11020512. [PMID: 36838478 PMCID: PMC9967251 DOI: 10.3390/microorganisms11020512] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2023] [Revised: 01/28/2023] [Accepted: 01/30/2023] [Indexed: 02/22/2023] Open
Abstract
Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray drying to produce a recombinant vaccine in microalgae. A transformant line of Chlamydomonas reinhardtii carrying a subunit vaccine against salmonid alphavirus was created via chloroplast engineering. The integrity of the recombinant protein after spray drying and its stability after 27 months storage at -80 °C, +4 °C and room temperature were assessed by immunoblotting. The protein withstood spray drying without significant losses. Long-term storage at +4 °C and room temperature resulted in 50% and 92% degradation, respectively. Optimizing spray drying and storage conditions should minimize degradation and favour short-term storage at positive temperatures. Using data on yield and productivity, the economics of spray drying- and freeze drying-based bioprocesses were compared. The drying stage corresponded to 41% of the total production cost. Process optimization, genetic engineering and new market strategies were identified as potential targets for cost reduction. Overall, this study successfully demonstrates the suitability of spray drying as a process option for recombinant protein production in microalgae at the industrial scale.
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Affiliation(s)
- Anaëlle Vilatte
- Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, UK
| | - Xenia Spencer-Milnes
- Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, UK
- Algal Research Group, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
| | - Harry Oliver Jackson
- Algal Research Group, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
| | - Saul Purton
- Algal Research Group, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
| | - Brenda Parker
- Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, UK
- Correspondence:
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15
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Masi A, Leonelli F, Scognamiglio V, Gasperuzzo G, Antonacci A, Terzidis MA. Chlamydomonas reinhardtii: A Factory of Nutraceutical and Food Supplements for Human Health. Molecules 2023; 28:molecules28031185. [PMID: 36770853 PMCID: PMC9921279 DOI: 10.3390/molecules28031185] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 01/17/2023] [Accepted: 01/20/2023] [Indexed: 01/27/2023] Open
Abstract
Chlamydomonas reinhardtii (C. reinhardtii) is one of the most well-studied microalgae organisms that revealed important information for the photosynthetic and metabolic processes of plants and eukaryotes. Numerous extensive studies have also underpinned its great potential as a biochemical factory, capable of producing various highly desired molecules with a direct impact on human health and longevity. Polysaccharides, lipids, functional proteins, pigments, hormones, vaccines, and antibodies are among the valuable biomolecules that are produced spontaneously or under well-defined conditions by C. reinhardtii and can be directly linked to human nutrition and diet. The aim of this review is to highlight the recent advances in the field focusing on the most relevant applications related to the production of important biomolecules for human health that are also linked with human nutrition and diet. The limitations and challenges are critically discussed along with the potential future applications of C. reinhardtii biomass and processed products in the field of nutraceuticals and food supplements. The increasing need for high-value and low-cost biomolecules produced in an environmentally and economy sustainable manner also underline the important role of C. reinhardtii.
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Affiliation(s)
- Annalisa Masi
- Institute of Crystallography, National Research Council, 00010 Montelibretti, Italy
| | - Francesca Leonelli
- Department of Chemistry, University of Rome “Sapienza”, 00185 Rome, Italy
| | - Viviana Scognamiglio
- Institute of Crystallography, National Research Council, 00010 Montelibretti, Italy
| | - Giulia Gasperuzzo
- Institute of Crystallography, National Research Council, 00010 Montelibretti, Italy
| | - Amina Antonacci
- Institute of Crystallography, National Research Council, 00010 Montelibretti, Italy
- Correspondence: (A.A.); (M.A.T.); Tel.: +39-0690675597 (A.A.); +30-2310013224 (M.A.T.)
| | - Michael A. Terzidis
- Department of Nutritional Sciences and Dietetics, International Hellenic University, Sindos Campus, 57400 Thessaloniki, Greece
- Correspondence: (A.A.); (M.A.T.); Tel.: +39-0690675597 (A.A.); +30-2310013224 (M.A.T.)
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16
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Production of Recombinant Biopharmaceuticals in Chlamydomonas reinhardtii. INTERNATIONAL JOURNAL OF PLANT BIOLOGY 2022. [DOI: 10.3390/ijpb14010004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
This review aimed to present Chlamydomonas reinhardtii as an alternative for heterologous protein production, especially for biopharmaceuticals, and its general characteristics when compared with other expression systems. The need to produce heterologous proteins for industrial interest, therapeutic ends, and diagnostic kits has led to the development of recombinant microalgal technology. This technology presents some interesting features, such as rapid growth and low transgene dispersion compared to plants, the ability to fold complex proteins compared to bacteria, and low production costs compared to other expression systems, such as yeast and mammalian cells. Overall, C. reinhardtii heterologous protein expression is coming of age with several research groups focused on developing an optimal producer strain.
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17
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Gaobotse G, Venkataraman S, Mmereke KM, Moustafa K, Hefferon K, Makhzoum A. Recent Progress on Vaccines Produced in Transgenic Plants. Vaccines (Basel) 2022; 10:1861. [PMID: 36366370 PMCID: PMC9698746 DOI: 10.3390/vaccines10111861] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2022] [Revised: 10/27/2022] [Accepted: 10/31/2022] [Indexed: 01/15/2024] Open
Abstract
The development of vaccines from plants has been going on for over two decades now. Vaccine production in plants requires time and a lot of effort. Despite global efforts in plant-made vaccine development, there are still challenges that hinder the realization of the final objective of manufacturing approved and safe products. Despite delays in the commercialization of plant-made vaccines, there are some human vaccines that are in clinical trials. The novel coronavirus (SARS-CoV-2) and its resultant disease, coronavirus disease 2019 (COVID-19), have reminded the global scientific community of the importance of vaccines. Plant-made vaccines could not be more important in tackling such unexpected pandemics as COVID-19. In this review, we explore current progress in the development of vaccines manufactured in transgenic plants for different human diseases over the past 5 years. However, we first explore the different host species and plant expression systems during recombinant protein production, including their shortcomings and benefits. Lastly, we address the optimization of existing plant-dependent vaccine production protocols that are aimed at improving the recovery and purification of these recombinant proteins.
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Affiliation(s)
- Goabaone Gaobotse
- Department of Biological Sciences & Biotechnology, Botswana International University of Science & Technology, Palapye, Botswana
| | - Srividhya Venkataraman
- Virology Laboratory, Department of Cell & Systems Biology, University of Toronto, Toronto, ON M5S 3B2, Canada
| | - Kamogelo M. Mmereke
- Department of Biological Sciences & Biotechnology, Botswana International University of Science & Technology, Palapye, Botswana
| | - Khaled Moustafa
- The Arabic Preprint Server/Arabic Science Archive (ArabiXiv)
| | - Kathleen Hefferon
- Department of Microbiology, Cornell University, Ithaca, NY 14850, USA
| | - Abdullah Makhzoum
- Department of Biological Sciences & Biotechnology, Botswana International University of Science & Technology, Palapye, Botswana
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18
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Saeed MU, Hussain N, Shahbaz A, Hameed T, Iqbal HMN, Bilal M. Bioprospecting microalgae and cyanobacteria for biopharmaceutical applications. J Basic Microbiol 2022; 62:1110-1124. [PMID: 34914840 DOI: 10.1002/jobm.202100445] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2021] [Revised: 11/19/2021] [Accepted: 11/27/2021] [Indexed: 02/05/2023]
Abstract
Microalgae and cyanobacteria have sparked a lot of interest due to their potential in various industries like biorefineries, biopharmaceuticals, food supplements, nutraceuticals, and other high-value products. Polysaccharides, vitamins, proteins, enzymes, and steroids are valuable products isolated from microalgae and cyanobacteria and potentially used in health and biomedical applications. Bioactive compounds derived from microalgae and cyanobacteria exhibit various pharmaceutical properties like antibacterial, anticancer, antiviral, antialgal, and antioxidant. From the properties listed above, the research for novel antibiotics has become particularly appropriate. In addition, the possible emergence of resistance against pathogens, as well as the potential decline in antibiotic efficacy, has prompted researchers to look for a new source of antibiotics. Microalgae and cyanobacteria have indicated a great and unexplored potential among these sources. For this reason, microalgae and cyanobacteria have been highlighted for their efficiency in different industrial sectors, as well as for their potential uses in the betterment of human and environmental health. This review gives an overview of bioactive compounds and metabolites with several biological properties isolated from microalgae and cyanobacteria for treating different animal and human diseases.
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Affiliation(s)
- Muhammad U Saeed
- Center for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan
| | - Nazim Hussain
- Center for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan
| | - Areej Shahbaz
- Center for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan
| | - Tooba Hameed
- School of Biochemistry & Biotechnology, University of the Punjab Lahore, Lahore, Pakistan
| | - Hafiz M N Iqbal
- Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico
| | - Muhammad Bilal
- School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, China
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19
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Ma K, Deng L, Wu H, Fan J. Towards green biomanufacturing of high-value recombinant proteins using promising cell factory: Chlamydomonas reinhardtii chloroplast. BIORESOUR BIOPROCESS 2022; 9:83. [PMID: 38647750 PMCID: PMC10992328 DOI: 10.1186/s40643-022-00568-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Accepted: 07/29/2022] [Indexed: 11/10/2022] Open
Abstract
Microalgae are cosmopolitan organisms in nature with short life cycles, playing a tremendous role in reducing the pressure of industrial carbon emissions. Besides, microalgae have the unique advantages of being photoautotrophic and harboring both prokaryotic and eukaryotic expression systems, becoming a popular host for recombinant proteins. Currently, numerous advanced molecular tools related to microalgal transgenesis have been explored and established, especially for the model species Chlamydomonas reinhardtii (C. reinhardtii hereafter). The development of genetic tools and the emergence of new strategies further increase the feasibility of developing C. reinhardtii chloroplasts as green factories, and the strong genetic operability of C. reinhardtii endows it with enormous potential as a synthetic biology platform. At present, C. reinhardtii chloroplasts could successfully produce plenty of recombinant proteins, including antigens, antibodies, antimicrobial peptides, protein hormones and enzymes. However, additional techniques and toolkits for chloroplasts need to be developed to achieve efficient and markerless editing of plastid genomes. Mining novel genetic elements and selectable markers will be more intensively studied in the future, and more factors affecting protein expression are urged to be explored. This review focuses on the latest technological progress of selectable markers for Chlamydomonas chloroplast genetic engineering and the factors that affect the efficiency of chloroplast protein expression. Furthermore, urgent challenges and prospects for future development are pointed out.
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Affiliation(s)
- Ke Ma
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China
| | - Lei Deng
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China
| | - Haizhen Wu
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
- Department of Applied Biology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
| | - Jianhua Fan
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
- Department of Applied Biology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
- School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, 832003, People's Republic of China.
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20
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El-Hadary MH, Elsaied HE, Khalil NM, Mikhail SK. Molecular taxonomical identification and phylogenetic relationships of some marine dominant algal species during red tide and harmful algal blooms along Egyptian coasts in the Alexandria region. ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2022; 29:53403-53419. [PMID: 35287194 PMCID: PMC9343293 DOI: 10.1007/s11356-022-19217-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Accepted: 02/10/2022] [Indexed: 06/14/2023]
Abstract
Harmful algal blooms (HABs) threaten the aquatic ecosystems due to either poisonous effects on living organisms or oxygen-consuming. So HABs' accurate identification, including red tide, is crucial. This study aimed to molecular identification of dominant species during tide period in nine stations along Alexandria region at Egyptian costs during one year. Samples were collected weekly before water discoloration but daily during red tide intensive growth from both 50 cm below the surface and 3 m depth over the bottom from the water surface. The red tide detection was highly from early August to half of September, since its highest peak with a maximum frequency inside the Eastern Harbor. The examined cultures samples isolated during red tide had four dominant species. Peroxidase profile showed an expression pattern of three loci (Px1, Px2, and Px3) in most species. The Px2 was the only heterozygous locus among the three loci in all species. Protein profiling showed that 17 bands out of 65 were specific to the species. The phylogenetic relationships derived from profiles of protein and 18S rRNA gene operon sequences for the four isolated species were mostly similar. We identified the four dominant HABs species as Aplanochytrium sp., Chlamydomonas sp., Cryptophyceae sp., and Psammodictyon sp. based on their 18S rRNA sequences and deposited them at DDBJ/EMBL/GenBank database. Aplanochytrium sp. is recorded as a red tide causative species for the first time in the screened region despite belonging to the defunct fungi.
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Affiliation(s)
- Mona H El-Hadary
- Department of Botany and Microbiology, Faculty of Science, Damanhour University, Damanhour, Al Beheria Governorate, Egypt.
| | - Hosam E Elsaied
- National Institutes of Oceanography and Fisheries (NIOF), Al kanater Elkhiria, Al Qalyubiyah, Egypt
| | - Nehma M Khalil
- National Institute of Oceanography and Fisheries (NIOF), Alexandria, Egypt
| | - Samia K Mikhail
- National Institute of Oceanography and Fisheries (NIOF), Alexandria, Egypt
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21
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Bolaños-Martínez OC, Mahendran G, Rosales-Mendoza S, Vimolmangkang S. Current Status and Perspective on the Use of Viral-Based Vectors in Eukaryotic Microalgae. Mar Drugs 2022; 20:md20070434. [PMID: 35877728 PMCID: PMC9318342 DOI: 10.3390/md20070434] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Revised: 06/25/2022] [Accepted: 06/27/2022] [Indexed: 11/29/2022] Open
Abstract
During the last two decades, microalgae have attracted increasing interest, both commercially and scientifically. Commercial potential involves utilizing valuable natural compounds, including carotenoids, polysaccharides, and polyunsaturated fatty acids, which are widely applicable in food, biofuel, and pharmaceutical industries. Conversely, scientific potential focuses on bioreactors for producing recombinant proteins and developing viable technologies to significantly increase the yield and harvest periods. Here, viral-based vectors and transient expression strategies have significantly contributed to improving plant biotechnology. We present an updated outlook covering microalgal biotechnology for pharmaceutical application, transformation techniques for generating recombinant proteins, and genetic engineering tactics for viral-based vector construction. Challenges in industrial application are also discussed.
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Affiliation(s)
- Omayra C. Bolaños-Martínez
- Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; (O.C.B.-M.); (G.M.)
- Center of Excellence in Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok 10330, Thailand
| | - Ganesan Mahendran
- Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; (O.C.B.-M.); (G.M.)
- Center of Excellence in Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok 10330, Thailand
| | - Sergio Rosales-Mendoza
- Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, Av. Dr. Manuel Nava 6, San Luis Potosí 78210, Mexico;
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, Av. Sierra Leona 550, Lomas 2a Sección, San Luis Potosí 78210, Mexico
| | - Sornkanok Vimolmangkang
- Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; (O.C.B.-M.); (G.M.)
- Center of Excellence in Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok 10330, Thailand
- Correspondence: ; Tel.: +662-218-8358
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22
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Wang Q, Zhuang J, Ni S, Luo H, Zheng K, Li X, Lan C, Zhao D, Bai Y, Jia B, Hu Z. Overexpressing CrePAPS Polyadenylate Activity Enhances Protein Translation and Accumulation in Chlamydomonas reinhardtii. Mar Drugs 2022; 20:276. [PMID: 35621927 PMCID: PMC9147819 DOI: 10.3390/md20050276] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 04/15/2022] [Accepted: 04/15/2022] [Indexed: 02/01/2023] Open
Abstract
The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3'-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform.
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Affiliation(s)
- Quan Wang
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
- Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
| | - Jieyi Zhuang
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Shuai Ni
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Haolin Luo
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Kaijie Zheng
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Xinyi Li
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Chengxiang Lan
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Di Zhao
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Yongsheng Bai
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
| | - Bin Jia
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
- Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Longhua Innovation Institute for Biotechnology, Shenzhen University, Shenzhen 518055, China
| | - Zhangli Hu
- Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518055, China; (Q.W.); (J.Z.); (S.N.); (H.L.); (K.Z.); (X.L.); (C.L.); (D.Z.); (Y.B.)
- Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Longhua Innovation Institute for Biotechnology, Shenzhen University, Shenzhen 518055, China
- Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
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23
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Garduño-González KA, Peña-Benavides SA, Araújo RG, Castillo-Zacarías C, Melchor-Martínez EM, Oyervides-Muñoz MA, Sosa-Hernández JE, Purton S, Iqbal HM, Parra-Saldívar R. Current challenges for modern vaccines and perspectives for novel treatment alternatives. J Drug Deliv Sci Technol 2022; 70:103222. [DOI: 10.1016/j.jddst.2022.103222] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
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24
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Molino JVD, Carpine R, Gademann K, Mayfield S, Sieber S. Development of a cell surface display system in Chlamydomonas reinhardtii. ALGAL RES 2022. [DOI: 10.1016/j.algal.2021.102570] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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25
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Ferrer-Miralles N, Saccardo P, Corchero JL, Garcia-Fruitós E. Recombinant Protein Production and Purification of Insoluble Proteins. Methods Mol Biol 2022; 2406:1-31. [PMID: 35089548 DOI: 10.1007/978-1-0716-1859-2_1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The efficient production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and its growth conditions to minimize the formation of insoluble protein aggregates should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.
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Affiliation(s)
- Neus Ferrer-Miralles
- Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Cerdanyola del Vallès, Spain
| | - Paolo Saccardo
- Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Cerdanyola del Vallès, Spain
| | - José Luis Corchero
- Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Cerdanyola del Vallès, Spain
| | - Elena Garcia-Fruitós
- Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Caldes de Montbui, Spain.
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Xia D, Qiu W, Wang X, Liu J. Recent Advancements and Future Perspectives of Microalgae-Derived Pharmaceuticals. Mar Drugs 2021; 19:703. [PMID: 34940702 PMCID: PMC8703604 DOI: 10.3390/md19120703] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Revised: 11/25/2021] [Accepted: 12/07/2021] [Indexed: 12/19/2022] Open
Abstract
Microalgal cells serve as solar-powered factories that produce pharmaceuticals, recombinant proteins (vaccines and drugs), and valuable natural byproducts that possess medicinal properties. The main advantages of microalgae as cell factories can be summarized as follows: they are fueled by photosynthesis, are carbon dioxide-neutral, have rapid growth rates, are robust, have low-cost cultivation, are easily scalable, pose no risk of human pathogenic contamination, and their valuable natural byproducts can be further processed. Despite their potential, there are many technical hurdles that need to be overcome before the commercial production of microalgal pharmaceuticals, and extensive studies regarding their impact on human health must still be conducted and the results evaluated. Clearly, much work remains to be done before microalgae can be used in the large-scale commercial production of pharmaceuticals. This review focuses on recent advancements in microalgal biotechnology and its future perspectives.
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Affiliation(s)
- Donghua Xia
- State Key Laboratory of Food Science and Technology, The Engineering Research Center for Biomass Conversion, Nanchang University, Nanchang 330047, China;
| | - Wen Qiu
- Eco-Environmental Protection Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China;
| | - Xianxian Wang
- Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany;
| | - Junying Liu
- State Key Laboratory of Food Science and Technology, The Engineering Research Center for Biomass Conversion, Nanchang University, Nanchang 330047, China;
- Pharmaceutical Manufacturing Technology Centre (PMTC), Bernal Institute, University of Limerick, V94T9PX Limerick, Ireland
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Abu-Ghosh S, Dubinsky Z, Verdelho V, Iluz D. Unconventional high-value products from microalgae: A review. BIORESOURCE TECHNOLOGY 2021; 329:124895. [PMID: 33713898 DOI: 10.1016/j.biortech.2021.124895] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Revised: 02/18/2021] [Accepted: 02/19/2021] [Indexed: 06/12/2023]
Abstract
Microalgae have gained significant importance in biotechnology development, providing valuable goods and services in multiple applications. Although there is a rising market for most of these applications, the incorporation and introduction of microalgae into new venues will extend in the near future. These advances are due to the vast biodiversity of microalgal species, recent genetic engineering tools, and culture techniques. There are three main possible approaches for novel algal compounds from: (1) recently isolated yet less known microalgae; (2) selectively stressed conditions; and (3) enzymatically adjusted compounds from conventional molecules. All these approaches can be combined in a specific manner. This review discusses the opportunities, potential and limitations of introducing novel microalgae-based products, and how the recent technologies can be deployed to make these products financially viable. To give an outlook to the future, an analysis of the developments and predicted future market that further enlarge the promise of cultivating microalgae for commercial purposes are considered.
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Affiliation(s)
- Said Abu-Ghosh
- The Mina and Everard Goodman, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
| | - Zvy Dubinsky
- The Mina and Everard Goodman, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
| | - Vitor Verdelho
- General Manager of the European Algae Biomass Association (EABA), Portugal
| | - David Iluz
- The Mina and Everard Goodman, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; Department of Environmental Sciences and Agriculture, Beit Berl Academic College, Israel; Talpiot academic College, Holon, Israel
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Chin-Fatt A, Menassa R. A V HH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7. FRONTIERS IN PLANT SCIENCE 2021; 12:686421. [PMID: 34122494 PMCID: PMC8193579 DOI: 10.3389/fpls.2021.686421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Accepted: 04/26/2021] [Indexed: 06/12/2023]
Abstract
Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.
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Affiliation(s)
- Adam Chin-Fatt
- Agriculture and Agri-Food Canada, London Research and Development Centre, London, ON, Canada
- Department of Biology, University of Western Ontario, London, ON, Canada
| | - Rima Menassa
- Agriculture and Agri-Food Canada, London Research and Development Centre, London, ON, Canada
- Department of Biology, University of Western Ontario, London, ON, Canada
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Reyes-Barrera KL, Soria-Guerra RE, López-Martínez R, Huerta L, Salinas-Jazmín N, Cabello-Gutiérrez C, Alpuche-Solís ÁG. The Entry Blocker Peptide Produced in Chlamydomonas reinhardtii Inhibits Influenza Viral Replication in vitro. FRONTIERS IN PLANT SCIENCE 2021; 12:641420. [PMID: 34054890 PMCID: PMC8149740 DOI: 10.3389/fpls.2021.641420] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Accepted: 03/23/2021] [Indexed: 06/01/2023]
Abstract
This year, a respiratory virus caused an emergency pandemic alert in health services around the world, showing the need for biotechnological approaches to fight these diseases. The influenza virus is one of the main viral agents that generate pandemic outbreaks. Currently, the majority of co-circulating influenza A virus (IAV) strains are adamantine- and oseltamivir-resistant strains, and the challenge is to find new antivirals for more efficient treatments. The antiviral entry blocker (EB) peptide is a promising candidate for blocking the virus entry into cells. The aim of this research was to express the EB peptide in the microalgae Chlamydomonas reinhardtii and test its antiviral activity against IAV in vitro. The EB peptide nucleotide sequence was introduced into the nuclear genome of microalgae using Agrobacterium tumefaciens transformation. The EB peptide amount produced in transformed microalgae was 4.99 ± 0.067% of the total soluble protein. In hemagglutination inhibition assays using influenza A/H1N1 pdm and influenza A H1N1/Virginia/ATCC/2009 strains, we reported that the EB peptide extract from the microalgae showed 100-fold higher efficiency than the EB synthetic peptide. In addition, both the EB peptide extract and synthetic peptide inhibited viral replication in MDCK cells (IC50 = 20.7 nM and IC50 = 754.4 nM, respectively); however, the EB peptide extract showed a 32-fold higher antiviral effectiveness than the synthetic peptide against influenza A/H1N1 pdm. Extracts from untransformed and transformed microalgae and synthetic peptide did not show cytotoxic effect on MDCK cell monolayers. Thus, C. reinhardtii may be a fast, safe, and effective expression platform for production of peptides with significant antiviral activity and can be used as a prophylactic treatment to reduce viral propagation.
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Affiliation(s)
- Karen Lizbeth Reyes-Barrera
- Laboratorio de Biología Molecular de Plantas, División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A.C., San Luis Potosí, Mexico
| | - Ruth Elena Soria-Guerra
- Laboratorio de Biotecnología Molecular de Células Vegetales, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
| | - Rogelio López-Martínez
- Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
| | - Leonor Huerta
- Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
| | - Nohemí Salinas-Jazmín
- Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
| | - Carlos Cabello-Gutiérrez
- Departamento de Investigación en Virología y Micología, Instituto Nacional de Enfermedades Respiratorias “Ismael Cosío Villegas”, Ciudad de México, Mexico
| | - Ángel Gabriel Alpuche-Solís
- Laboratorio de Biología Molecular de Plantas, División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A.C., San Luis Potosí, Mexico
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Liu X, Xie X, Du H, Sanganyado E, Wang W, Aslam M, Chen J, Chen W, Liang H. Bioinformatic analysis and genetic engineering approaches for recombinant biopharmaceutical glycoproteins production in microalgae. ALGAL RES 2021. [DOI: 10.1016/j.algal.2021.102276] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
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Expression and purification of a novel single-chain diabody (scDb-hERG1/β1) from Pichia pastoris transformants. Protein Expr Purif 2021; 184:105879. [PMID: 33826963 DOI: 10.1016/j.pep.2021.105879] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2020] [Revised: 03/01/2021] [Accepted: 03/31/2021] [Indexed: 01/07/2023]
Abstract
In the last decades, protein engineering has developed particularly in biotechnology and pharmaceutical field. In particular, the engineered antibody subclass has arisen. The single chain diabody format (scDb), conjugating small size with antigen specificity, offers versatility representing a gold standard for a variety of applications, spacing from research to diagnostics and therapy. Along with such advantages, comes the challenge of optimizing their production, improving expression systems, purification procedures and stability. All such parameters are detrimental for protein production in general and above all for recombinant antibody expression, which has to be fine-tuned, choosing a proper protein-expression host and adjusting expression protocols accordingly. In the present paper, we present data regarding the production and purification of a single chain diabody directed against the macromolecular complex hERG1/β1 integrin. We focus on the expression of clones deriving from the transformation of Pichia pastoris yeast cells. In particular, we compare two different clones arose from two separate transformation processes, demonstrating that both are suitable for proper protein expression. Moreover, we have set up an expression protocol and compared the yields obtained using two purification machines: Akta Pure and Akta Start, with a positive outcome.
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Macedo-Osorio KS, Martínez-Antonio A, Badillo-Corona JA. Pas de Trois: An Overview of Penta-, Tetra-, and Octo-Tricopeptide Repeat Proteins From Chlamydomonas reinhardtii and Their Role in Chloroplast Gene Expression. FRONTIERS IN PLANT SCIENCE 2021; 12:775366. [PMID: 34868174 PMCID: PMC8635915 DOI: 10.3389/fpls.2021.775366] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 10/26/2021] [Indexed: 05/05/2023]
Abstract
Penta-, Tetra-, and Octo-tricopeptide repeat (PPR, TPR, and OPR) proteins are nucleus-encoded proteins composed of tandem repeats of 35, 34, and 38-40 amino acids, respectively. They form helix-turn-helix structures that interact with mRNA or other proteins and participate in RNA stabilization, processing, maturation, and act as translation enhancers of chloroplast and mitochondrial mRNAs. These helical repeat proteins are unevenly present in plants and algae. While PPR proteins are more abundant in plants than in algae, OPR proteins are more abundant in algae. In Arabidopsis, maize, and rice there have been 450, 661, and 477 PPR proteins identified, respectively, which contrasts with only 14 PPR proteins identified in Chlamydomonas reinhardtii. Likewise, more than 120 OPR proteins members have been predicted from the nuclear genome of C. reinhardtii and only one has been identified in Arabidopsis thaliana. Due to their abundance in land plants, PPR proteins have been largely characterized making it possible to elucidate their RNA-binding code. This has even allowed researchers to generate engineered PPR proteins with defined affinity to a particular target, which has served as the basis to develop tools for gene expression in biotechnological applications. However, fine elucidation of the helical repeat proteins code in Chlamydomonas is a pending task. In this review, we summarize the current knowledge on the role PPR, TPR, and OPR proteins play in chloroplast gene expression in the green algae C. reinhardtii, pointing to relevant similarities and differences with their counterparts in plants. We also recapitulate on how these proteins have been engineered and shown to serve as mRNA regulatory factors for biotechnological applications in plants and how this could be used as a starting point for applications in algae.
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Affiliation(s)
- Karla S. Macedo-Osorio
- Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, México City, México
- Biological Engineering Laboratory, Genetic Engineering Department, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional-Unidad Irapuato, Irapuato, México
- División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Xochimilco, México City, México
- *Correspondence: Karla S. Macedo-Osorio,
| | - Agustino Martínez-Antonio
- Biological Engineering Laboratory, Genetic Engineering Department, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional-Unidad Irapuato, Irapuato, México
| | - Jesús A. Badillo-Corona
- Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, México City, México
- Jesús A. Badillo-Corona,
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Lim S, Kim DS, Ko K. Expression of a Large Single-Chain 13F6 Antibody with Binding Activity against Ebola Virus-Like Particles in a Plant System. Int J Mol Sci 2020; 21:E7007. [PMID: 32977599 PMCID: PMC7582593 DOI: 10.3390/ijms21197007] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2020] [Revised: 09/17/2020] [Accepted: 09/20/2020] [Indexed: 11/16/2022] Open
Abstract
Pathogenic animal and human viruses present a growing and persistent threat to humans worldwide. Ebola virus (EBOV) causes zoonosis in humans. Here, two structurally different anti-Ebola 13F6 antibodies, recognizing the heavily glycosylated mucin-like domain (MLD) of the glycoprotein (GP), were expressed in transgenic Nicotiana tabacum plants and designed as inexpensive and effective diagnostic antibodies against Ebola virus disease (EVD). The first was anti-EBOV 13F6 full size antibody with heavy chain (HC) and light chain (LC) (monoclonal antibody, mAb 13F6-FULL), while the second was a large single-chain (LSC) antibody (mAb 13F6-LSC). mAb 13F6-LSC was constructed by linking the 13F6 LC variable region (VL) with the HC of mAb 13F6-FULL using a peptide linker and extended to the C-terminus using the endoplasmic reticulum (ER) retention motif KDEL. Agrobacterium-mediated plant transformation was employed to express the antibodies in N. tabacum. PCR, RT-PCR, and immunoblot analyses confirmed the gene insertion, transcription, and protein expression of these antibodies, respectively. The antibodies tagged with the KDEL motif displayed high-mannose type N-glycan structures and efficient binding to EBOV-like particles (VLPs). Thus, various forms of anti-EBOV plant-derived mAbs 13F6-FULL and LSC with efficient binding affinity to EBOV VLP can be produced in the plant system.
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Affiliation(s)
- Sohee Lim
- Department of Medicine, College of Medicine, Chung-Ang University, Seoul 06974, Korea;
| | - Do-Sun Kim
- Vegetable Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Jeonju 55365, Korea;
| | - Kisung Ko
- Department of Medicine, College of Medicine, Chung-Ang University, Seoul 06974, Korea;
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Hadiatullah H, Wang H, Liu YX, Fan ZC. Chlamydomonas reinhardtii-derived multimer Mytichitin-CB possesses potent antibacterial properties. Process Biochem 2020. [DOI: 10.1016/j.procbio.2020.05.010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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Gutiérrez CL, Muñoz C, San Martín M, Cadoret JP, Henríquez V. Chloroplast Dual Divergent Promoter Plasmid for Heterologous Protein Expression in Tetraselmis suecica (Chlorophyceae, Chlorodendrales). JOURNAL OF PHYCOLOGY 2020; 56:1066-1076. [PMID: 32359200 DOI: 10.1111/jpy.13013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Revised: 02/24/2020] [Accepted: 04/14/2020] [Indexed: 06/11/2023]
Abstract
The eukaryotic green microalga Tetraselmis suecica is commonly used for aquaculture purposes because of its high stress tolerance and ease of culture in a wide spectrum of environments; they are therefore suitable candidates for biotechnology applications. To date, no data are available regarding chloroplast transformation vectors based on specific endogenous promoters and homologous targeting regions. We report on the identification of Tetraselmis suecica genes encoding the ribulose bisphosphate carboxylase/oxygenase large subunit protein, the photosystem II D1 protein and the ATP synthase CF1-beta subunit protein together with their untranslated regions (5'UTR, 3'UTR). The full-length ORFs of the putative genes with their regulatory sequences were obtained. We were also able to identify the downstream 3' end of the large subunit ribosomal RNA gene (23S) along with the 5S RNA end-to-end with the psbA gene on the complementary strand. The intergenic region between these genes appears to be a good target site for the integration of target proteins. Moreover, we identified a back-to-back promoter region among the rbcL and atpB genes. To assess the bidirectionality activities of both promoters, a dual reporter vector was constructed for Tetraselmis suecica transformation containing the cat and TurboGFP genes driven by the 5'rbcL/5'atpB divergent promoter. The vector included the 23S-5S and psbA nucleotide sequences as flanking regions. These flanking regions provided suitable insertion sites within the chloroplast genome for cassette integration via homologous recombination. Simultaneous expression of the chloramphenicol-resistant conferring gene and the gene coding for TurboGFP driven by 5'rbcL/5'atpB showed a potent natural bidirectional promoter as a reliable genetic tool.
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Affiliation(s)
- Carla L Gutiérrez
- Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
| | - Carla Muñoz
- Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
| | - Margarita San Martín
- Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
| | | | - Vitalia Henríquez
- Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
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Shin JH, Choi J, Jeon J, Kumar M, Lee J, Jeong WJ, Kim SR. The establishment of new protein expression system using N starvation inducible promoters in Chlorella. Sci Rep 2020; 10:12713. [PMID: 32728100 PMCID: PMC7391781 DOI: 10.1038/s41598-020-69620-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Accepted: 06/22/2020] [Indexed: 11/09/2022] Open
Abstract
Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.
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Affiliation(s)
- Jun-Hye Shin
- Department of Life Science, Sogang University, Seoul, South Korea
| | - Juyoung Choi
- Department of Life Science, Sogang University, Seoul, South Korea
| | - Jeongmin Jeon
- Department of Life Science, Sogang University, Seoul, South Korea
| | - Manu Kumar
- Department of Life Science, Sogang University, Seoul, South Korea
| | - Juhyeon Lee
- Department of Life Science, Sogang University, Seoul, South Korea
| | - Won-Joong Jeong
- Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea
| | - Seong-Ryong Kim
- Department of Life Science, Sogang University, Seoul, South Korea.
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Towards a new avenue for producing therapeutic proteins: Microalgae as a tempting green biofactory. Biotechnol Adv 2020; 40:107499. [DOI: 10.1016/j.biotechadv.2019.107499] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Revised: 10/02/2019] [Accepted: 12/17/2019] [Indexed: 02/08/2023]
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Rosales-Mendoza S, Solís-Andrade KI, Márquez-Escobar VA, González-Ortega O, Bañuelos-Hernandez B. Current advances in the algae-made biopharmaceuticals field. Expert Opin Biol Ther 2020; 20:751-766. [DOI: 10.1080/14712598.2020.1739643] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Affiliation(s)
- Sergio Rosales-Mendoza
- Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
| | - Karla I. Solís-Andrade
- Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
| | - Verónica A. Márquez-Escobar
- Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
| | - Omar González-Ortega
- Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México
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Barolo L, Abbriano RM, Commault AS, George J, Kahlke T, Fabris M, Padula MP, Lopez A, Ralph PJ, Pernice M. Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae. Cells 2020; 9:E633. [PMID: 32151094 PMCID: PMC7140410 DOI: 10.3390/cells9030633] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 02/28/2020] [Accepted: 02/28/2020] [Indexed: 12/15/2022] Open
Abstract
Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.
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Affiliation(s)
- Lorenzo Barolo
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
| | - Raffaela M. Abbriano
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
| | - Audrey S. Commault
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
| | - Jestin George
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
| | - Tim Kahlke
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
| | - Michele Fabris
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
- CSIRO Synthetic Biology Future Science Platform, Brisbane, QLD 4001, Australia
| | - Matthew P. Padula
- School of Life Sciences and Proteomics Core Facility, Faculty of Science, University of Technology Sydney, Ultimo NSW 2007, Sydney, Australia;
| | - Angelo Lopez
- Department of Chemistry, University of York, York, YO10 5DD, UK;
| | - Peter J. Ralph
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
| | - Mathieu Pernice
- Climate Change Cluster, University of Technology Sydney, Broadway Campus, Ultimo NSW 2007, Sydney, Australia; (R.M.A.); (A.S.C.); (J.G.); (T.K.); (M.F.); (P.J.R.)
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Siddiqui A, Wei Z, Boehm M, Ahmad N. Engineering microalgae through chloroplast transformation to produce high‐value industrial products. Biotechnol Appl Biochem 2020; 67:30-40. [DOI: 10.1002/bab.1823] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2019] [Accepted: 09/16/2019] [Indexed: 12/18/2022]
Affiliation(s)
- Ayesha Siddiqui
- Agricultural Biotechnology DivisionNational Institute for Biotechnology & Genetic Engineering (NIBGE) Faisalabad Pakistan
| | - Zhengyi Wei
- Institute of Agricultural BiotechnologyJilin Academy of Agricultural Sciences Changchun Jilin Province People's Republic of China
| | - Marko Boehm
- Botanical InstituteChristian‐Albrechts‐University Kiel Germany
| | - Niaz Ahmad
- Agricultural Biotechnology DivisionNational Institute for Biotechnology & Genetic Engineering (NIBGE) Faisalabad Pakistan
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Pang X, Tong Y, Li F, Wei X, Chen X, Liu J, Chen D. Expression and characterization of human lactoferrin with tandem zinc finger protein in Chlamydomonas reinhardtii. ALGAL RES 2019. [DOI: 10.1016/j.algal.2019.101635] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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M U N, Mehar JG, Mudliar SN, Shekh AY. Recent Advances in Microalgal Bioactives for Food, Feed, and Healthcare Products: Commercial Potential, Market Space, and Sustainability. Compr Rev Food Sci Food Saf 2019; 18:1882-1897. [PMID: 33336956 DOI: 10.1111/1541-4337.12500] [Citation(s) in RCA: 78] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 07/24/2019] [Accepted: 08/13/2019] [Indexed: 12/20/2022]
Abstract
To combat food scarcity as well as to ensure nutritional food supply for sustainable living of increasing population, microalgae are considered as innovative sources for adequate nutrition. Currently, the dried biomass, various carotenoids, phycocyanin, phycoerythrin, omega fatty acids, and enzymes are being used as food additives, food coloring agents, and food supplements. Apart from nutritional importance, microalgae are finding the place in the market as "functional foods." When compared to the total market size of food and feed products derived from all the possible sources, the market portfolio of microalgae-based products is still smaller, but increasing steadily. On the other hand, the genetic modification of microalgae for enhanced production of commercially important metabolites holds a great potential. However, the success of commercial application of genetically modified (GM) algae will be defined by their safety to human health and environment. In view of this, the present study attempts to highlight the industrially important microalgal metabolites, their production, and application in food, feed, nutraceuticals, pharmaceuticals, and cosmeceuticals. The current and future market trends for microalgal products have been thoroughly discussed. Importantly, the safety pertaining to microalgae cultivation and consumption, and regulatory issues for GM microalgae have also been covered.
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Affiliation(s)
- Nethravathy M U
- Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Inst. (CFTRI), Mysore, 570020, India
| | - Jitendra G Mehar
- Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Inst. (CFTRI), Mysore, 570020, India.,Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India
| | - Sandeep N Mudliar
- Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Inst. (CFTRI), Mysore, 570020, India.,Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India
| | - Ajam Y Shekh
- Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Inst. (CFTRI), Mysore, 570020, India.,Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India
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Fu W, Nelson DR, Mystikou A, Daakour S, Salehi-Ashtiani K. Advances in microalgal research and engineering development. Curr Opin Biotechnol 2019; 59:157-164. [PMID: 31252302 DOI: 10.1016/j.copbio.2019.05.013] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Revised: 05/17/2019] [Accepted: 05/22/2019] [Indexed: 02/07/2023]
Abstract
Microalgae have been investigated for the photosynthetic production of natural products with industrial and biomedical applications. Their rapid growth offers an advantage over higher plants, while their complex metabolic capacities allow for the production of various molecules. Despite their potentials, molecular techniques are underdeveloped in microalgae compared to higher plants, fungi, and bacteria. However, recent advances in genome sequencing, strain development, and genome editing technologies, are providing thrust to enhance research on microalgal species that have branched out from several focal model organisms to encompass a great diversity of species. In this review, we highlight the recent, significant advances in microalgal research, with a focus on the development of new resources that can enhance work on model and non-model species.
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Affiliation(s)
- Weiqi Fu
- Laboratory of Algal, Systems, and Synthetic Biology, Division of Science and Math, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates; Center for Systems Biology and Faculty of Industrial Engineering, Mechanical Engineering and Computer Science, School of Engineering and Natural Sciences, University of Iceland, 101 Reykjavík, Iceland
| | - David R Nelson
- Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates
| | - Alexandra Mystikou
- Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates
| | - Sarah Daakour
- Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates
| | - Kourosh Salehi-Ashtiani
- Laboratory of Algal, Systems, and Synthetic Biology, Division of Science and Math, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates; Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
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Tevatia R, Payne S, Allen J, White D, Clemente TE, Cerutti H, Demirel Y, Blum P. A synthetic cdo/csad taurine pathway in the green unicellular alga Chlamydomonas reinhardtii. ALGAL RES 2019. [DOI: 10.1016/j.algal.2019.101491] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
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Hosts for Hostile Protein Production: The Challenge of Recombinant Immunotoxin Expression. Biomedicines 2019; 7:biomedicines7020038. [PMID: 31108917 PMCID: PMC6630761 DOI: 10.3390/biomedicines7020038] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2019] [Revised: 05/07/2019] [Accepted: 05/13/2019] [Indexed: 12/12/2022] Open
Abstract
For the recombinant expression of toxin-based drugs, a crucial step lies not only in the choice of the production host(s) but also in the accurate design of the protein chimera. These issues are particularly important since such products may be toxic to the expressing host itself. To avoid or limit the toxicity to productive cells while obtaining a consistent yield in chimeric protein, several systems from bacterial to mammalian host cells have been employed. In this review, we will discuss the development of immunotoxin (IT) expression, placing special emphasis on advantages and on potential drawbacks, as one single perfect host for every chimeric protein toxin or ligand does not exist.
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Kwon KC, Lamb A, Fox D, Porphy Jegathese SJ. An evaluation of microalgae as a recombinant protein oral delivery platform for fish using green fluorescent protein (GFP). FISH & SHELLFISH IMMUNOLOGY 2019; 87:414-420. [PMID: 30703550 DOI: 10.1016/j.fsi.2019.01.038] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/19/2018] [Revised: 01/15/2019] [Accepted: 01/25/2019] [Indexed: 05/15/2023]
Abstract
Recombinant proteins produced by biological systems such as bacteria, yeasts, mammalian and insect cell cultures are widely used for clinical or industrial purposes. Most therapeutic protein drugs require purification, cold chain, and injection, which make them prohibitively expensive and hinders their widespread use. Here, we describe a new economical oral vaccination platform using algae and evaluated its potential for the delivery of recombinant drugs using GFP expressed in the chloroplast of algal cells. The transplastomic algae expressing recombinant GFPs were freeze-dried for long-term storage at ambient temperature and for easy handling in feeding. GFPs bioencapsulated by lyophilized Chlamydomonas reinhardtii were found intact without degradation for several months at ambient temperature. The expression level of GFP in the lyophilized algae was estimated at 0.47 μg/mg dry weight. The GFPs bioencapsulated and orally delivered to Danio rerio were immunostained and observed in the intestinal tissues using a confocal microscope. Furthermore, the uptaken GFPs in the intestine were detected in the blood using ELISA and the detected level was 5.4 ng of GFP/μl of serum. These results demonstrate that microalgae can be a viable protein production and oral delivery system to vaccinate fish. The results give greater justification to continue exploring the concept of microalgal-based oral vaccines. The potential of the technology would greatly benefit aquaculture farmers by providing them with affordable, environmentally sustainable, and user-friendly vaccines.
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Affiliation(s)
- Kwang-Chul Kwon
- MicroSynbiotiX Ltd, 11011 N Torrey Pines Rd Ste. #135, La Jolla, CA, 92037, USA.
| | - Antonio Lamb
- MicroSynbiotiX Ltd, 11011 N Torrey Pines Rd Ste. #135, La Jolla, CA, 92037, USA
| | - David Fox
- Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA
| | - Simon Jegan Porphy Jegathese
- MicroSynbiotiX Ltd, University College, Cork, Food Science Building, Level 4, Lab 442, Microbiology Department, Cork, Republic of Ireland
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Current state-of-the-art in plant-based antibody production systems. Biotechnol Lett 2019; 41:335-346. [DOI: 10.1007/s10529-019-02651-z] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Accepted: 01/18/2019] [Indexed: 12/26/2022]
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Kwon YM, Kim KW, Choi TY, Kim SY, Kim JYH. Manipulation of the microalgal chloroplast by genetic engineering for biotechnological utilization as a green biofactory. World J Microbiol Biotechnol 2018; 34:183. [PMID: 30478596 DOI: 10.1007/s11274-018-2567-8] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2018] [Accepted: 11/23/2018] [Indexed: 12/16/2022]
Abstract
The chloroplast is an essential organelle in microalgae for conducting photosynthesis, thus enabling the photoautotrophic growth of microalgae. In addition to photosynthesis, the chloroplast is capable of various biochemical processes for the synthesis of proteins, lipids, carbohydrates, and terpenoids. Due to these attractive characteristics, there has been increasing interest in the biotechnological utilization of microalgal chloroplast as a sustainable alternative to the conventional production platforms used in industrial biotechnology. Since the first demonstration of microalgal chloroplast transformation, significant development has occurred over recent decades in the manipulation of microalgal chloroplasts through genetic engineering. In the present review, we describe the advantages of the microalgal chloroplast as a production platform for various bioproducts, including recombinant proteins and high-value metabolites, features of chloroplast genetic systems, and the development of transformation methods, which represent important factors for gene expression in the chloroplast. Furthermore, we address the expression of various recombinant proteins in the microalgal chloroplast through genetic engineering, including reporters, biopharmaceutical proteins, and industrial enzymes. Finally, we present many efforts and achievements in the production of high-value metabolites in the microalgal chloroplast through metabolic engineering. Based on these efforts and advances, the microalgal chloroplast represents an economically viable and sustainable platform for biotechnological applications in the near future.
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Affiliation(s)
- Yong Min Kwon
- Department of Applied Research, National Marine Biodiversity Institute of Korea, Jangsan-ro 101-75, Seocheon, Chungcheongnamdo, 33662, Republic of Korea
| | - Kyung Woo Kim
- Department of Applied Research, National Marine Biodiversity Institute of Korea, Jangsan-ro 101-75, Seocheon, Chungcheongnamdo, 33662, Republic of Korea
| | - Tae-Young Choi
- Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea, Jangsan-ro 101-75, Seocheon, Chungcheongnamdo, 33662, Republic of Korea
| | - Sun Young Kim
- Department of Applied Research, National Marine Biodiversity Institute of Korea, Jangsan-ro 101-75, Seocheon, Chungcheongnamdo, 33662, Republic of Korea
| | - Jaoon Young Hwan Kim
- Department of Applied Research, National Marine Biodiversity Institute of Korea, Jangsan-ro 101-75, Seocheon, Chungcheongnamdo, 33662, Republic of Korea.
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Macedo-Osorio KS, Pérez-España VH, Garibay-Orijel C, Guzmán-Zapata D, Durán-Figueroa NV, Badillo-Corona JA. Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons. PLANT MOLECULAR BIOLOGY 2018; 98:303-317. [PMID: 30225747 DOI: 10.1007/s11103-018-0776-z] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/02/2018] [Accepted: 08/31/2018] [Indexed: 05/21/2023]
Abstract
Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3'-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.
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Affiliation(s)
- Karla S Macedo-Osorio
- Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto SN, Col. Barrio la Laguna Ticoman, Mexico City, Mexico
| | - Víctor H Pérez-España
- Universidad Autónoma del Estado de Hidalgo, Escuela Superior de Apan, Carretera Apan Calpulalpan km 8, Col. Chimalpa-Tlalayote, Apan, Hidalgo, Mexico
| | - Claudio Garibay-Orijel
- Labcitec, Camino a Atzacoalco 99, Col. Constitución de la República, Mexico City, Mexico
| | - Daniel Guzmán-Zapata
- Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto SN, Col. Barrio la Laguna Ticoman, Mexico City, Mexico
| | - Noé V Durán-Figueroa
- Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto SN, Col. Barrio la Laguna Ticoman, Mexico City, Mexico
| | - Jesús A Badillo-Corona
- Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto SN, Col. Barrio la Laguna Ticoman, Mexico City, Mexico.
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