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Schilling T, Biedendieck R, Moran-Torres R, Saaranen MJ, Ruddock LW, Daniel R, van Dijl JM. Toward Antibody Production in Genome-Minimized Bacillus subtilis Strains. ACS Synth Biol 2025; 14:740-755. [PMID: 40013841 PMCID: PMC11934139 DOI: 10.1021/acssynbio.4c00688] [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] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2024] [Revised: 01/10/2025] [Accepted: 02/13/2025] [Indexed: 02/28/2025]
Abstract
Bacillus subtilis is a bacterial cell factory with outstanding protein secretion capabilities that has been deployed as a workhorse for the production of industrial enzymes for more than a century. Nevertheless, the production of other proteins with B. subtilis, such as antibody formats, has thus far been challenging due to specific requirements that relate to correct protein folding and disulfide bond formation upon export from the cytoplasm. In the present study, we explored the possibility of producing functional antibody formats, such as scFvs and scFabs, using the genome-reduced Midi- and MiniBacillus strain lineage. The applied workflow included selection of optimal chassis strains, appropriate expression vectors, signal peptides, growth media, and analytical methods to verify the functionality of the secreted antibody fragments. The production of scFv fragments was upscaled to the 1 L bioreactor level. As demonstrated for a human C-reactive protein-binding scFv antibody by mass spectrometry, biolayer interferometry, circular dichroism, free thiol cross-linking with N-ethylmaleimide, and nano-differential scanning fluorimetry, MidiBacillus strains can secrete fully functional, natively folded, disulfide-bonded, and thermostable antibody fragments. We therefore conclude that genome-reduced B. subtilis chassis strains are capable of secreting high-quality antibody fragments.
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Affiliation(s)
- Tobias Schilling
- University
Medical Center Groningen, Department of Medical Microbiology, University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700RB Groningen, The Netherlands
| | - Rebekka Biedendieck
- Braunschweig
Centre of Systems Biology (BRICS) and Institute of Microbiology, Technische Universität Braunschweig, Rebenring 56, 38106 Braunschweig, Germany
| | - Rafael Moran-Torres
- Theoretical
Biophysics, Humboldt-Universität
zu Berlin, 10115 Berlin, Germany
| | - Mirva J. Saaranen
- Faculty
of Biochemistry and Molecular Medicine, Protein and Structural Biology
Research Unit, University of Oulu, Aapistie 7B, 90220 Oulu, Finland
| | - Lloyd W. Ruddock
- Faculty
of Biochemistry and Molecular Medicine, Protein and Structural Biology
Research Unit, University of Oulu, Aapistie 7B, 90220 Oulu, Finland
| | - Rolf Daniel
- Institute
of Microbiology and Genetics, Department of Genomic and Applied Microbiology, Georg-August-Universität Göttingen, Grisebachstr. 8, 37077 Göttingen, Germany
| | - Jan Maarten van Dijl
- University
Medical Center Groningen, Department of Medical Microbiology, University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700RB Groningen, The Netherlands
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2
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Santos‐Beneit F. What is the role of microbial biotechnology and genetic engineering in medicine? Microbiologyopen 2024; 13:e1406. [PMID: 38556942 PMCID: PMC10982607 DOI: 10.1002/mbo3.1406] [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: 01/12/2024] [Revised: 02/26/2024] [Accepted: 03/12/2024] [Indexed: 04/02/2024] Open
Abstract
Microbial products are essential for developing various therapeutic agents, including antibiotics, anticancer drugs, vaccines, and therapeutic enzymes. Genetic engineering techniques, functional genomics, and synthetic biology unlock previously uncharacterized natural products. This review highlights major advances in microbial biotechnology, focusing on gene-based technologies for medical applications.
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Affiliation(s)
- Fernando Santos‐Beneit
- Institute of Sustainable ProcessesValladolidSpain
- Department of Chemical Engineering and Environmental Technology, School of Industrial EngineeringUniversity of ValladolidValladolidSpain
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3
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Zhou D, Wang X, Zou J, Song J, Su P, Yang Y, Wu L. Determination of [Glu 1]-fibrinopeptide B purity by gas chromatography - isotope dilution mass spectrometry. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2024; 16:1741-1747. [PMID: 38372017 DOI: 10.1039/d3ay02114a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/20/2024]
Abstract
The present work assessed the purity of [Glu1]-fibrinopeptide B (GFB) as a model peptide using gas chromatography - isotope dilution mass spectrometry. GFB and various isotope-labeled amino acids were hydrolyzed in HCl and then derivatized using optimized procedures. The primary impurity in GFB was also identified and used to correct the final result. A method repeatability of 0.5% was achieved and linear calibrations were obtained for five amino acids. The LOD and LOQ were 0.041 to 0.096 μg g-1, and 0.16 to 0.56 μg g-1, respectively. The purity of GFB was found to be (0.715 ± 0.012) g g-1. This technique exhibited comparable accuracy to that obtainable from liquid chromatography - isotope dilution mass spectrometry but at lower cost. This method could be employed as a reference technique or in fields such as clinical diagnostics or bio-pharmaceutical peptide purity analysis.
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Affiliation(s)
- Dongmei Zhou
- National Institute of Metrology, P.R. China, No. 18, North Third Ring East Road, Beijing, China.
| | - Xianxia Wang
- National Institute of Metrology, P.R. China, No. 18, North Third Ring East Road, Beijing, China.
| | - Jun Zou
- China Quality Certification Centre, Building 3, Zone 9, No. 188, South Fourth Ring West Road, Fengtai District, Beijing, China.
| | - Jiayi Song
- Beijing University of Chemical Technology, North Third Ring Road 15, Chaoyang District, Beijing, China.
| | - Ping Su
- Beijing University of Chemical Technology, North Third Ring Road 15, Chaoyang District, Beijing, China.
| | - Yi Yang
- Beijing University of Chemical Technology, North Third Ring Road 15, Chaoyang District, Beijing, China.
| | - Liqing Wu
- National Institute of Metrology, P.R. China, No. 18, North Third Ring East Road, Beijing, China.
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4
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Schilling T, Ferrero-Bordera B, Neef J, Maaβ S, Becher D, van Dijl JM. Let There Be Light: Genome Reduction Enables Bacillus subtilis to Produce Disulfide-Bonded Gaussia Luciferase. ACS Synth Biol 2023; 12:3656-3668. [PMID: 38011677 PMCID: PMC10729301 DOI: 10.1021/acssynbio.3c00444] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2023] [Revised: 11/09/2023] [Accepted: 11/17/2023] [Indexed: 11/29/2023]
Abstract
Bacillus subtilis is a major workhorse for enzyme production in industrially relevant quantities. Compared to mammalian-based expression systems, B. subtilis presents intrinsic advantages, such as high growth rates, high space-time yield, unique protein secretion capabilities, and low maintenance costs. However, B. subtilis shows clear limitations in the production of biopharmaceuticals, especially proteins from eukaryotic origin that contain multiple disulfide bonds. In the present study, we deployed genome minimization, signal peptide screening, and coexpression of recombinant thiol oxidases as strategies to improve the ability of B. subtilis to secrete proteins with multiple disulfide bonds. Different genome-reduced strains served as the chassis for expressing the model protein Gaussia Luciferase (GLuc), which contains five disulfide bonds. These chassis lack extracellular proteases, prophages, and key sporulation genes. Importantly, compared to the reference strain with a full-size genome, the best-performing genome-minimized strain achieved over 3000-fold increased secretion of active GLuc while growing to lower cell densities. Our results show that high-level GLuc secretion relates, at least in part, to the absence of major extracellular proteases. In addition, we show that the thiol-disulfide oxidoreductase requirements for disulfide bonding have changed upon genome reduction. Altogether, our results highlight genome-engineered Bacillus strains as promising expression platforms for proteins with multiple disulfide bonds.
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Affiliation(s)
- Tobias Schilling
- Department
of Medical Microbiology, University of Groningen,
University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700RB Groningen, The Netherlands
| | - Borja Ferrero-Bordera
- Institute
of Microbiology Department of Microbial Proteomics, University of Greifswald, D-17489 Greifswald, Germany
| | - Jolanda Neef
- Department
of Medical Microbiology, University of Groningen,
University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700RB Groningen, The Netherlands
| | - Sandra Maaβ
- Institute
of Microbiology Department of Microbial Proteomics, University of Greifswald, D-17489 Greifswald, Germany
| | - Dörte Becher
- Institute
of Microbiology Department of Microbial Proteomics, University of Greifswald, D-17489 Greifswald, Germany
| | - Jan Maarten van Dijl
- Department
of Medical Microbiology, University of Groningen,
University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700RB Groningen, The Netherlands
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5
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Kim MS, Jeong DE, Choi SK. Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains. Microb Cell Fact 2022; 21:259. [PMID: 36517844 PMCID: PMC9753358 DOI: 10.1186/s12934-022-01989-w] [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: 08/19/2022] [Accepted: 12/09/2022] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND Owing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems. RESULTS Here, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains. CONCLUSION The Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.
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Affiliation(s)
- Man Su Kim
- grid.249967.70000 0004 0636 3099Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 South Korea ,grid.412786.e0000 0004 1791 8264Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon, 34113 South Korea
| | - Da-Eun Jeong
- grid.249967.70000 0004 0636 3099Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 South Korea
| | - Soo-Keun Choi
- grid.249967.70000 0004 0636 3099Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141 South Korea ,grid.412786.e0000 0004 1791 8264Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon, 34113 South Korea
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6
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Krüger A, Welsch N, Dürwald A, Brundiek H, Wardenga R, Piascheck H, Mengers HG, Krabbe J, Beyer S, Kabisch JF, Popper L, Hübel T, Antranikian G, Schweder T. A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis. Appl Microbiol Biotechnol 2022; 106:5137-5151. [PMID: 35802157 PMCID: PMC9329435 DOI: 10.1007/s00253-022-12062-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2022] [Revised: 06/24/2022] [Accepted: 06/28/2022] [Indexed: 11/29/2022]
Abstract
Abstract
Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1β, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. Key points • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-12062-2.
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Affiliation(s)
- Anna Krüger
- Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany
| | - Norma Welsch
- Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany.,Institute of Marine Biotechnology, Walther-Rathenau-Str. 49, 17489, Greifswald, Germany
| | - Alexandra Dürwald
- Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany
| | - Henrike Brundiek
- Enzymicals AG, Walther-Rathenau-Straße 49a, 17489, Greifswald, Germany
| | - Rainer Wardenga
- Enzymicals AG, Walther-Rathenau-Straße 49a, 17489, Greifswald, Germany
| | - Henning Piascheck
- Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany
| | - Hendrik G Mengers
- Institute of Applied Microbiology - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany
| | - Jana Krabbe
- Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany.,Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745, Jena, Germany
| | - Sandra Beyer
- Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany.,Bioprocess Center, Eppendorf AG, Rudolf-Schulten-Str. 5, 52428, Jülich, Germany
| | - Johannes F Kabisch
- Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany.,Department of Biotechnology and Food Science, NTNU, Sem Sælands vei 6, 7034, Trondheim, Norway
| | - Lutz Popper
- Stern Enzym GmbH & Co. KG, Kurt-Fischer-Str. 55, 22926, Ahrensburg, Germany
| | - Tanno Hübel
- Miltenyi Biotec GmbH, Robert-Koch-Str. 1, 17166, Teterow, Germany
| | - Garabed Antranikian
- Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany
| | - Thomas Schweder
- Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany. .,Institute of Marine Biotechnology, Walther-Rathenau-Str. 49, 17489, Greifswald, Germany.
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7
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Microbial protein cell factories fight back? Trends Biotechnol 2021; 40:576-590. [PMID: 34924209 DOI: 10.1016/j.tibtech.2021.10.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2021] [Revised: 10/01/2021] [Accepted: 10/01/2021] [Indexed: 01/26/2023]
Abstract
The biopharmaceutical market is growing faster than ever, with two production systems competing for market dominance: mammalian cells and microorganisms. In recent years, based on the rise of antibody-based therapies, new biotherapeutic approvals have favored mammalian hosts. However, not only has extensive research elevated our understanding of microbes to new levels, but emerging therapeutic molecules also facilitate their use; thus, is it time for microbes to fight back? In this review, we answer this timely question by cross-comparing four microbial production hosts and examining the innovations made to both their secretion and post-translational modification (PTM) capabilities. Furthermore, we discuss the impact of tools, such as omics and systems biology, as well as alternative production systems and emerging biotherapeutics.
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8
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Fermentative production of alternative antimicrobial peptides and enzymes. BIOCATALYSIS AND AGRICULTURAL BIOTECHNOLOGY 2021. [DOI: 10.1016/j.bcab.2021.102189] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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9
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Harwood CR, Kikuchi Y. The ins and outs of Bacillus proteases: activities, functions and commercial significance. FEMS Microbiol Rev 2021; 46:6354784. [PMID: 34410368 PMCID: PMC8767453 DOI: 10.1093/femsre/fuab046] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Accepted: 08/17/2021] [Indexed: 12/23/2022] Open
Abstract
Because the majority of bacterial species divide by binary fission, and do not have distinguishable somatic and germline cells, they could be considered to be immortal. However, bacteria ‘age’ due to damage to vital cell components such as DNA and proteins. DNA damage can often be repaired using efficient DNA repair mechanisms. However, many proteins have a functional ‘shelf life’; some are short lived, while others are relatively stable. Specific degradation processes are built into the life span of proteins whose activities are required to fulfil a specific function during a prescribed period of time (e.g. cell cycle, differentiation process, stress response). In addition, proteins that are irreparably damaged or that have come to the end of their functional life span need to be removed by quality control proteases. Other proteases are involved in performing a variety of specific functions that can be broadly divided into three categories: processing, regulation and feeding. This review presents a systematic account of the proteases of Bacillus subtilis and their activities. It reviews the proteases found in, or associated with, the cytoplasm, the cell membrane, the cell wall and the external milieu. Where known, the impacts of the deletion of particular proteases are discussed, particularly in relation to industrial applications.
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Affiliation(s)
- Colin R Harwood
- Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University NE2 4AX, Newcastle upon Tyne, UK
| | - Yoshimi Kikuchi
- Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., Kawasaki 210-8681, JAPAN
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10
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Appelbaum M, Schweder T. Metabolic Engineering of
Bacillus
– New Tools, Strains, and Concepts. Metab Eng 2021. [DOI: 10.1002/9783527823468.ch13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
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11
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The "beauty in the beast"-the multiple uses of Priestia megaterium in biotechnology. Appl Microbiol Biotechnol 2021; 105:5719-5737. [PMID: 34263356 PMCID: PMC8390425 DOI: 10.1007/s00253-021-11424-6] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Revised: 06/17/2021] [Accepted: 06/21/2021] [Indexed: 01/05/2023]
Abstract
Abstract Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. Key points • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.
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12
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Irla M, Drejer EB, Brautaset T, Hakvåg S. Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus. Microb Cell Fact 2020; 19:151. [PMID: 32723337 PMCID: PMC7389648 DOI: 10.1186/s12934-020-01409-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2019] [Accepted: 07/20/2020] [Indexed: 01/06/2023] Open
Abstract
BACKGROUND The suitability of bacteria as microbial cell factories is dependent on several factors such as price of feedstock, product range, production yield and ease of downstream processing. The facultative methylotroph Bacillus methanolicus is gaining interest as a thermophilic cell factory for production of value-added products from methanol. The aim of this study was to expand the capabilities of B. methanolicus as a microbial cell factory by establishing a system for secretion of recombinant proteins. RESULTS Native and heterologous signal peptides were tested for secretion of α-amylases and proteases, and we have established the use of the thermostable superfolder green fluorescent protein (sfGFP) as a valuable reporter protein in B. methanolicus. We demonstrated functional production and secretion of recombinant proteases, α-amylases and sfGFP in B. methanolicus MGA3 at 50 °C and showed that the choice of signal peptide for optimal secretion efficiency varies between proteins. In addition, we showed that heterologous production and secretion of α-amylase from Geobacillus stearothermophilus enables B. methanolicus to grow in minimal medium with starch as the sole carbon source. An in silico signal peptide library consisting of 169 predicted peptides from B. methanolicus was generated and will be useful for future studies, but was not experimentally investigated any further here. CONCLUSION A functional system for recombinant production of secreted proteins at 50 °C has been established in the thermophilic B. methanolicus. In addition, an in silico signal peptide library has been generated, that together with the tools and knowledge presented in this work will be useful for further development of B. methanolicus as a host for recombinant protein production and secretion at 50 °C.
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Affiliation(s)
- Marta Irla
- Department of Biotechnology and Food Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, Norway
| | - Eivind B Drejer
- Department of Biotechnology and Food Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, Norway
| | - Trygve Brautaset
- Department of Biotechnology and Food Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, Norway
| | - Sigrid Hakvåg
- Department of Biotechnology and Food Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
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13
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Efficient synthesis of 2-phenylethanol from L-phenylalanine by engineered Bacillus licheniformis using molasses as carbon source. Appl Microbiol Biotechnol 2020; 104:7507-7520. [DOI: 10.1007/s00253-020-10740-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2020] [Revised: 05/31/2020] [Accepted: 06/09/2020] [Indexed: 01/07/2023]
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14
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Fang CW, Tsai LC, Fu YS, Cheng TY, Wu PC. Gel-based Microemulsion Design and Evaluation for Topical Application of Rivastigmine. Curr Pharm Biotechnol 2019; 21:298-304. [PMID: 31729297 DOI: 10.2174/1389201020666191113144636] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Revised: 07/04/2019] [Accepted: 10/24/2019] [Indexed: 11/22/2022]
Abstract
OBJECTIVE The aim of the present study was to design nanocarriers for the topical application of rivastigmine. METHODS The effect of cosurfactants, hydrophilic gel and loading amount on the permeability of rivastigmine through rat skin was evaluated. Skin irritation tests and stability tests were performed to evaluate the utility of tested formulations. RESULTS The results showed that the microemulsion formation and characteristics of drug-loaded formulations were related to many parameters of the components. When using microemulsion systems as a vehicle, the permeation rate remarkably increased about 13.2~24.3-fold and the lag time was significantly shortened from 24 h to 4.7 h. Formulations containing a cosurfactant of Diethylene Glycol Monobutyl Ether (DEGBE) showed higher enhancement effect, while increasing the loading dose from 0.5% to 5% further increased the flux about 2.1-fold and shortened the lag time. CONCLUSION The drug-loaded experimental formulation did not cause skin irritation and had good stability at 20ºC and 40ºC storage for at least 3 months. The result showed that gel-based microemulsion formulation could be a promising approach for topical administration.
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Affiliation(s)
- Chih-Wen Fang
- Division of Pharmacy, Zuoying Branch of Kaohsiung Armed Forces General Hospital, 553 Junxiao Road, Kaohsiung City 813, Taiwan, China
| | - Ling-Chun Tsai
- School of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan, China
| | - Yaw-Syan Fu
- Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan, China
| | - Ting-Yu Cheng
- School of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan, China
| | - Pao-Chu Wu
- School of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan, China.,Department of Medical Research, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan, China
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15
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Cobos-Puc L, Rodríguez-Herrera R, Cano-Cabrera JC, Aguayo-Morales H, Silva-Belmares SY, Gallegos ACF, Hernández JLM. Classical and New Pharmaceutical Uses of Bacterial Penicillin G Acylase. Curr Pharm Biotechnol 2019; 21:287-297. [PMID: 31713475 DOI: 10.2174/1389201020666191111151642] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Revised: 10/28/2019] [Accepted: 10/30/2019] [Indexed: 12/16/2022]
Abstract
BACKGROUND β-lactam antibiotics are the most used worldwide for the treatment of bacterial infections. The consumption of these classes of drugs is high, and it is increasing around the world. To date, the best way to produce them is using penicillin G Acylase (PGA) as a biocatalyst. OBJECTIVE This manuscript offers an overview of the most recent advances in the current tools to improve the activity of the PGA and its pharmaceutical application. RESULTS Several microorganisms produce PGA, but some bacterial strains represent the primary source of this enzyme. The activity of bacterial PGA depends on its adequate expression and carbon or nitrogen source, as well as a specific pH or temperature depending on the nature of the PGA. Additionally, the PGA activity can be enhanced by immobilizing it to a solid support to recycle it for a prolonged time. Likewise, PGAs more stable and with higher activity are obtained from bacterial hosts genetically modified. CONCLUSION PGA is used to produce b-lactam antibiotics. However, this enzyme has pharmaceutical potential to be used to obtain critical molecules for the synthesis of anti-tumor, antiplatelet, antiemetic, antidepressive, anti-retroviral, antioxidant, and antimutagenic drugs.
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Affiliation(s)
- Luis Cobos-Puc
- Department of Biotechnology, Faculty of Chemistry, Autonomous University of Coahuila, Saltillo, Coahuila, Mexico
| | - Raúl Rodríguez-Herrera
- Department of Food Research, Faculty of Chemistry, Autonomous University of Coahuila, Saltillo, Coahuila, Mexico
| | - Juan C Cano-Cabrera
- Department of Biotechnology, Faculty of Chemistry, Autonomous University of Coahuila, Saltillo, Coahuila, Mexico
| | - Hilda Aguayo-Morales
- Department of Biotechnology, Faculty of Chemistry, Autonomous University of Coahuila, Saltillo, Coahuila, Mexico
| | - Sonia Y Silva-Belmares
- Department of Food Research, Faculty of Chemistry, Autonomous University of Coahuila, Saltillo, Coahuila, Mexico
| | - Adriana C F Gallegos
- Department of Food Research, Faculty of Chemistry, Autonomous University of Coahuila, Saltillo, Coahuila, Mexico
| | - José L M Hernández
- Department of Food Research, Faculty of Chemistry, Autonomous University of Coahuila, Saltillo, Coahuila, Mexico
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16
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Murray EM, Allen CF, Handy TE, Huffine CA, Craig WR, Seaton SC, Wolfe AL. Development of a Robust and Quantitative High-Throughput Screening Method for Antibiotic Production in Bacterial Libraries. ACS OMEGA 2019; 4:15414-15420. [PMID: 31572841 PMCID: PMC6761686 DOI: 10.1021/acsomega.9b01461] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Accepted: 08/28/2019] [Indexed: 05/10/2023]
Abstract
Over the past 30 years, there has been a dramatic rise in the number of infections caused by multidrug-resistant bacteria, which have proliferated due to the misuse and overuse of antibiotics. Over this same time period, however, there has also been a decline in the number of antibiotics with novel mechanisms of action coming to market. Therefore, there is a growing need for an increase in the speed at which new antibiotics are discovered and developed. Natural products produced by bacteria have been and continue to be a robust source of novel antibiotics; however, new and complementary methods for screening large bacterial libraries for novel antibiotic production are needed due to the current agar methods being limited in scope, time consuming, and prone to error. Herein, we describe a rapid, robust, and quantitative high-throughput liquid culture screening method for antibiotic production by bacteria. This method has the ability to screen both mono- and coculture mixtures of bacteria in vitro and be adapted to other phenotypic natural product analyses. Over 260 bacterial species were screened in monoculture, and 38 and 34% were found to produce antibiotics capable of inhibition of Staphylococcus aureus or Escherichia coli, respectively, with 8 and 4% being classified as strong producers (≥30% growth inhibition), respectively. Bacteria found to not produce antibiotics in monoculture were also screened in coculture using an adaptation of this method. Of the more than 270 cocultures screened, 14 and 30% were found to produce antibiotics capable of inhibition of S. aureus or E. coli, respectively. Of those bacteria found to produce antibiotics in monoculture, 43 bacteria were subjected to 16S rRNA sequencing and found to be majority Pseudomonas (37%), Serratia (19%), and Bacillus (14%) bacteria, but two novel producers, Herbaspirillum and Kluyvera, were also found.
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Affiliation(s)
- Elizabeth M. Murray
- Department
of Biology and Department of Chemistry, University of
North Carolina Asheville, One University Heights, Asheville, North Carolina 28804, United States
| | - Catherine F. Allen
- Department
of Biology and Department of Chemistry, University of
North Carolina Asheville, One University Heights, Asheville, North Carolina 28804, United States
| | - Tess E. Handy
- Department
of Biology and Department of Chemistry, University of
North Carolina Asheville, One University Heights, Asheville, North Carolina 28804, United States
| | - Clair A. Huffine
- Department
of Biology and Department of Chemistry, University of
North Carolina Asheville, One University Heights, Asheville, North Carolina 28804, United States
| | - Whitney R. Craig
- Department
of Biology and Department of Chemistry, University of
North Carolina Asheville, One University Heights, Asheville, North Carolina 28804, United States
| | - Sarah C. Seaton
- Indigo Ag, 500 Rutherford Avenue, Boston, Massachusetts 02129, United States
| | - Amanda L. Wolfe
- Department
of Biology and Department of Chemistry, University of
North Carolina Asheville, One University Heights, Asheville, North Carolina 28804, United States
- E-mail:
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17
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Hackl E, Darkwah J, Smith G, Ermolina I. Effect of Arginine on the Aggregation of Protein in Freeze-Dried Formulations Containing Sugars and Polyol: II. BSA Reconstitution and Aggregation. AAPS PharmSciTech 2018; 19:2934-2947. [PMID: 29980982 DOI: 10.1208/s12249-018-1114-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2018] [Accepted: 06/24/2018] [Indexed: 11/30/2022] Open
Abstract
The current paper continues our study on the ability of L-arginine to prevent/reduce the aggregation of proteins that results from the various stresses during the lyophilisation and/or storage of lyophilized protein-based products. The first part of our study, i.e. formulation development, was devoted to the rational design and optimization of an L-arginine containing lyophilized formulation which can resist the natural tendency of L-arginine to absorb atmosphere moisture. Mannitol and trehalose were chosen among other excipients to be included in the protein-based formulation, as mannitol in a combination with L-arginine has been shown to reduce moisture sorption while trehalose provides a degree of lyoprotection. In the present study, a number of formulations, which comprised bovine serum albumin (BSA) with and without L-arginine, and with five different ratios of trehalose-to-mannitol (from 30:70 to 80:20) were lyophilised and assessed. The internal structures and the moisture sorption/retention of the lyophilized formulations were characterised. To study the effect of L-arginine on BSA solid-phase stability, the lyophilized powder was exposed to accelerated storage conditions (high moisture (75% RH) and temperature (22 or 45 °C)) for up to 24 h. The lyophilized BSA formulations were then reconstituted and solution-state protein aggregation assessed by turbidimetry at 360 nm and fluorescence spectroscopy using the thioflavin T assay. It was demonstrated that L-arginine can be used in protein-based freeze-dried formulations to significantly reduce the aggregation of protein during the manufacturing, storage and subsequent reconstitution. The results also revealed the importance of a sufficient amount of mannitol in the arginine-containing formulations.
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18
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Drejer EB, Hakvåg S, Irla M, Brautaset T. Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae. Microorganisms 2018; 6:microorganisms6020042. [PMID: 29748477 PMCID: PMC6027425 DOI: 10.3390/microorganisms6020042] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2018] [Revised: 05/02/2018] [Accepted: 05/03/2018] [Indexed: 01/17/2023] Open
Abstract
Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B.methanolicus, B.coagulans, B.smithii, B.licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production.
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Affiliation(s)
- Eivind B Drejer
- Department of Biotechnology and Food Science, NTNU: Norwegian University of Science and Technology, 7491 Trondheim, Norway.
| | - Sigrid Hakvåg
- Department of Biotechnology and Food Science, NTNU: Norwegian University of Science and Technology, 7491 Trondheim, Norway.
| | - Marta Irla
- Department of Biotechnology and Food Science, NTNU: Norwegian University of Science and Technology, 7491 Trondheim, Norway.
| | - Trygve Brautaset
- Department of Biotechnology and Food Science, NTNU: Norwegian University of Science and Technology, 7491 Trondheim, Norway.
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