The high level food-grade expression of glutamate decarboxylase in Bacillus subtilis through combination of genomic integration and free plasmid

Abstract

γ-Aminobutyric acid (GABA) is a bioactive compound with diverse physiological functions. It has a wide range of applications in food and medicine and mainly biosynthesized through glutamate decarboxylase (GAD) catalysis. Bacillus subtilis is recognized for its robust secretion capabilities and high food safety standards, making it a prevalent choice for recombinant protein expression. In traditional industrial enzyme production in B. subtilis, antibiotics are required to sustain plasmid stability. However, the incorporation of antibiotics fails to align with the criteria applicable to enzymes for use in the food industry. To eliminate the need for antibiotics in the production of GAD preparations, we constructed a marker-free recombinant strain B. subtilis WS9D-GAD. This strain was developed based on antibiotic-free multi-copy gene expression vector pUBDAL-amyL and d-alanine racemase (dal)—deficient B. subtilis WS9D, ultimately enabling the food-grade expression of GAD. To enhance GAD expression levels, we integrated the gadA expression cassette into the genome of B. subtilis using the Cre/lox system method. Additionally, strain WS9C6D-GAD was generated through the co-expression of free plasmid and genome integration, which carried the free plasmid pUBDAL-gadA and featured six copies of the gadA expression cassette within its genome. The enzyme activity during shake flask fermentation reached 28.15 U mL⁻¹, while the enzyme activity in high-density 3-L fermenter culture reached 199.49 U mL⁻¹, marking the highest level of food-grade GAD expression with a multitude of potential applications. This study presents an effective strategy for the expression of food-grade industrial enzymes in B. subtilis.

Keywords

Glutamate decarboxylase / Bacillus subtilis / Cre/lox system / Food-grade expression

References

1.	Hong J, Kim K. Crystal structure of γ-aminobutyrate aminotransferase in complex with a PLP-GABA adduct from Corynebacterium glutamicum. Biochem Biophys Res Commun, 2019, 514: 601-606,
2.	Hong AR, Kim YA, Bae JH, et al.. A possible link between parathyroid hormone secretion and local regulation of GABA in human parathyroid adenomas. J Clin Endocrinol Metab, 2016, 101: 2594-2601,
3.	Xu N, Wei L, Liu J. Biotechnological advances and perspectives of gamma-aminobutyric acid production. World J Microbiol Biotechnol, 2017, 33: 64,
4.	Piao X, Jiang S, Wang J, et al.. Pingchuan formula attenuates airway mucus hypersecretion via regulation of the PNEC-GABA-IL13-Muc5ac axis in asthmatic mice. Biomed Pharmacother, 2021, 140,
5.	Strandwitz P, Kim KH, Terekhova D, et al.. GABA-modulating bacteria of the human gut microbiota. Nat Microbiol, 2019, 4: 396-403,
6.	Sarasa SB, Mahendran R, Muthusamy G, Thankappan B, Selta DRF, Angayarkanni J. A brief review on the non-protein amino acid, gamma-amino butyric acid (GABA): its production and role in microbes. Curr Microbiol, 2020, 77: 534-544,
7.	Luo H, Liu Z, Xie F, et al.. Microbial production of gamma-aminobutyric acid: applications, state-of-the-art achievements, and future perspectives. Crit Rev Biotechnol, 2021, 41: 491-512,
8.	Yuan H, Zhang W, Xiao G, Zhan J. Efficient production of gamma-aminobutyric acid by engineered Saccharomyces cerevisiae with glutamate decarboxylases from Streptomyces. Biotechnol Appl Biochem, 2020, 67(2): 240-248,
9.	Jia M, Zhu Y, Wang L, Sun T, Pan H, Li H. pH auto-sustain-based fermentation supports efficient gamma-aminobutyric acid production by Lactobacillus brevis CD0817. Fermentation, 2022, 8(5): 208,
10.	Huang Y, Su L, Wu J. Pyridoxine supplementation improves the activity of recombinant glutamate decarboxylase and the enzymatic production of gama-aminobutyric acid. PLoS ONE, 2016, 11(7), pmcid: 4954698
11.	He W, Mu W, Jiang B, Yan X, Zhang T. Food-grade expression of d-psicose 3-epimerase with tandem repeat genes in Bacillus subtilis. J Agric Food Chem, 2016, 64: 5701-5707,
12.	Song Y, Nikoloff JM, Zhang D. Improving protein production on the level of regulation of both expression and secretion pathways in Bacillus subtilis. J Microbiol Biotechnol, 2015, 25: 963-977,
13.	Meissner L, Kauffmann K, Wengeler T, Mitsunaga H, Fukusaki E, Büchs J. Influence of nitrogen source and pH value on undesired poly (γ-glutamic acid) formation of a protease producing Bacillus licheniformis strain. J Ind Microbiol Biotechnol, 2015, 42: 1203-1215,
14.	Popov M, Petrov S, Kirilov K, Ivanov GNI. Segregational instability in E. coli of expression plasmids carrying human interferon gamma gene and its 3′-end truncated variants. Biotechnol Biotechnol Equip, 2009, 23: 840-843,
15.	Peubez I, Chaudet N, Mignon C, et al.. Antibiotic-free selection in E. coli: new considerations for optimal design and improved production. Microb Cell Fact, 2010, 9: 65, pmcid: 2941680
16.	Yang S, Kang Z, Cao W, Du G, Chen J. Construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in Bacillus subtilis. J Biotechnol, 2016, 219: 40-47,
17.	Li R, Takala TM, Qiao M, Xu H, Saris PEJ. Nisin-selectable food-grade secretion vector for Lactococcus lactis. Biotechnol Lett, 2011, 33: 797-803,
18.	Emond E, Lavallée R, Drolet G, Moineau S, LaPointe G. Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis. Appl Environ Microbiol, 2001, 67: 1700-1709, pmcid: 92788
19.	Xia Y, Chen W, Zhao J, Tian F, Zhang H, Ding X. Construction of a new food-grade expression system for Bacillus subtilis based on theta replication plasmids and auxotrophic complementation. Appl Microbiol Biotechnol, 2007, 76: 643-650,
20.	Wang Y, Weng J, Waseem R, Yin X, Zhang R, Shen Q. Bacillus subtilis genome editing using ssDNA with short homology regions. Nucleic Acids Res, 2012, 40, pmcid: 3384351
21.	Wenzel M, Altenbuchner J. Development of a markerless gene deletion system for Bacillus subtilis based on the mannose phosphoenolpyruvate-dependent phosphotransferase system. Microbiology (NY), 2015, 161: 1942-1949,
22.	Yan X, Yu H, Hong Q, Li S. Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microbiol, 2008, 74: 5556-5562, pmcid: 2546623
23.	Makarova KS, Wolf YI, Iranzo J, et al.. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol, 2020, 18: 67-83,
24.	Togawa Y, Nunoshiba T, Hiratsu K. Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus. Mol Genet Genomics, 2018, 293: 277-291,
25.	Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF. Stability of biocatalysts. Curr Opin Chem Biol, 2007, 11: 220-225,
26.	Chen H, Luo J, Zheng P, et al.. Application of Cre-lox gene switch to limit the cry expression in rice green tissues. Sci Rep, 2017, 7: 14505, pmcid: 5673937
27.	Zhang K, Su L, Duan X, Liu L, Wu J. High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system. Microb Cell Fact, 2017, 16: 32, pmcid: 5319110
28.	You C, Zhang X, Zhang YHP. Simple cloning via direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis. Appl Environ Microbiol, 2012, 78: 1593-1595, pmcid: 3294473
29.	Ling Q. Heterologous expression and fermentation optimization of E. coli glutamate decarboxylase in B. subtilis, and its application in preparation of GABA. Jiangnan University; 2021. https://doi.org/10.27169/d.cnki.gwqgu.2020.000254.
30.	Chen J, Jin Z, Gai Y, Sun J, Zhang D. A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker. Bioresour Bioprocess, 2017, 4: 9,
31.	Liu D, Zhang L, Xue W, Wang Y, Ju J, Zhao B. Knockout of the alanine racemase gene in Aeromonas hydrophila HBNUAh01 results in cell wall damage and enhanced membrane permeability. Fems Microbiol Lett, 2015, 362: fnv89,
32.	Wangyang D, Bo J, Wanmeng M, Tao Z. Construction of a recombinant Bacillus subtilis and food-grade expression of glutamate decarboxylase. J Food Sci Biotechnol, 2020, 39: 24-31,
33.	Zhang R, Yang Y, Wang J, Lin Y, Yan Y. Synthetic symbiosis combining plasmid displacement enables rapid construction of phenotype-stable strains. Metab Eng, 2019, 55: 85-91,
34.	Liu Y, Liu L, Li J, Du G, Chen J. Synthetic biology toolbox and chassis development in Bacillus subtilis. Trends Biotechnol, 2019, 37: 548-562,
35.	Lambert JM, Bongers RS, Kleerebezem M. Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Appl Environ Microbiol, 2007, 73: 1126-1135,

Funding

the National Natural Science Foundation of China(31901633); the Natural Science Foundation of Jiangsu Province(BE2023686); the Fundamental Research Funds for the Central Universities(JUSRP122012); Shenzhen Institute of Synthetic Biology Scientific Research Program(DWKF20210004)