芽胞杆菌耐受胁迫条件的机制及工业应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0183

摘要/Abstract

摘要：

芽胞杆菌作为一种极具潜力的底盘菌株，能够在多种工农业废物和极端环境中生长，也能生产出多种工业化产品，如食品、饲料、益生菌、植物生长促进剂、酶和生物活性化合物等。然而，尽管具有生产高效、成本低廉等优点，芽胞杆菌在发酵生产中依然存在几个瓶颈问题，导致其工业生产的巨大潜力难以得到充分利用。其中一个关键问题在于，发酵生产过程中的生长以及生产效率较易受到多种胁迫条件的限制，进而导致发酵生产的不彻底、不完全。因此，探究芽胞杆菌胁迫响应的影响因子及改造策略，发掘其多种代谢活动与生长性状间的联系，就可以增强芽胞杆菌的抗胁迫能力，进而提高芽胞杆菌在工业应用中的质与量。本文首先分析了芽胞杆菌的多种应激反应机制，为进一步提高芽胞杆菌胁迫耐受能力、构建一个高效生产且具有良好抗逆性能的底盘菌株，总结阐述了非理性设计筛选抗性菌株的多种策略，以及抗逆基因线路和高耐受性微生物底盘的多种构建方法。为推动芽胞杆菌胁迫耐受机制的研究及工业应用领域的拓展提供策略和思路。

关键词: 芽胞杆菌, 胁迫耐受, 改造策略, 工业应用

Abstract:

Bacillus, as a highly potential chassis strain, can grow in various industrial and agricultural waste and extreme conditions. They can also produce a variety of industrial products such as food, feed, probiotics, plant growth promoters, enzymes, and bioactive compounds. However, despite their advantages in efficient production and low cost, Bacillus still face several bottlenecks in fermentation production, limiting the full exploitation of their industrial potential. One of the key issues is that the growth and production efficiency during fermentation are easily restricted by various stress conditions, leading to incomplete and inefficient fermentation production. Therefore, exploring the influencing factors and modification strategies of Bacillus stress response, and exploring the relationship between its various metabolic activities and growth traits, can enhance the tolerance of Bacillus to stress and improve their quality and quantity in industrial applications.This article first analyzes the various stress response mechanisms of Bacillus, aiming to further enhance its stress tolerance and to construct a high-efficiency production strain with excellent resistance, and then summarizes the irrational design strategies for screening resistant strains. It also elaborates on the construction methods of stress-resistant gene circuits and high-tolerance microbial chassis. These efforts provide strategies and insights to advance research on stress tolerance mechanisms in Bacillus and expand its industrial applications.

Key words: Bacillus, tolerance to stress, modification strategy, industrial applications

韩钟娆, 霍毅欣, 郭淑元. 芽胞杆菌耐受胁迫条件的机制及工业应用[J]. 生物技术通报, 2024, 40(8): 24-38.

HAN Zhong-rao, HUO Yi-xin, GUO Shu-yuan. Mechanism and Industrial Application of Bacillus Tolerance to Stress Conditions[J]. Biotechnology Bulletin, 2024, 40(8): 24-38.

图/表 9

参考文献 105

[1]	Su Y, Liu C, Fang H, et al. Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine[J]. Microb Cell Fact, 2020, 19(1): 173. doi: 10.1186/s12934-020-01436-8 pmid: 32883293
[2]	Popp PF, Benjdia A, Strahl H, et al. The epipeptide YydF intrinsically triggers the cell envelope stress response of Bacillus subtilis and causes severe membrane perturbations[J]. Front Microbiol, 2020, 11: 151.
[3]	Herrmann LW, Letti LAJ, Penha RO, et al. Bacillus genus industrial applications and innovation: first steps towards a circular bioeconomy[J]. Biotechnol Adv, 2024, 70: 108300.
[4]	Nadezhdin E, Murphy N, Dalchau N, et al. Stochastic pulsing of gene expression enables the generation of spatial patterns in Bacillus subtilis biofilms[J]. Nat Commun, 2020, 11: 950. doi: 10.1038/s41467-020-14431-9 pmid: 32075967
[5]	Łubkowska B, Jeżewska-Frąckowiak J, Sroczyński M, et al. Analysis of industrial Bacillus species as potential probiotics for dietary supplements[J]. Microorganisms, 2023, 11(2): 488.
[6]	Stefanic P, Belcijan K, Kraigher B, et al. Kin discrimination promotes horizontal gene transfer between unrelated strains in Bacillus subtilis[J]. Nat Commun, 2021, 12(1): 3457. doi: 10.1038/s41467-021-23685-w pmid: 34103505
[7]	Luna-Bulbarela A, Romero-Gutiérrez MT, Tinoco-Valencia R, et al. Response of Bacillus velezensis 83 to interaction with Colletotrichum gloeosporioides resembles a Greek phalanx-style formation: a stress resistant phenotype with antibiosis capacity[J]. Microbiol Res, 2024, 280: 127592.
[8]	Rodriguez Ayala F, Bartolini M, Grau R. The stress-responsive alternative Sigma factor SigB of Bacillus subtilis and its relatives: an old friend with new functions[J]. Front Microbiol, 2020, 11: 1761. doi: 10.3389/fmicb.2020.01761 pmid: 33042030
[9]	Neissi A, Majidi Zahed H, Roshan R. Probiotic performance of B. subtilis MS. 45 improves aquaculture of rainbow trout Oncorhynchus mykiss during acute hypoxia stress[J]. Sci Rep, 2024, 14: 3720.
[10]	Wang RF, Li HB, Qin Z, et al. Antifungal activity and application of Bacillus tequilensis A13 in biocontrol of Rehmannia glutinosa root-rot disease[J]. Chem Biol Technol Agric, 2023, 10(1): 20.
[11]	Xiao JC, Cao MY, Lai KY, et al. Unveiling key metabolic pathways in Bacillus subtilis-mediated salt tolerance enhancement in Glycyrrhiza uralensis Fisch. through multi-omics analysis[J]. Environ Exp Bot, 2024, 219: 105631.
[12]	Zhu XK, Yang BT, Hao ZP, et al. Dietary supplementation with Weissella cibaria C-10 and Bacillus amyloliquefaciens T-5 enhance immunity against Aeromonas veronii infection in crucian carp(Carassiu auratus)[J]. Microb Pathog, 2022, 167: 105559.
[13]	Bozsó Z, Lapat V, Ott PG, et al. Disparate effects of two clerodane diterpenes of giant goldenrod(Solidago gigantea ait.)on Bacillus spizizenii[J]. Int J Mol Sci, 2024, 25(3): 1531.
[14]	Odo EO, Ikwuegbu JA, Obeagu EI, et al. Analysis of the antibacterial effects of turmeric on particular bacteria[J]. Medicine, 2023, 102(48): e36492.
[15]	Raz C, Shemesh M, Argov-Argaman N. The role of milk fat globule size in modulating the composition of postbiotics produced by Bacillus subtilis and their effect on mammary epithelial cells[J]. Food Chem, 2023, 427: 136730.
[16]	Woodley JM. Protein engineering of enzymes for process applications[J]. Curr Opin Chem Biol, 2013, 17(2): 310-316. doi: 10.1016/j.cbpa.2013.03.017 pmid: 23562542
[17]	Rath H, Reder A, Hoffmann T, et al. Management of osmoprotectant uptake hierarchy in Bacillus subtilis via a SigB-dependent antisense RNA[J]. Front Microbiol, 2020, 11: 622.
[18]	Kashef N, Hamblin MR. Can microbial cells develop resistance to oxidative stress in antimicrobial photodynamic inactivation?[J]. Drug Resist Updat, 2017, 31: 31-42.
[19]	Tian HY, Liu JW, Zhang YX, et al. Stress response and signalling of a low-temperature bioaugmentation system in decentralized wastewater treatment: degradation characteristics, community structure, and bioaugmented mechanisms[J]. J Environ Manage, 2023, 342: 118257.
[20]	Garre A, Egea JA, Iguaz A, et al. Relevance of the induced stress resistance when identifying the critical microorganism for microbial risk assessment[J]. Front Microbiol, 2018, 9: 1663. doi: 10.3389/fmicb.2018.01663 pmid: 30087669
[21]	Bombaywala S, Purohit HJ, Dafale NA. Mobility of antibiotic resistance and its co-occurrence with metal resistance in pathogens under oxidative stress[J]. J Environ Manage, 2021, 297: 113315.
[22]	Garre A, Huertas JP, González-Tejedor GA, et al. Mathematical quantification of the induced stress resistance of microbial populations during non-isothermal stresses[J]. Int J Food Microbiol, 2018, 266: 133-141. doi: S0168-1605(17)30517-2 pmid: 29216553
[23]	Shuryak I. Review of microbial resistance to chronic ionizing radiation exposure under environmental conditions[J]. J Environ Radioact, 2019, 196: 50-63.
[24]	Jia HY, Fan YS, Feng XD, et al. Enhancing stress-resistance for efficient microbial biotransformations by synthetic biology[J]. Front Bioeng Biotechnol, 2014, 2: 44. doi: 10.3389/fbioe.2014.00044 pmid: 25368869
[25]	Tibocha-Bonilla JD, Zuñiga C, Lekbua A, et al. Predicting stress response and improved protein overproduction in Bacillus subtilis[J]. NPJ Syst Biol Appl, 2022, 8(1): 50. doi: 10.1038/s41540-022-00259-0 pmid: 36575180
[26]	Davidson JF, Whyte B, Bissinger PH, et al. Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae[J]. Proc Natl Acad Sci USA, 1996, 93(10): 5116-5121. pmid: 8643537
[27]	Campos Guillen J, Jones GH, Saldaña Gutiérrez C, et al. Critical minireview: the fate of tRNA^Cys during oxidative stress in Bacillus subtilis[J]. Biomolecules, 2017, 7(1): 6.
[28]	Zhang QQ, Qian H, Li PY, et al. Insight into the evolution of microbial community and antibiotic resistance genes in anammox process induced by copper after recovery from oxytetracycline stress[J]. Bioresour Technol, 2021, 330: 124945.
[29]	Angelini LL, Dos Santos RAC, Fox G, et al. Pulcherrimin protects Bacillus subtilis against oxidative stress during biofilm development[J]. NPJ Biofilms Microbiomes, 2023, 9(1): 50.
[30]	Kikuchi K, Galera-Laporta L, Weatherwax C, et al. Electrochemical potential enables dormant spores to integrate environmental signals[J]. Science, 2022, 378(6615): 43-49. doi: 10.1126/science.abl7484 pmid: 36201591
[31]	Sun YJ, Hürlimann S, Garner E. Growth rate is modulated by monitoring cell wall precursors in Bacillus subtilis[J]. Nat Microbiol, 2023, 8(3): 469-480.
[32]	Arnaouteli S, Bamford NC, Stanley-Wall NR, et al. Bacillus subtilis biofilm formation and social interactions[J]. Nat Rev Microbiol, 2021, 19(9): 600-614. doi: 10.1038/s41579-021-00540-9 pmid: 33824496
[33]	Giammarinaro PI, Young MKM, Steinchen W, et al. Diadenosine tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis[J]. Nat Microbiol, 2022, 7(9): 1442-1452. doi: 10.1038/s41564-022-01193-x pmid: 35953658
[34]	Kei A. Anti-sigma factor-mediated cell surface stress responses in Bacillus subtilis[J]. Genes Genet Syst, 2018, 92(5): 223-234.
[35]	Jia ZX, Zhou JW, Han JZ, et al. Proteomics-based analysis of the stress response of Bacillus cereus spores under ultrasound and electrolyzed water treatment[J]. Ultrason Sonochem, 2023, 98: 106523.
[36]	Castro-Cerritos KV, Yasbin RE, Robleto EA, et al. Role of ribonucleotide reductase in Bacillus subtilis stress-associated mutagenesis[J]. J Bacteriol, 2017, 199(4): e00715-e00716.
[37]	Zhao H, Sachla AJ, Helmann JD. Mutations of the Bacillus subtilis YidC1(SpoIIIJ)insertase alleviate stress associated with σM-dependent membrane protein overproduction[J]. PLoS Genet, 2019, 15(10): e1008263.
[38]	Korza G, Camilleri E, Green J, et al. Analysis of the mRNAs in spores of Bacillus subtilis[J]. J Bacteriol, 2019, 201(9): e00007-e00019.
[39]	Zhang YP, Zhu Y, Zhu Y, et al. The importance of engineering physiological functionality into microbes[J]. Trends Biotechnol, 2009, 27(12): 664-672. doi: 10.1016/j.tibtech.2009.08.006 pmid: 19793618
[40]	Hecker M, Völker U. General stress response of Bacillus subtilis and other bacteria[J]. Adv Microb Physiol, 2001, 44: 35-91. pmid: 11407115
[41]	Yeak KYC, Tempelaars M, Wu JL, et al. SigB modulates expression of novel SigB regulon members via Bc1009 in non-stressed and heat-stressed cells revealing its alternative roles in Bacillus cereus[J]. BMC Microbiol, 2023, 23(1): 37.
[42]	Krajnc M, Stefanic P, Kostanjšek R, et al. Systems view of Bacillus subtilis pellicle development[J]. NPJ Biofilms Microbiomes, 2022, 8(1): 25.
[43]	Dervyn E, Planson AG, Tanaka K, et al. Greedy reduction of Bacillus subtilis genome yields emergent phenotypes of high resistance to a DNA damaging agent and low evolvability[J]. Nucleic Acids Res, 2023, 51(6): 2974-2992.
[44]	Lenz P, Bakkes PJ, Müller C, et al. Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay[J]. Microb Cell Fact, 2023, 22(1): 203.
[45]	Matavacas J, Anand D, von Wachenfeldt C. New insights into the disulfide stress response by the Bacillus subtilis Spx system at a single-cell level[J]. Mol Microbiol, 2023, 120(1): 75-90.
[46]	Ho TD, Hastie JL, Intile PJ, et al. The Bacillus subtilis extracytoplasmic function σ factor σ(V)is induced by lysozyme and provides resistance to lysozyme[J]. J Bacteriol, 2011, 193(22): 6215-6222.
[47]	Helmann JD. Bacillus subtilis extracytoplasmic function(ECF)sigma factors and defense of the cell envelope[J]. Curr Opin Microbiol, 2016, 30: 122-132. doi: S1369-5274(16)30002-9 pmid: 26901131
[48]	Kessenikh AG, Novoyatlova US, Bazhenov SV, et al. Constructing of Bacillus subtilis-based lux-biosensors with the use of stress-inducible promoters[J]. Int J Mol Sci, 2021, 22(17): 9571.
[49]	Radeck J, Fritz G, Mascher T. The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network[J]. Curr Genet, 2017, 63(1): 79-90. doi: 10.1007/s00294-016-0624-0 pmid: 27344142
[50]	Gingichashvili S, Duanis-Assaf D, Shemesh M, et al. The adaptive morphology of Bacillus subtilis biofilms: a defense mechanism against bacterial starvation[J]. Microorganisms, 2019, 8(1): 62.
[51]	Piepenbreier H, Sim A, Kobras CM, et al. From modules to networks: a systems-level analysis of the bacitracin stress response in Bacillus subtilis[J]. mSystems, 2020, 5(1): e00687-19.
[52]	Losick RM. Bacillus subtilis: a bacterium for all seasons[J]. Curr Biol, 2020, 30(19): R1146-R1150.
[53]	Yaraguppi DA, Bagewadi ZK, Patil NR, et al. Iturin: a promising cyclic lipopeptide with diverse applications[J]. Biomolecules, 2023, 13(10): 1515.
[54]	Guo Y, Liu YY, Yang ZJ, et al. Enhanced production of poly-γ-glutamic acid by Bacillus subtilis using stage-controlled fermentation and viscosity reduction strategy[J]. Appl Biochem Biotechnol, 2024, 196(3): 1527-1543.
[55]	Wu YZ, Kawabata H, Kita K, et al. Constitutive glucose dehydrogenase elevates intracellular NADPH levels and luciferase luminescence in Bacillus subtilis[J]. Microb Cell Fact, 2022, 21(1): 266.
[56]	Amjad Zanjani FS, Afrasiabi S, Norouzian D, et al. Hyaluronic acid production and characterization by novel Bacillus subtilis harboring truncated Hyaluronan Synthase[J]. AMB Express, 2022, 12(1): 88.
[57]	Xu F, Liu C, Xia MM, et al. Characterization of a riboflavin-producing mutant of Bacillus subtilis isolated by droplet-based microfluidics screening[J]. Microorganisms, 2023, 11(4): 1070.
[58]	Tian JH, Xing BW, Li MY, et al. Efficient large-scale and scarless genome engineering enables the construction and screening of Bacillus subtilis biofuel overproducers[J]. Int J Mol Sci, 2022, 23(9): 4853.
[59]	Sánchez-Morán H, Kaar JL, Schwartz DK. Supra-biological performance of immobilized enzymes enabled by chaperone-like specific non-covalent interactions[J]. Nat Commun, 2024, 15(1): 2299. doi: 10.1038/s41467-024-46719-5 pmid: 38485940
[60]	Cui SX, Xia HZ, Chen TC, et al. Cell membrane and electron transfer engineering for improved synthesis of menaquinone-7 in Bacillus subtilis[J]. iScience, 2020, 23(3): 100918.
[61]	Shi L, Lin Y, Song JA, et al. Engineered Bacillus subtilis for the production of tetramethylpyrazine,(R, R)-2, 3-butanediol and acetoin[J]. Fermentation, 2023, 9(5): 488.
[62]	Wu YK, Chen TC, Liu YF, et al. Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis[J]. Nucleic Acids Res, 2020, 48(2): 996-1009.
[63]	宁雪伟. 地衣芽胞杆菌厌氧基础生理代谢特性研究[D]. 无锡: 江南大学, 2023.
	Ning XW. Study on anaerobic basic physiological and metabolic characteristics of Bacillus licheniformis[D]. Wuxi: Jiangnan University, 2023.
[64]	Voigt B, Schroeter R, Jürgen B, et al. The response of Bacillus licheniformis to heat and ethanol stress and the role of the SigB regulon[J]. Proteomics, 2013, 13(14): 2140-2161.
[65]	Dong ZX, Chen XL, Cai K, et al. Exploring the metabolomic responses of Bacillus licheniformis to temperature stress by gas chromatography/mass spectrometry[J]. J Microbiol Biotechnol, 2018, 28(3): 473-481.
[66]	Hoi LT, Voigt B, Jürgen B, et al. The phosphate-starvation response of Bacillus licheniformis[J]. Proteomics, 2006, 6(12): 3582-3601.
[67]	Guo LF, Lu L, Wang HC, et al. Effects of Fe²⁺ addition to sugarcane molasses on poly-γ-glutamic acid production in Bacillus licheniformis CGMCC NO. 23967[J]. Microb Cell Fact, 2023, 22(1): 37.
[68]	Zhang Y, Hu JM, Zhang Q, et al. Enhancement of alkaline protease production in recombinant Bacillus licheniformis by response surface methodology[J]. Bioresour Bioprocess, 2023, 10(1): 27.
[69]	Zhang AD, Ma YD, Deng Y, et al. Enhancing protease and amylase activities in Bacillus licheniformis XS-4 for traditional soy sauce fermentation using ARTP mutagenesis[J]. Foods, 2023, 12(12): 2381.
[70]	Moon JH, Ajuna HB, Won SJ, et al. The anti-termite activity of Bacillus licheniformis PR2 against the subterranean termite, Reticulitermes speratus kyushuensis morimoto(Isoptera: Rhinotermitidae)[J]. Forests, 2023, 14(5): 1000.
[71]	Rahimnahal S, Meimandipour A, Fayazi J, et al. Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis(KRLr₁)[J]. Front Microbiol, 2023, 14: 1132760.
[72]	Muff JL, Sokolovski F, Walsh-Korb Z, et al. Surgical treatment of short bowel syndrome-the past, the present and the future, a descriptive review of the literature[J]. Children, 2022, 9(7): 1024.
[73]	Boutonnet C, Ginies C, Alpha-Bazin B, et al. S-layer is a key element in metabolic response and entry into the stationary phase in Bacillus cereus AH187[J]. J Proteom, 2023, 289: 105007.
[74]	Jana A, Kakkar N, Halder SK, et al. Efficient valorization of feather waste by Bacillus cereus IIPK35 for concomitant production of antioxidant keratin hydrolysate and milk-clotting metallo-serine keratinase[J]. J Environ Manage, 2022, 324: 116380.
[75]	Wang XM, Han MN, Jiang JP, et al. Isolation of a Bacillus cereus strain HBL-AI and its application for production of Trans-4-hydroxy-l-proline[J]. Lett Appl Microbiol, 2021, 72(1): 53-59.
[76]	M LBK, Muni Kumar D, Sowjanya M, et al. Industrial applications of alkaline protease with novel properties from Bacillus cereus strain S8[J]. J Adv Zool, 2023, 44(S-3): 1314-1322.
[77]	Li WC, Huang XX, Liu H, et al. Improvement in bacterial cellulose production by co-culturing Bacillus cereus and Komagataeibacter xylinus[J]. Carbohydr Polym, 2023, 313: 120892.
[78]	Abdelkader I, Ben Mabrouk S, Hadrich B, et al. Optimization using response surface methodology of phospholipase C production from Bacillus cereus suitable for soybean oil degumming[J]. Prep Biochem Biotechnol, 2023, 53(10): 1165-1175.
[79]	Hyder S, Gondal AS, Sehar A, et al. Use of ginger extract and bacterial inoculants for the suppression of Alternaria solani causing early blight disease in tomato[J]. BMC Plant Biol, 2024, 24(1): 131.
[80]	Gomis-Cebolla J, Berry C. Bacillus thuringiensis as a biofertilizer in crops and their implications in the control of phytopathogens and insect pests[J]. Pest Manag Sci, 2023, 79(9): 2992-3001.
[81]	Guo HY, Yu XL, Liu ZY, et al. Deltamethrin transformation by Bacillus thuringiensis and the associated metabolic pathways[J]. Environ Int, 2020, 145: 106167.
[82]	Glenwright H, Pohl S, Navarro F, et al. The identification of intrinsic chloramphenicol and tetracycline resistance genes in members of the Bacillus cereus group(sensu lato)[J]. Front Microbiol, 2017, 7: 2122.
[83]	Banerjee S, Misra A, Chaudhury S, et al. A Bacillus strain TCL isolated from Jharia coalmine with remarkable stress responses, chromium reduction capability and bioremediation potential[J]. J Hazard Mater, 2019, 367: 215-223. doi: S0304-3894(18)31178-6 pmid: 30594722
[84]	Driessen JLSP, Johnsen J, Pogrebnyakov I, et al. Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles(DDGS)[J]. Metab Eng Commun, 2023, 16: e00223.
[85]	Kataoka N, Tajima T, Kato J, et al. Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host[J]. AMB Express, 2011, 1(1): 10. doi: 10.1186/2191-0855-1-10 pmid: 21906347
[86]	Yan SM, Wu G. Bottleneck in secretion of α-amylase in Bacillus subtilis[J]. Microb Cell Fact, 2017, 16(1): 124.
[87]	Yamamoto J, Chumsakul O, Toya Y, et al. Constitutive expression of the global regulator AbrB restores the growth defect of a genome-reduced Bacillus subtilis strain and improves its metabolite production[J]. DNA Res, 2022, 29(3): dsac015.
[88]	Repar J, Šućurović S, Zahradka K, et al. Stress resistance of Escherichia coli and Bacillus subtilis is modulated by auxins[J]. Can J Microbiol, 2013, 59(11): 766-770. doi: 10.1139/cjm-2013-0266 pmid: 24206360
[89]	Ragul K, Syiem I, Sundar K, et al. Characterization of probiotic potential of Bacillus species isolated from a traditional brine pickle[J]. J Food Sci Technol, 2017, 54(13): 4473-4483. doi: 10.1007/s13197-017-2928-6 pmid: 29184254
[90]	Deka P, Goswami G, Das P, et al. Bacterial exopolysaccharide promotes acid tolerance in Bacillus amyloliquefaciens and improves soil aggregation[J]. Mol Biol Rep, 2019, 46(1): 1079-1091.
[91]	Zeng ZB, Zhang JB, Li Y, et al. Probiotic potential of Bacillus licheniformis and Bacillus pumilus isolated from Tibetan yaks, China[J]. Probiotics Antimicrob Proteins, 2022, 14(3): 579-594.
[92]	Xia Y, Sun LC, Liang ZY, et al. Chromosome segment scanning for gain- or loss-of-function screening(CHASING)and its application in metabolic engineering[J]. bioRxiv, 2024. DOI: 10.1101/2024.01.31.578163.
[93]	Vinayavekhin N, Vangnai AS. The effects of disruption in membrane lipid biosynthetic genes on 1-butanol tolerance of Bacillus subtilis[J]. Appl Microbiol Biotechnol, 2018, 102(21): 9279-9289. doi: 10.1007/s00253-018-9298-5 pmid: 30141082
[94]	Zhang CL, Wang C, Zhao S, et al. Role of c-di-GMP in improving stress resistance of alginate-chitosan microencapsulated Bacillus subtilis cells in simulated digestive fluids[J]. Biotechnol Lett, 2021, 43(3): 677-690.
[95]	Wang JY, Wang WS, Wang HZ, et al. Improvement of stress tolerance and riboflavin production of Bacillus subtilis by introduction of heat shock proteins from thermophilic Bacillus strains[J]. Appl Microbiol Biotechnol, 2019, 103(11): 4455-4465.
[96]	Avila-Pérez M, Vreede J, Tang YF, et al. In vivo mutational analysis of YtvA from Bacillus subtilis: mechanism of light activation of the general stress response[J]. J Biol Chem, 2009, 284(37): 24958-24964. doi: 10.1074/jbc.M109.033316 pmid: 19581299
[97]	He K, Xin YP, Shan Y, et al. Phosphorylation residue T175 in RsbR protein is required for efficient induction of sigma B factor and survival of Listeria monocytogenes under acidic stress[J]. J Zhejiang Univ Sci B, 2019, 20(8): 660-669.
[98]	Morawska LP, Detert Oude Weme RGJ, Frenzel E, et al. Stress-induced activation of the proline biosynthetic pathway in Bacillus subtilis: a population-wide and single-cell study of the osmotically controlled proHJ promoter[J]. Microb Biotechnol, 2022, 15(9): 2411-2425.
[99]	El-Khoury T, Nguyen HA, Candusso MP, et al. UbK is involved in the resistance of Bacillus subtilis to oxidative stress[J]. Curr Microbiol, 2020, 77(12): 4063-4071. doi: 10.1007/s00284-020-02239-1 pmid: 33044618
[100]	Van Beilen J, Blohmke CJ, Folkerts H, et al. RodZ and PgsA play intertwined roles in membrane homeostasis of Bacillus subtilis and resistance to weak organic acid stress[J]. Front Microbiol, 2016, 7: 1633. pmid: 27818647
[101]	Shen J, Liu ZY, Yu HN, et al. Systematic stress adaptation of Bacillus subtilis to tetracycline exposure[J]. Ecotoxicol Environ Saf, 2020, 188: 109910.
[102]	Niu TF, Lv XQ, Liu YF, et al. The elucidation of phosphosugar stress response in Bacillus subtilis guides strain engineering for high N-acetylglucosamine production[J]. Biotechnol Bioeng, 2021, 118(1): 383-396.
[103]	Yang W, Yan HX, Dong GH, et al. Comparative transcriptomics reveal different genetic adaptations of biofilm formation in Bacillus subtilis isolate 1JN2 in response to Cd²⁺ treatment[J]. Front Microbiol, 2022, 13: 1002482.
[104]	Muto A, Fujihara A, Ito KI, et al. Requirement of transfer-messenger RNA for the growth of Bacillus subtilis under stresses[J]. Genes Cells, 2000, 5(8): 627-635. pmid: 10947848
[105]	Wu CD, Huang J, Zhou RQ. Progress in engineering acid stress resistance of lactic acid bacteria[J]. Appl Microbiol Biotechnol, 2014, 98(3): 1055-1063. doi: 10.1007/s00253-013-5435-3 pmid: 24337395

工业产品 Industrial application	菌株 Strain	菌株特性 Characteristics	产量 Titer	参考文献 Reference
乙偶姻	B. subtilis 168-phaCBA	将聚羟基丁酸合成途径引入B. subtilis 168，显著提升菌株乙偶姻耐受能力，进而提升乙偶姻工业生产产量	70.14 g/L	[54]
甲萘醌-7	B. subtilis 20-QT	枯草芽胞杆菌中共过表达基因tatAD-CD和qcrA-C	410 mg/L	[60]
鲨肌醇	B. subtilis KW018	敲除基因iolHIJ、iolX 和iolR且过表达基因amyE	22 g/L	[55]
透明质酸	B. subtilis RBSTr3	通过改造透明质酸合酶（HAS）获得高产透明质酸突变菌株	728 mg/L	[56]
核黄素	B. subtilis U3	利用基于液滴的微流体技术筛选分离枯草芽胞杆菌高产核黄素突变体	24.3 g/L	[57]
2,3-丁二醇	B. subtilis BS-ppb11	枯草芽胞杆菌中过表达2,3-丁二醇脱氢酶	96.5 g/L	[61]
聚γ-谷氨酸	B. subtilis GXC-36	使用阶段控制发酵和黏度降低策略优化发酵流程，提升聚γ-谷氨酸工业发酵产量	22.17 g/L	[54]
N-乙酰葡糖胺	B. subtilis 168 BNDR122	敲除基因nagP、gamP、gamA、gamR、nagA、nagB、ldh、alsRSD、pta、ackA、glcK、pckA、pyk、lacA和amyE	131.6 g/L	[62]
异丁醇	B. subtilis GI10	敲除非必须基因134.3 kb后获得异丁醇高产菌株	201.7 mg/L	[58]

工业产品 Industrial application	菌株 Strain	菌株特性 Characteristics	产量 Titer	参考文献 Reference
乙偶姻	B. subtilis 168-phaCBA	将聚羟基丁酸合成途径引入B. subtilis 168，显著提升菌株乙偶姻耐受能力，进而提升乙偶姻工业生产产量	70.14 g/L	[54]
甲萘醌-7	B. subtilis 20-QT	枯草芽胞杆菌中共过表达基因tatAD-CD和qcrA-C	410 mg/L	[60]
鲨肌醇	B. subtilis KW018	敲除基因iolHIJ、iolX 和iolR且过表达基因amyE	22 g/L	[55]
透明质酸	B. subtilis RBSTr3	通过改造透明质酸合酶（HAS）获得高产透明质酸突变菌株	728 mg/L	[56]
核黄素	B. subtilis U3	利用基于液滴的微流体技术筛选分离枯草芽胞杆菌高产核黄素突变体	24.3 g/L	[57]
2,3-丁二醇	B. subtilis BS-ppb11	枯草芽胞杆菌中过表达2,3-丁二醇脱氢酶	96.5 g/L	[61]
聚γ-谷氨酸	B. subtilis GXC-36	使用阶段控制发酵和黏度降低策略优化发酵流程，提升聚γ-谷氨酸工业发酵产量	22.17 g/L	[54]
N-乙酰葡糖胺	B. subtilis 168 BNDR122	敲除基因nagP、gamP、gamA、gamR、nagA、nagB、ldh、alsRSD、pta、ackA、glcK、pckA、pyk、lacA和amyE	131.6 g/L	[62]
异丁醇	B. subtilis GI10	敲除非必须基因134.3 kb后获得异丁醇高产菌株	201.7 mg/L	[58]

工业产品 Industrial application	菌株 Strain	菌株特性 Characteristics	产量或酶活性 Titer or enzymatic activity	参考文献 Reference
聚-γ-谷氨酸	B. licheniformis CGMCC NO. 23967	发酵体系中添加FeSO₄·7H₂O可提高地衣芽胞杆菌聚-γ-谷氨酸的生产效率，这种添加导致细胞内代谢物丰度增加，包括氨基酸，有机酸和关键的TCA循环中间体等含量的上调	70.436 g/L	[67]
碱性蛋白酶	B. licheniformis BL10	利用单因素实验和响应面法开发和优化发酵条件，提升生产效率	39 233.6 U/mL	[68]
淀粉酶	B. licheniformis XS-4	采用常压室温等离子体（ARTP）诱导突变，获得地衣芽胞杆菌高产淀粉酶突变株	15 U/mL	[69]
几丁质酶	B. licheniformis PR2	地衣芽胞杆菌PR2可以分泌几丁质酶用于工蚁生物防治	82.3 U/mL	[70]
角质蛋白酶	B. licheniformis	使用Ni-NTA色谱法纯化后获得较高浓度角质蛋白酶	222.01 U/mL	[71]

工业产品 Industrial application	菌株 Strain	菌株特性 Characteristics	产量或酶活性 Titer or enzymatic activity	参考文献 Reference
聚-γ-谷氨酸	B. licheniformis CGMCC NO. 23967	发酵体系中添加FeSO₄·7H₂O可提高地衣芽胞杆菌聚-γ-谷氨酸的生产效率，这种添加导致细胞内代谢物丰度增加，包括氨基酸，有机酸和关键的TCA循环中间体等含量的上调	70.436 g/L	[67]
碱性蛋白酶	B. licheniformis BL10	利用单因素实验和响应面法开发和优化发酵条件，提升生产效率	39 233.6 U/mL	[68]
淀粉酶	B. licheniformis XS-4	采用常压室温等离子体（ARTP）诱导突变，获得地衣芽胞杆菌高产淀粉酶突变株	15 U/mL	[69]
几丁质酶	B. licheniformis PR2	地衣芽胞杆菌PR2可以分泌几丁质酶用于工蚁生物防治	82.3 U/mL	[70]
角质蛋白酶	B. licheniformis	使用Ni-NTA色谱法纯化后获得较高浓度角质蛋白酶	222.01 U/mL	[71]

工业产品 Industrial application	菌株 Strain	菌株特性 Characteristics	产量或酶活性 Titer or enzymatic activity	参考文献 Reference
碱性蛋白酶	B. cereus S8	由于其热稳定性和在碱性pH值下的活性较好，蜡样芽胞杆菌菌株S8表达的蛋白酶降解蛋白效率较高	20 U/mg	[76]
细菌纤维素	B. cereus	共培养蜡样芽胞杆菌和木糖驹形氏杆菌（Komagataeibacter xylinus）提高细菌生产纤维素的效率	4.4 g/L	[77]
反式-4-羟基-l-脯氨酸	B. cereus HBL-AI	从空气中分离出生产反式-4-羟基-l-脯氨酸（trans-Hyp）的蜡状芽胞杆菌HBL-AI，仅使用l-脯氨酸作为碳源进行筛选分离培养	46.2 g/L	[75]
角蛋白酶	B. cereus IIPK35	通过固态发酵（SSF）技术提升了蜡状芽胞杆菌IIPK35的角蛋白酶生产效率	648.28 U/gds	[74]
磷脂酶C	B. cereus PLC_Bc	利用响应曲面法（RMS）和效应面法设计优化蜡样芽胞杆菌的磷脂酶C 生产效率	51 U/mL	[78]