Surfactin is a lipopeptide biosurfactant with good physicochemical properties and biological activity.However, natural bacterial strains exhibit low surfactin yield and high costs, severely limits its industrial application.Therefore, Bacillus subtilis ATCC 21332 served as the starting strain, homologous recombination and CRISPR-Cas9 gene editing, combined with fermentation condition optimization, were employed to enhance surfactin expression and yield.Homologous recombination was used to replace the native PsrfA promoter with three strong promoters.The highest yield was achieved with Pveg promoter, reached 3.59 g/L, which was 28.2% higher than the wild type.To further increase yield, seven key genes were knocked out by CRISPR-Cas9, including srfA synthase negative regulatory factors (phoP, codY, sinI), spore synthesis key genes (spoIIIE, spoIVB), and biofilm synthesis key genes (sipW, tasA).The results showed that knocking out the phoP gene resulted in the greatest increase, reached 4.17 g/L, which was 49% higher than the wild-type.Fermentation optimization identified the following optimal conditions:4% inoculum (volume fraction), 30 mL/250 mL loading, 37 ℃, initial pH 7, and addition of 2 g/L glutamic acid.Under these conditions, engineered strains achieved approximately 1.5-2.0-fold improvements in surfactin production.The highest yield was obtained from B.subtilis ATCC 21332- Δ sipW, reached 5.98 g/L after 48 h of fermentation, representing a 113.7% increase over the wild type.This study offers theoretical support for elucidating the biosynthetic mechanism of surfactin and lays a foundation for industrial production and application.
[1] CHAUHAN V, PANDEY A, MAHAJAN G, et al.Synergistic exploration of surfactin-capped silver nanoparticles:Bioinformatics insights, antibacterial potency, and anticancer activity[J].3 Biotech, 2024, 15(1):13.
[2] WEI X Y, WANG H F, LIU S J, et al.Surfactin’s impact on gut microbiota and intestinal tumor cells[J].International Journal of Biological Macromolecules, 2025, 287:138607.
[3] CHEN X Y, LU Y J, SHAN M Y, et al.A mini-review:Mechanism of antimicrobial action and application of surfactin[J].World Journal of Microbiology and Biotechnology, 2022, 38(8):143.
[4] ZHANG Q, LIU P, YANG C J, et al.Effect of orally surfactin on lactogenic immunity in sows[J].Journal of Nanjing Agricultural University, 2024, 47(6):1122-1129.
[5] DEGANG L, AURICCHIO G, DE OLIVEIRA SCHMIDT V K, et al.Oil displacement properties of surfactin:A comparative study[J].Tenside Surfactants Detergents, 2023, 60(5):414-418.
[6] HU Y F, YANG X, TAI B W, et al.Bacillus amyloliquefaciens A-1 inhibiting fungal spoilage in agricultural products is improved by metabolic engineering of enhancing surfactin yield[J].Food Research International, 2024, 175:113752.
[7] WANG H F, WEI X Y, LI D D, et al.Impact of surfactin on the physicochemical properties of dough and quality of corresponding steamed bread[J].Journal of the Science of Food and Agriculture, 2025, 105(5):3122-3132.
[8] BUENO-MANCEBO J, BARRENA R, ARTOLA A, et al.Surfactin as an ingredient in cosmetic industry:Benefits and trends[J].International Journal of Cosmetic Science, 2024, 46(5):702-716.
[9] SUN H G, BIE X M, LU F X, et al.Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter[J].Canadian Journal of Microbiology, 2009, 55(8):1003-1006.
[10] WILLENBACHER J, MOHR T, HENKEL M, et al.Substitution of the native srfA promoter by constitutive P in two B.subtilis strains and evaluation of the effect on surfactin production[J].Journal of Biotechnology, 2016, 224:14-17.
[11] SU Y M, GAO L, LI C Y, et al.Regulation mechanism and bioactivity characteristic of surfactin homologues with C14 and C15 fatty acid chains[J].Microbial Cell Factories, 2024, 23(1):94.
[12] VLAMAKIS H, CHAI Y R, BEAUREGARD P, et al.Sticking together:Building a biofilm the Bacillus subtilis way[J].Nature Reviews Microbiology, 2013, 11(3):157-168.
[13] 王军强, 王连国, 郭荣君.枯草芽胞杆菌B006产表面活性素的培养条件优化[J].生物技术通报, 2017, 33(4):214-221.
WANG J Q, WANG L G, GUO R J, et al.Optimization of culture conditions for the enhancement of surfactin production from Bacillus substilis B006[J].Biotechnology Bulletin, 2017, 33(4):214-221.
[14] 姬杨, 冯红.短小芽孢杆菌BA06产表面活性素培养基组分的优化[J].四川大学学报(自然科学版), 2016, 53(4):925-930.
JI Y, FENG H.Optimization of fermentation medium for producing surfactin by Bacillus pumilus BA06[J].Journal of Sichuan University (Natural Science Edition), 2016, 53(4):925-930.
[15] 李萍, 武刚, 黄会琴, 等.产表面活性素芽孢杆菌SFP03的发酵工艺优化[J].发酵科技通讯, 2024, 53(2):67-73.
LI P, WU G, HUANG H Q, et al.Optimization of surfactin production by Bacillus sp.SFP03 using response surface methodology[J].Bulletin of Fermantation Science and Technology, 2024, 53(2):67-73.
[16] 周大袁, 林佳辉, 李霜.利用溶氧调控型启动子Pvgb构建产surfactin的重组枯草芽孢杆菌[J].生物加工过程, 2020, 18(6):690-695.
ZHOU D Y, LIN J H, LI S.Constructing recombinant Bacillus subtilis producing surfactin using aeration-inducible promoter Pvgb[J].Chinese Journal of Bioprocess Engineering, 2020, 18(6):690-695.
[17] LIU Z Z, WANG Y F, LIU S H, et al.Boosting the heterologous expression of d-allulose 3-epimerase in Bacillus subtilis through protein engineering and catabolite-responsive element box engineering[J].Journal of Agricultural and Food Chemistry, 2022, 70(38):12128-12134.
[18] BISWAS I, GRUSS A, EHRLICH S D, et al.High-efficiency gene inactivation and replacement system for gram-positive bacteria[J].Journal of Bacteriology, 1993, 175(11):3628-3635.
[19] ALTENBUCHNER J.Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system[J].Applied and Environmental Microbiology, 2016, 82(17):5421-5427.
[20] 艾嘉, 陆兆新, 别小妹, 等.高效液相色谱法测定发酵液中表面活性素的含量[J].食品科学, 2009, 30(6):188-190.
AI J, LU Z X, BIE X M, et al.High performance liquid chromatography analysis of surfactin content in fermentation broth of Bacillus amyloliquefaciens ES-2[J].Food Science, 2009, 30(6):188-190.
[21] 董丽红, 郭庆港, 王培培, 等.PhoR/PhoP双组份对枯草芽胞杆菌NCD-2菌株中surfactin合成的影响[J].植物病理学报, 2018, 48(1):119-127.
DONG L H, GUO Q G, WANG P P, et al.The effect of PhoR/PhoP two-component regulatory system on surfactin production in Bacillus subtilis NCD-2[J].Acta Phytopathologica Sinica, 2018, 48(1):119-127.
[22] GUO Z H, SUN J Y, MA Q Y, et al.Improving surfactin production in Bacillus subtilis 168 by metabolic engineering[J].Microorganisms, 2024, 12(5):998.
[23] WANG M M, YU H M, LI X, et al.Single-gene regulated non-spore-forming Bacillus subtilis:Construction, transcriptome responses, and applications for producing enzymes and surfactin[J].Metabolic Engineering, 2020, 62:235-248.
[24] HIGGINS D, DWORKIN J.Recent progress inBacillus subtilissporulation[J].FEMS Microbiology Reviews, 2012, 36(1):131-148.
[25] COSBY W M, VOLLENBROICH D, LEE O H, et al. Altered srf expression in Bacillus subtilis resulting from changes in culture pH is dependent on the Spo0K oligopeptide permease and the ComQX system of extracellular control[J].Journal of Bacteriology, 1998, 180(6):1438-1445.
[26] 陈亮, 毛晶晶, 宇光海, 等.响应面法优化Bacillus subtilis抗菌脂肽发酵工艺条件[J].食品科技, 2015, 40(4):17-21.
CHEN L, MAO J J, YU G H, et al.Optimization of fermentation conditions for enhanced antimicrobial lipopeptide production by Bacillus subtilis LM2303 using response surface methodology[J].Food Science and Technology, 2015, 40(4):17-21.
[27] LI G Y, LI X L, QI J J, et al.Optimization of fermentation condition of surfactin from Bacillus subtilis by response surface methodology[J].Science and Technology of Food Industry, 2022, 43(12):146-154.
