为解决L-色氨酸发酵过程中乙酸、乳酸等抑制性副产物积累对对菌体活力和色氨酸积累造成抑制的问题,利用2株色氨酸生产菌,即大肠杆菌TRTH和谷氨酸棒杆菌TQ2223进行混合发酵,利用谷氨酸棒杆菌代谢大肠杆菌产生的乙酸、乳酸等副产物,降低乙酸等副产物对大肠杆菌发酵的负面影响。通过单因素试验确定了混菌发酵的最佳接种间隔时间为14 h,谷氨酸棒杆菌TQ2223接种量为7.5%,培养温度为36 ℃,pH 7.0。在最佳工艺条件下进行了30 L发酵罐小试验证,结果显示,与普通发酵工艺相比,混菌发酵工艺的乙酸积累量减少84.8%,乳酸积累量减少82.9%,L-色氨酸产量提高13.6%,糖酸转化率提高19.1%。为发酵法高效生产L-色氨酸提供了新方案,为混菌发酵在氨基酸发酵中的应用提供了理论依据。
In this study, Escherichia coli TRTH and Corynebacterium glutamicum TQ2223 were used for mixed fermentation in order to solve the problem related to the fermentation by-products (acetic acid and lactic acid) formation during L-tryptophan fermentation. During fermentation, acetic acid and lactic acid produced by E. coli were metabolized by C. glutamicum to reduce their negative effects on E. coli fermentation. The optimum condition for mixed fermentation was to inoculate 7.5% C. glutamicum TQ2223 after a 14 h interval, followed by incubating at 36℃ and pH 7.0. By using a 30 L fermenter, it was found that compared with conventional fermentation, mixed fermentation under the optimum condition decreased accumulated acetic acid and lactic acid by 84.8% and 82.9%, respectively. Moreover, the yield of L-tryptophan increased by 13.6%, and the conversion rate of sugar to acid increased by 19.1%. Overall, this study provides a new strategy for efficient L-tryptophan fermentation and builds a theoretical basis for applying mixed bacterial fermentation in amino acid fermentation.
[1] 王继雯, 岳丹丹,赵俊杰,等. 两株芽孢杆菌混菌发酵产芽孢条件的优化[J]. 中国酿造, 2017, 36(5): 95-99.
[2] 王恩旭. 维生素C一步混菌发酵体系的设计与构建[D]. 天津:天津大学, 2017.
[3] KEDIA G, WANG R, PATEL H. Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties [J]. Process Biochemistry, 2007, 42(1): 65-70.
[4] 徐颖宣, 徐尔尼,冯乃宪,等. 微生物混菌发酵应用研究进展[J]. 中国酿造, 2008, 27(9): 1-4.
[5] 刘莉娜. 大肠杆菌L-色氨酸生产菌株基因工程改造及代谢调控研究[D]. 无锡:江南大学, 2018.
[6] WANG J, CHENG L K, WANG J, et al. Genetic engineering of Escherichia coli to enhance production of L-tryptophan [J]. Applied Microbiology & Biotechnology, 2013, 97(17): 7 587-7 596.
[7] LIU Q, CHENG Y, XIE X, et al. Modification of tryptophan transport system and its impact on production of L-tryptophan in Escherichia coli [J]. Bioresource Technology, 2012, 114(2): 549-554.
[8] 王健. 直接发酵法生产L-色氨酸的研究[D]. 天津:天津科技大学, 2005.
[9] 黄静, 史建明,霍文婷,等. 氮源对L-色氨酸发酵的影响[J]. 食品与发酵工业, 2011, 37(5): 21-25.
[10] ZHAO Z J, ZOU C, ZHU Y X, et al. Development of L-tryptophan production strains by defined genetic modification in Escherichia coli [J]. Journal of Industrial Microbiology & Biotechnology, 2011, 38(12): 1 921-1 929.
[11] 张晓云, 叶勤. 大肠杆菌乙酸产生及其控制研究[J]. 生物技术通报, 2009(10): 66-69.
[12] SHILOACH J, FASS R. Growing E.coli to high cell density-A historical perspective on method development[J]. Biotechnol Adv, 2005, 23(5):345-357.
[13] 阮红. 谷氨酸棒状杆菌在乙酸盐和葡萄糖基质上的蓝白生长筛选[J]. 浙江大学学报(农业与生命科学版), 2002, 28(2): 141-146.
[14] SGOBBA E, STUMPF A K, VORTMANN M, et al. Synthetic Escherichia coli-Corynebacterium glutamicum consortia for L-lysine production from starch and sucrose [J]. Bioresour Technol, 2018, 260:302-310.
[15] 杨梦晨, 户红通,蔡萌萌,等. L-色氨酸清液发酵工艺研究[J]. 食品与发酵科技, 2017, 53(2): 29-33.
[16] 程立坤, 黄静,秦永锋,等. 代谢副产物乙酸对L-色氨酸发酵的影响[J]. 微生物学通报, 2010, 37(2): 166-173.
[17] 刘子强, 张震,蔡萌萌,等. 稀糖分罐发酵对L-色氨酸发酵的影响[J]. 中国酿造, 2018, 37(4): 132-136.
[18] REPASKE D R, ADLER J. Change in intracellular pH of Escherichia coli mediates the chemotactic response to certain attractants and repellents [J]. Journal of Bacteriology, 1981, 145(3): 1 196-1 208.
[19] KRÄMER R. Genetic and physiological approaches for the production of amino acids [J]. Journal of Biotechnology, 1996, 45(1): 1-21.
[20] 阮红, GERSTMEIR R, SCHINKE S,等. amrG1基因在谷氨酸棒杆菌乙酸活化中的作用[J]. 中国科学, 2004, 34(3): 223-231.
[21] 王健, 张蓓,张克旭,等. L-色氨酸产生菌TQ2223的途径分析[J]. 无锡轻工大学学报:食品与生物技术, 2003, 22(5): 15-18.
