Pyruvic acid is an important organic acid with wide applications in polymer, cosmetic, food additive, pharmaceutical and other fields. Saccharomyces cerevisiae is considered as the best candidate microorganism for pyruvic acid production. However, pyruvic acid produced during glycolysis could be catalyzed by pyruvate decarboxylase in the cytoplasm to degrade into CO2 and acetaldehyde, resulting in the loss of carbon metabolic flux. In order to redirect more carbon metabolic flux to pyruvic acid synthesis, the deletions of the three structural genes of pyruvate decarboxylase (pdc1, pdc5 and pdc6) were performed, which significantly promoted pyruvic acid accumulation. Nevertheless, poor growth of the mutant strain XY-156 was also observed. Compared with the non-evolved strain XY-156, the evolved strain XY-156A obtained by adaptive evolution showed significant improvement in cell growth, glucose consumption and pyruvate accumulation. Fed-batch fermentation was further performed in a 2 L stirred bioreactor, and up to 105 g/L pyruvic acid was obtained within 76 h, with a productivity of 1.38 g/(L·h) and a yield of 0.5 g/g glucose. The results showed that the combination of inactivating pyruvate decarboxylase with adaptive evolution could achieve efficient pyruvic acid accumulation in S. cerevisiae, and could lay a solid foundation for the industrial production of biological pyruvic acid.
[1] LI Y, CHEN J, LUN S Y. Biotechnological production of pyruvic acid[J]. Appl Microbiol Biotechnol, 2001, 57(4): 451-459.
[2] MALEKI N, EITEMAAN M. Recent progress in the microbial production of pyruvic acid[J]. Fermentation, 2017, 3(1): 8.
[3] SONG Y, LI J, SHIN H, et al. Biotechnological production of alpha-keto acids: Current status and perspectives[J]. Bioresourcetechnology, 2016, 219: 716-724.
[4] XU Ping, QIU Jianhua, GAO Cao, et al. Biotechnological routes to pyruvate production[J]. Journal of Bioscience and Bioengineering, 2008, 105(3): 169-175.
[5] LI Shubo, CHEN Xiulai, LIU Liming, et al. Pyruvate production in Candida glabrata: Manipulation and optimization of physiological function[J]. Critical Reviews in Biotechnology, 2016, 36(1): 1-10.
[6] LIU Liming, XU Qinglong, LI Yin, et al. Enhancement of pyruvate production by osmotic‐tolerant mutant of Torulopsis glabrata[J]. Biotechnology and bioengineering, 2007, 97(4): 825-832.
[7] ZHANG Jian, GAO Nianfa. Application of response surface methodology in medium optimization for pyruvic acid production of Torulopsis glabrata TP19 in batch fermentation[J]. Journal of Zhejiang University SCIENCE B, 2007, 8(2): 98-104.
[8] ZELIĆ B, GERHARZ T, BOTT M, et al. Fed-batch process for pyruvate production by recombinant Escherichia coli YYC202 strain[J]. Engineering in Life Sciences, 2003, 3(7): 299-305.
[9] ZELIĆ B, GOSTOVIĆ S, VUORILEHTO K, et al. Process strategies to enhance pyruvate production with recombinant Escherichia coli: From repetitive fed-batch to in situ product recovery with fully integrated electrodialysis[J]. Biotechnology and Bioengineering, 2004, 85(6): 638-646.
[10] 韩全乡, 王宗生,刘金淑. 医院真菌感染128例分析[J]. 实用预防科学, 2004, 11(1): 180-181.
[11] XU Da, ZUO Jiakun, CHEN Zhaoguo, et al. Different activated methyl cycle pathways affect the pathogenicity of avian pathogenic Escherichia coli[J]. Veterinary microbiology, 2017, 211: 160-168.
[12] PLEISSNER D, DIETZ D, DUUREN J B J H V, et al. Biotechnological production of organic acids from renewable resources[J]. Advances in Biochemical Engineering/Biotechnology, 2017, 166: 373-410.
[13] BARNETT J A. The utilization of sugars by yeasts[J]. Advances in Carbohydrate Chemistry and Biochemistry, 1976, 32: 125-234.
[14] VAN MARIS A J A, GEERTMAN J M A, VERMEULEN A, et al. Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-Independent, glucose-tolerant, and pyruvate-hyperproducing yeast[J]. Appl Environ Microbiol, 2004, 70(1): 159-166.
[15] FLIKWEERT M T, DE SWAAF M, VAN DIJKEN J P, et al. Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae[J]. FEMS Microbiology Letters, 1999, 174(1): 73-79.
[16] 巩继贤, 郑辉杰, 郑宗宝. 微生物进化工程育种技术进展与展望[J]. 生物加工过程, 2010, 8(2): 69-76.
[17] SAUER U. Evolutionary engineering of industrially important microbial phenotypes[J]. Adv Biochem Eng Biotechnol, 2001,73: 129-169.
[18] GUIMARAES P M R, FRANCOIS J, PARROU J L, et al. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant[J]. Appl Environ Microbiol, 2008, 74(6): 1 748-1 756.
[19] FLIKWEERT M T, VAN DER ZANDEN L, JANSSEN W M T M, et al. Pyruvate decarboxylase: An indispensable enzyme for growth of Saccharomyces cerevisiae on glucose[J]. Yeast, 1996, 12(3): 247-257.
[20] 肖冬光, 刘青, 李静, 等. 酿酒酵母单倍体制备方法的优化[J]. 酿酒科技, 2004(4): 21-22.
[21] 罗贞贞, 陈小玲, 练梅华, 等. 酿酒酵母Mbp1缺陷型菌株的构建及其乙醇发酵特性[J]. 微生物学通报, 2015, 42(11): 2 108-2 114.
[22] 闫道江, 王彩霞, 周杰民, 等. 酿酒酵母产苹果酸的还原TCA 路径构建及发酵调控[J]. 生物工程学报, 2013, 29(10): 1 484-1 493.
[23] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(1-2): 248-254.
[24] WANG Zhikun, GAO Cuijuan, WANG Qian, et al. Production of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering[J]. Biochemical Engineering Journal, 2012, 67: 126-131.
[25] VAN MARIS A J A, LUTTIK M A H, WINKLER A A, et al. Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae[J]. Appl Environ Microbiol, 2003, 69(4): 2 094-2 099.
[26] OUD B, FLORES C L, GANCEDO C, et al. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae[J]. Microbial Cell Factories, 2012, 11(1): 131.
[27] PRONK J T, STEENSMA H Y, VAN DIJKEN J P. Pyruvate metabolism in Saccharomyces cerevisiae[J]. Yeast, 1996, 12: 1 607-1 633.
[28] WANG Depei, WANG Lu, HOU Li, et al. Metabolic engineering of Saccharomyces cerevisiae for accumulating pyruvic acid[J]. Annals of Microbiology, 2015, 65(4): 2 323-2 331.
