为探究丙酮酸羧化酶及微量元素Ca2+和生物素对酿酒酵母积累琥珀酸的作用,敲除了琥珀酸脱氢酶编码基因(SDH2)并过量表达来自米根霉的丙酮酸羧化酶基因(RoPYC)。琥珀酸产量由(0.011±0.002)g/L提高至(0.841±0.020)g/L,较表达前提高了75.45%。随后,外源添加不同浓度Ca2+和生物素考察其对琥珀酸发酵的影响,结果发现,Ca2+的添加促进了菌体的生长但不利于琥珀酸的积累,而外源添加浓度为16、32、64、96 μg/L生物素时,琥珀酸产量分别提高75.04%、84.26%、69.28%、66.79%。其中生物素质量浓度为32 μg/L时,琥珀酸产量达到最大为(0.964±0.02)g/L,且丙酮酸羧化酶(pyruvate carboxylase,PC)酶活提高14.42%,说明PC酶活的提高促进了酿酒酵母中琥珀酸的积累。该研究为解决酿酒酵母琥珀酸合成过程中前体碳代谢流不足问题提供了新的解决思路。
This study was constructed to explore the effects of pyruvate carboxylase as well as trace elements Ca2+ and biotin on succinic acid accumulation in Saccharomyces cerevisiae. The succinate dehydrogenase gene (SDH2) of S. cerevisiae was knocked out and pyruvate carboxylase gene (RoPYC) from Rhizopus oryzae was over-expressed. The results showed that the production of succinic acid increased by 75.45% from (0.011±0.002)g/L to (0.841±0.020)g/L. Moreover, it was found that Ca2+ promoted the growth of the cells but did not have effects on succinic acid accumulation. In comparison, 16, 32, 64, and 96 μg/L biotin enhanced the production of succinic acid by 75.04%, 84.26%, 69.28%, and 66.79%, respectively. Additionally, succinic acid production reached a maximum of (0.964±0.02)g/L when adding 32 μg/L biotin, and the activity of pyruvate carboxylase increased by 14.42%. This indicated that the increment of pyruvate carboxylase activity promoted the accumulation of succinic acid in S. cerevisiae. This study provides a new solution to the insufficient carbon flow in succinic acid synthesis in S. cerevisiae.
[1] ZEIKUS J G, JAIN M K, ELANKOVAN P. Biotechnology of succinic acid production and markets for derived industrial products[J]. Applied Microbiology and Biotechnology, 1999, 51: 545-552.
[2] MILLS E L, PIERCE K A, JEDRYCHOWSKI M P,et al. Accumulation of succinate controls activation of adipose tissue thermogenesis[J]. Nature, 2018.
[3] LIU R, LIANG L, CAO W, et al. Succinate production by metabolically engineered Escherichia coli using sugarcane bagasse hydrolysate as the carbon source[J]. Bioresource Technology, 2013, 135(C): 574-577.
[4] LIANG L, LIU R, LI F, et al. Repetitive succinic acid production from lignocellulose hydrolysates by enhancement of ATP supply in metabolically engineered Escherichia coli[J]. Bioresource Technology, 2013, 143(C): 405-412.
[5] MAO Y, LI G, CHANG Z, et al. Metabolic engineering of Corynebacterium glutamicum for efficient production of succinate from lignocellulosic hydrolysate[J]. Biotechnology for Biofuels, 2018, 11(1):95.
[6] WANG J, WANG H, YANG L,et al. A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid[J]. Journal of Industrial Microbiology and Biotechnology, 2018, 45: 253-269.
[7] CUI Z, GAO C, LI J, et al. Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH[J]. Metabolic Engineering, 2017, 42: 126-133.
[8] AHN J H, JANG Y S, LEE S Y. Production of succinic acid by metabolically engineered microorganisms[J]. Current Opinion in Biotechnology, 2016, 42: 54-66.
[9] 郑美娟,余华顺.酵母菌耐高渗机理研究[J].酿酒科技,2008(10):37-39.
[10] 徐国强,吴满珍.微生物发酵生产C4二元羧酸研究进展[J].中国生物工程杂志,2014,34(8):97-104
[11] ABBOTT D A, ZELLE R M, PRONK J T,et al. Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: Current status and challenges[J]. FEMS Yeast Research, 2009, 9(8): 1 123-1 136.
[12] KUBO Y, TAKAGI H, NAKAMORI S. Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain[J]. Journal of Bioscience and Bioengineering, 2000, 90(6): 619-624.
[13] OTERO J M, CIMINI D, PATIL K R, et al. Industrial systems biology of Saccharomyces cerevisiae enables novel succinic acid cell factory[J]. PLoS One, 2013, 8(1):e54 144.
[14] RAAB A M, GEBHARDT G, BOLOTINA N, et al. Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid[J]. Metabolic Engineering, 2010, 12(6): 518-525.
[15] YAN D, WANG C, ZHOU J, et al. Construction of reductive pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low pH value[J]. Bioresource Technology, 2014, 156: 232-239.
[16] XU G, WU M, JIANG L.Site-saturation engineering of proline 474 in pyruvate carboxylase from Rhizopus oryzae to elevate fumaric acid production in engineered Saccharomyces cerevisiae cells[J]. Biochemical Engineering Journal, 2017, 117(part_PB): 36-42.
[17] 徐国强.代谢工程改造酿酒酵母生产延胡索酸[D].无锡:江南大学,2012.
[18] XU G, ZOU W, CHEN X, et al. Fumaric acid production in Saccharomyces cerevisiae by in silico aided metabolic engineering[J]. PLoS One, 2012, 7(12): 1-10.
[19] KURO Y, TAKAG H, NAKAMORI S. Effect of gene disruption of succinate dehydrogenase on succinate production in a Sake Yeast Strain[J]. Journal of Bioscience and Bioengineering, 2000, 90(6): 619-624.
[20] KOFFAS M A G, JUNG G Y, AON J C, et al. Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum [J]. Applied and Environmental Microbiology, 2002, 68(11): 5 422-5 428.
[21] LIN H, SAN K Y, BENNETT G N.Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli[J]. Applied Microbiology and Biotechnology, 2005, 67(4): 515-523.
[22] KENEALY W, ZAADY E, DU PREEZ J C, et al. Biochemical aspects of fumaric acid accumulation by Rhizopus arrhizus[J]. Applied and Environmental Microbiology, 1986, 52(1): 128-33.
[23] MCNEILLIE E M, CLEGG R A, ZAMMIT V A. Regulation of acetyl-CoA carboxylase in rat mammary gland. effects of incubation with Ca2+, Mg2+ and ATP on enzyme activity in tissue extracts[J]. The Biochemical Journal, 1981, 200(3): 639-644.
[24] LIETZAN A D, LIN Y, MAURICE M S T. The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase[J]. Archives of Biochemistry and Biophysics, 2014, 562: 70-79.
[25] LITSANOV B, KABUS A, BROCKER M, et al. Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum[J]. Microb. Biotechnol., 2012, 5(1): 116-128
