Construction and Characterization of the Pathways of Synthesizing Lactate in Vitro Related to NADP（H）

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.035

Abstract

Abstract: The regulating circles of the metabolic pathways in cells in vivo are complicated,and the intuitive researches may be carried out if the target metabolic pathways could be moved to the cells in vitro. Selecting 10 thermophilic enzymes,synthesizing pathways in vitro were constructed,which achieved the cycle balance of energy ATP and redox cofactors NAHP（H）,as well as the produced lactate from glucose metabolism. The preliminary study revealed this system’s optimal reaction condition was pH 7 and 50℃. Under this condition,adding small amount of NADP⁺,12.4 g/L glucose was converted to 9.5 g/L lactate within 8 hours.

Key words: synthesis in vitro, metabolic pathway, genetic engineering, multi-enzyme synthesis

MAO Jia-ling, XU Lin, YAN Ming. Construction and Characterization of the Pathways of Synthesizing Lactate in Vitro Related to NADP（H）[J]. Biotechnology Bulletin, 2016, 32(9): 260-266.

References

[1] Banuelos M, Gancedo C. In situ study of the glycolytic pathway in Saccharomyces cerevisiae[J]. Arch Microbiol, 1978, 117：197-201.
[2] 许可, 吕波, 李春. 无细胞的合成生物技术—多酶催化与生物合成[J]. 中国科学（化学）, 2015, 45（5）：429-437.
[3] Fessner WD. Systems Biocatalysis：Development and engineering of cell-free“artificial metabolisms”for preparative multi-enzymatic synthesis[J]. New Biotechnology, 2015, 32（6）：658-664.
[4] Welch P, Scopes RK. Studies on cell-free metabolism：Ethanol production by a yeast glycolytic system reconstituted from purified enzymes[J]. Journal of Biotechnology, 1985, 2：257-273.
[5] Peralta-Yahya P, Zhang FZ, del Cardaryre S, et al. Microbial engineering for the production of advanced biofuels[J]. Nature, 2012, 488（7411）：320-328.
[6] 孙继国, 韩增叶, 葛喜珍, 等. 基于重组大肠杆菌无细胞体系生产吡咯喹啉醌[J]. 生物技术通报, 2014（4）：164-168.
[7] 李卫星, 邢卫红. 发酵法乳酸精制技术研究进展[J]. 化工进展, 2009, 3：491-495.
[8] Woodward J, Orr M, Cordary K, et al. Biotechnology：enzymatic production of biohydrogen[J]. Nature, 2000, 405：1014-1015
[9] Ye XT, Honda K, Sakai T, et al. Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway[J]. Microbial Cell Factories, 2012, 11：120.
[10] 王立梅, 齐斌. L-乳酸应用及生产技术研究进展[J]. 食品科学, 2007, 10：608-612.
[11] 翁云宣. 聚乳酸合成、生产、加工及应用研究综述[J]. 塑料工业, 2007, 6（35）：69-73.
[12] Iddar A, Valverde F, Assobhei O, et al. Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria[J]. International Microbiology, 2005, 8（4）：251-258.
[13] Skander E, Christian S, Saskia B, et al. Exploration of extremop-hiles for high temperature biotechnological processes[J]. In Environmental Microbiology Extremophiles, Current Opinion in Microbiology, 2015, 25：113-119.
[14] Partow S, Siewers V, Bjorn S, et al. Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae[J]. Yeast, 2010（27）：955-964.
[15] 萨姆布鲁克J, 拉塞尔DW. 分子克隆实验指南[M]. 第3版. 黄培堂, 译. 北京：科学出版社, 2002：1601-1604.
[16] Bradford MM. 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）：248-254.
[17] Johannes TW, Woodywer RD, Zhao H. Efficient regeneration of NADPH using an engineered phosphite dehydrogenase[J]. Biotechnol Bioengineering, 2007, 96：18-26.