Glycerol Dehydratase-reactivating Factor Increasing the Recombinant <em>Escherichia coli</em> Strains' 3-Hydroxypropinic Acid Synthesis Capability

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China Biotechnology ›› 2011, Vol. 31 ›› Issue (06) : 75-80.

Glycerol Dehydratase-reactivating Factor Increasing the Recombinant Escherichia coli Strains' 3-Hydroxypropinic Acid Synthesis Capability

QUAN Guo-yan^1,2, FANG Hui-ying^1,2, ZHUGE Bin^1,2, ZHANG Bo^1,2, YAO Jia-jia^1,2, ZHUGE Jian^1,3

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Abstract

Glycerol dehydratase was the critical enzyme in the metabolic pathway of converting glycerol to 3-hydroxypropionic acid. It was subjected to suicide inactivation by the natural substrate glycerol during catalysis. The inactivated glycerol dehydratase will influence the production of 3-hydroxypropionic acid. Therefore, the dhaB gene encoding glycerol dehydratase and gdrA, gdrB gene encoding glycerol dehydratase-reactivating factor from Klebsiella pneumoniae, the aldH gene encoding aldehyde dehydrogenase from Saccharomyces cerevisiae W303 were amplified by PCR. dhaB, gdrA, gdrB and aldH gene was employed to construct the recombinant strain Escherichia coli JM109/pEtac-dhaB-tac-aldH and E. coli JM109/pEtac-dhaB-gdrAB-tac-aldH. The effects of gdrA, gdrB expression on 3-hydroxypropionic acid production from glycerol were investigated. When cultivated aerobically on medium, the recombinant E. coli JM109/pEtac-dhaB-gdrAB-tac-aldH and E. coli JM109/pEtac-dhaB-tac-aldH produced 3-HP at a maximum of 4.0 g/L and 0.54 g/L. The results showed that the gdrA, gdrB expression could reactivated the activity of the inactivated glycerol dehydratase. Compare with E. coli JM109/pEtac-dhaB-tac-aldH, E. coli cells coexpressing both gdrA and gdrB with the glycerol dehydratase genes showed that the production of 3-HP was increased by 6.4fold.

Key words

Glycerol / Recombinat Escherichia coli / 3-hydroxypropionic acid / Glycerol dehydratase / Glycerol dehydratase-reactivating factor

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QUAN Guo-yan, FANG Hui-ying, ZHUGE Bin, ZHANG Bo, YAO Jia-jia, ZHUGE Jian. Glycerol Dehydratase-reactivating Factor Increasing the Recombinant Escherichia coli Strains' 3-Hydroxypropinic Acid Synthesis Capability[J]. China Biotechnology, 2011, 31(06): 75-80

References

[1] 张鸿达, 刘成, 高卫华,等. 微生物发酵法生产3-羟基丙酸的研究进展. 化工进展, 2007, 26(1): 33-37 Zhang H D, Liu C, Gao W H, et al. Chemical Industry and Engineering Progress, 2007, 26(1): 33-37.
[2] Werpy T, Petersen G. Top value added chemicals from biomass. Energy efficiency and renewable energy, Washington D C, 2004.
[3] Zhu B, Li J, He Y, et al. Effect of steric hindrance on hydrogen-bonding interaction between polyesters and natural polyphenol catechin. J Appl Polym Sci, 2004 91: 3565-3573.
[4] Andersen G, Bjrnberg O, Polakova S, et al. A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes. J Mol Biol, 2008, 380(4): 656-666.
[5] Tetsuo Toraya. Radical catalysis of B₁₂ enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases. Cellular and Molecular Life Sciences, 2000, 57: 106-127.
[6] Yamanishil M, Yunoki1 M, Tobimatsul T, et al. The crystal structure of coenzyme B₁₂-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol. Eur J Biochem, 2002, 269: 4484-4494.
[7] Takamasa Tobimatsu, Hideki Kajiura, Michio Yunoki, et al. Identification and expression of the genes encoding a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase. Journal of Bacteriology, 1999, 181(13): 4110-4113.
[8] Takamasa Tobimatsu, Hideki Kajiura, Tetsuo Toraya. Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratese. Arch Microbial, 2000, 174: 81-88.
[9] Der-lng Liao, Lisa Reiss, Ivan Turner, et al. Structure of glycerol dehydratase reactivase: A new type of molecular chaperone. Structure, 2003, 11: 109-119.
[10] 沈微, 王正祥, 唐雪明,等. 古细菌Pyrococcus furiosus高嗜热α-淀粉酶基因在大肠杆菌中的分泌表达. 中国酿造, 2003, 1: 12-14. Shen W, Wang Z X, Tang X M, et al. China Brewing, 2003, 1: 12-14.
[11] Ausubel F M, Brent R, Kingston R E, et al. Short Protocols in Molecular Biology. Beijing: Science Press, 1988.
[12] 黄瑞, 张晓梅, 王璐,等. 酿酒酵母乙醛脱氢酶的克隆与表达. 工业微生物, 2009, 39(1): 22-27. Huang R, Zhang X M, Wang L, et al. Industrial Microbiolgy, 2009, 39(1): 22-27.
[13] 李雯君, 方柏山, 洪燕,等. 甘油脱水酶再激活酶的克隆表达及活性鉴定. 生物工程学报, 2006, 22(6): 951-955. Li W J, Fang B S, Hong Y, et al. Chin J Biotech, 2006, 22(6): 951-955.
[14] 张晓梅, 唐雪明, 诸葛斌,等. 产1,3-丙二醇新型重组大肠杆菌的构建. 生物工程学报, 2005, 21(5): 743-747. Zhang X M, Tang X M, Zhuge B, et al. Chin J Biotech, 2005, 21: 743-747.
[15] Bradford M M. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248-254.
[16] Gancedo C, Gancedo J M, Sols A. Glycerol metabolism in yeasts: Pathways of utilization and production. Eur J Biochem, 1968, 5(2): 165-172.
[17] Saigal D, Cunningham S J, Farres J, et al. Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation. J Bacteriol, 1991, 173(10): 3199-3208.
[18] Subramanian M R, Chelladurai R, Ji-Eun J, et al. Production of 3-hydroxypropionic acid from glycerol by a novel recombinant Escherichia coli BL21 strain. Process Biochem, 2008, 43: 1440-1446.