Construction of a Recombinant Strain Producing High-yield Dihydroxyacetone

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.025

Abstract

Abstract: This work aims to construct a recombinant strain containing gene sldAB over-expressing membrane-bound glycerol dehydrogenase for raising the yield of dihydroxyacetone（DHA）. Firstly, using genomic DNA of Gluconobacter oxydans ATCC621H as a template, sldAB amplified by PCR was ligated to the plasmid pBBR1MCS-2 and the expression vector pBBR1MCS-2-sldAB was constructed;subsequently the plasmid pBBR1MCS-2-sldAB was transformed into the G. oxydans ATCC621H by electrotransfer, and the recombinant strain GOX205 was obtained. Results showed that the recombinant strain was constructed successfully, and the activity of glycerol dehydrogenase increased by 26% compared with the original strain. When initial concentration 100 g/L of glycerol as carbon source, the growth of GOX205 was significantly improved compared with the original strain, the final DHA concentration was 94.1 g/L and increased by 19.7%, and the residue of glycerol decreased 15.1 g/L. Therefore, this study provides a basis for further improving the biotransformation process of DHA production.

Key words: 1, 3-dihydroxyacetone, recombinant strain, glycerol, over-expression

Xu Xiaojing, Zhao Qiong, Wang Xianghe. Construction of a Recombinant Strain Producing High-yield Dihydroxyacetone[J]. Biotechnology Bulletin, 2015, 31(8): 174-179.

References

[1] Mishra R, Jain SR, Kumar A. Microbial production of dihydroxyace-tone[J]. Biotechnol Adv, 2008, 26（4）：293-303.
[2] Ohrem Leonhard H, Voβ H. Process model of the oxidation of glycerol with Gluconobacter oxydans[J]. Process Biochemistry, 1996, 31（3）：295-301.
[3] Raska J, Skopal F, Komers K, et al. Kinetics of glycerol biotransformation to dihydroxyacetone by immobilized Gluconobacter oxydans and effect of reaction conditions[J]. Collect Czechoslov Chem Commun, 2007, 72（9）：1269-1283.
[4] 魏胜华. 氧化葡萄糖杆菌转化甘油制备二羟基丙酮的过程工程研究[D]. 上海：华东理工大学, 2006.
[5] Claret C, Salmon JM, Romieu C, et al. Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol[J]. Appl Microbiol Biotechnol, 1994, 41：359-365.
[6] Deppenmeier U, Hoffmeister M, Prust C. Biochemistry and biotechnological applications of Gluconobacter strains[J]. Appl Microbiol Biotechnol, 2002, 3：233-242.
[7] Wethmar M, Deckwer W. Semisynthetic culture medium for growth and dihydroxyacetone production by Gluconobacter oxydans[J]. Biotechnology Techniques, 1999, 13（4）：283-287.
[8] Wei S, Song Q, Wei D. Production of Gluconobacter oxydans cells from low-cost culture medium for conversion of glycerol to dihydroxyacetone[J]. Prep Biochem Biotechnol, 2007, 37（2）：113-121.
[9] Svitel J, Sturdik E. Product yield and by-product formation in glycerol conversion to dihydroxyacetone by Gluconobacter oxydans[J]. J Ferment Bioeng, 1994, 78：351-355.
[10] Wei S, Song Q, Wei D. Repeated use of immobilized Gluconobacter oxydans cells for conversion of glycerol to dihydroxyacetone[J]. Prep Biochem Biotechnol, 2007, 37（1）：67-76.
[11] Hekmat D, Bauer R, Neff V. Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans[J]. Process Biochemistry, 2007, 42：71-76.
[12] Hekmat D, Bauer R, Fricke J. Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans[J]. Bioprocess Biosyst Eng, 2003, 26：109-116.
[13] 徐美娟, 饶志明, 沈微, 等. 一株以甘油为底物产二羟丙酮（DHA）微生物菌种的筛选及鉴定[J]. 微生物学通报, 2008, 35（3）：336-340.
[14] 马丽娟. 生物法生产1, 3-二羟基丙酮产品研究[D]. 天津：天津大学, 2009.
[15] 魏东芝, 马兴元, 郑宇. 生产二羟基丙酮的基因工程菌, 其构建方法机器用途[P]. CN 101092604A, 2007-12-26.
[16] Li MH, Wu J, Liu X, et al. Enhanced production of dihydroxyacetone from glycerol by overexpression of glycerol dehydrogenase in an alcohol dehydrogenase-deficient mutant of Gluconobacter oxydans[J]. Bioresource Technology, 2010, 101：8294-8299.
[17] Bories A, Claret C, Soueaille P. Kinetic study and optimization of the production of dihydroxyacetone from glycerol using Gluconobacter oxydans[J]. Process Biochem, 1991, 26：243-248.
[18] Gupta A, Singh VK, Qazi GN, et al. Gluconobacter oxydans：Its biotechnological applications[J]. J Mol Microbiol Biotechnol, 2001, 3：445-456.
[19] Shinjoh M, Tomiyama N, Miyazaki T, et al. Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase, that needs product of upstream gene, sldB, for activity[J]. Biosci Biochem, 2002, 66：2314-2322.
[20] Gatgens C, Degner U, Bringer-Meyer S, et al. Biotransformation of glycerol to dihydroxyacetone by recombinant Gluconobacter oxydans DSM 2343[J]. Appl Microbiol Biotechnol, 2007, 76（3）：553-559.
[21] 陈菁, 陈建华, 周怡雯, 等. 高效液相色谱法检测发酵液中二羟基丙酮和甘油的含量[J]. 中国生化药物杂志, 2007, 28（3）：170-172.
[22] Claret C, Bories A, Soucaille P. Glycerol inhibition of growth and dihydroxyacetone production by Gluconobacter oxydans[J]. Current Microbiology, 1992, 25：149-155.
[23] Bauer R, Katsikis N, Varga S, et al. Study of the inhibitory effect of the product dihydroxyacetone on Gluconobacter oxydans in a semi-continuous two-stage repeated-fed-batch process[J]. Bioprocess and Biosystems Engineering, 2005, 28（1）：37-43.
[24] Habe H, Shimada Y, Yakushi T, et al. Microbial production of glyceric acid, an organic acid that can be mass produced from glycerol[J]. Appl Environ Microbiol, 2009, 75（24）：7760-7766.
[25] Habe H, Fukuoka T, Morita T, et al. Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans[J]. Biosci Biotechnol Biochem, 2010, 74（7）：1391-1395.