Modification of Pichia pastoris for Erythritol Production by Metabolic Engineering

doi:10.13560/j.cnki.biotech.bull.1985.2023-0138

Abstract

Abstract:

The aim of this study is to construct an erythritol-producing strain using Pichia pastoris as the chassis cell. An erythritol-producing P. pastoris strain was developed by regulating the expression of phosphofructokinase gene（pfk）in glycolysis pathway, knocking out the genes associated with the production of by-products arabitol and ribitol, and overexpressing 4-phosphate erythrose phosphorylase, erythrose reductase and sugar alcohol phosphatase genes derived from different organisms. Next, we investigated the effects of overexpressions of erythrose reductase and two key enzymes transketolase（TKL）and ribulose-phosphate epimerase（RPE）involved in pentose phosphate pathway on the erythritol production. The results showed that strain C8 harboring pyp1 gene derived from Saccharomyces cerevisiae and yidA gene derived from Escherichia coli had the ability to produce erythritol. The erythritol production of the C8 strain in shake-flask fermentation was 30 mg/L. Furthermore, the overexpression of tkl and rpe genes enhanced the erythritol production of C10 strain by about 40-fold. The erythritol production of C10 reached 1.2 and 10.6 g/L in the shake-flask and high-cell-density fermentations, respectively. Further the overexpression of erythrose reductase did not cause the erythritol production increased, while caused the production of by-products increased. In this study, the erythritol synthetic pathway was successfully constructed in P. pastoris for the first time, which laid a foundation for engineering P. pastoris for efficient production of erythritol and other high-value compounds.

Key words: erythritol, Pichia pastoris, metabolic engineering, fermentation

ZHAO Si-jia, WANG Xiao-lu, SUN Ji-lu, TIAN Jian, ZHANG Jie. Modification of Pichia pastoris for Erythritol Production by Metabolic Engineering[J]. Biotechnology Bulletin, 2023, 39(8): 137-147.

Figures/Tables 11

References 34

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基因Gene name	功能Function	来源Source	蛋白分子量Protein molecular weight/kD
pyp1	糖醇磷酸酶基因Sugar alcohol phosphatase gene	S. cerevisiae	30.7
yidA	去磷酸化酶基因Dphosphorylase gene	E. coli MG1655	32.9
err1	赤藓糖还原酶基因Erythrose reductase gene	T. reesei	39.5
er（NADH）	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica	39.4
er（NADPH）	赤藓糖还原酶基因Erythrose reductase gene	C. magnoliae JH110	34.6
er1	赤藓糖还原酶基因Erythrose reductase gene	M. megachiliensis	39.6
er3	赤藓糖还原酶基因Erythrose reductase gene	M. megachiliensis	39.8
ER10	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica CLIB122	39.1
ER25	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica CLIB122	38.2
JX885	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica	31.4
rpe1	差向异构酶基因Epimerase gene	S. cerevisiae	29.2

基因Gene name	功能Function	来源Source	蛋白分子量Protein molecular weight/kD
pyp1	糖醇磷酸酶基因Sugar alcohol phosphatase gene	S. cerevisiae	30.7
yidA	去磷酸化酶基因Dphosphorylase gene	E. coli MG1655	32.9
err1	赤藓糖还原酶基因Erythrose reductase gene	T. reesei	39.5
er（NADH）	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica	39.4
er（NADPH）	赤藓糖还原酶基因Erythrose reductase gene	C. magnoliae JH110	34.6
er1	赤藓糖还原酶基因Erythrose reductase gene	M. megachiliensis	39.6
er3	赤藓糖还原酶基因Erythrose reductase gene	M. megachiliensis	39.8
ER10	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica CLIB122	39.1
ER25	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica CLIB122	38.2
JX885	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica	31.4
rpe1	差向异构酶基因Epimerase gene	S. cerevisiae	29.2

化合物名称Compound name	最高浓度Maximum concentration/(g·L^-1)
赤藓糖醇 Erythritol	0
阿拉伯糖醇 Arabitol	1.4
核糖醇 Ribitol	5.6

化合物名称Compound name	最高浓度Maximum concentration/(g·L^-1)
赤藓糖醇 Erythritol	0
阿拉伯糖醇 Arabitol	1.4
核糖醇 Ribitol	5.6

化合物名称 Compound name	最高浓度Maximum concentration/（g·L^-1）
化合物名称 Compound name	C11	C12
赤藓糖醇 Erythritol	1.2	1.2
核糖醇 Ribitol	1.6	4.0
阿拉伯糖醇 Arabitol	4.9	5.1