代谢工程改造黑曲霉生产葡萄糖二酸

doi:10.13560/j.cnki.biotech.bull.1985.2022-0280

摘要/Abstract

摘要：

葡萄糖二酸是葡萄糖的一种二元羧酸衍生物，是重要的平台化合物，被应用于医药、化工等领域。本研究以黑曲霉为底盘细胞，通过表达来自恶臭假单胞菌Pseudomonas putida KT2440的糖醛酸脱氢酶基因ppudh，成功在黑曲霉中实现了葡萄糖二酸的合成，产量为18.74 mg/L；在此基础上通过共过表达黑曲霉自身来源的肌醇加氧酶（anmioxA）和肌醇-1-磷酸合酶（aninoA）、酿酒酵母Saccharomyces cerevisiae S288C来源的羧酸转运蛋白（scJEN1），强化了合成通路和外泌途径，将产量提高至102.10 mg/L；通过表达来自乳酸乳球菌Lactococcus lactis subsp. cremoris MG1363的NADH氧化酶（llnox），建立NAD⁺辅因子循环系统，使产量进一步提高至115.65 mg/L；利用RNA干扰技术对竞争支路中的关键酶磷酸果糖激酶（pfkA）和葡萄糖-6-磷酸脱氢酶（zwf）进行弱化表达，葡萄糖二酸最终的产量达到313.65 mg/L。本研究为微生物高效生产葡萄糖二酸和下游相关产品生产奠定基础。

关键词: 葡萄糖二酸, 黑曲霉, 辅因子循环系统, RNA干扰

Abstract:

D-glucaric acid, a dicarboxylic acid derivative of glucose, is an important platform compound and is used in medicine, chemical industry and other fields. Using Aspergillus niger as the chassis cell for D-glucaric acid production, expressing the uronate dehydrogenase gene ppudh from Pseudomonas putida KT2440 in A. niger, D-glucaric acid biosynthesis with a titer of 18.74 mg/L was successfully achieved. Strengthening D-glucaric acid biosynthesis secretion, by overexpressing the endogenous inositol oxygenase（anmioxA）and inositol-1-phosphate synthase（aninoA）as well as the carboxylate transporter（scJEN1）from Saccharomyces cerevisiae S288C, D-glucaric acid production was improved significantly with a titer of 102.10 mg/L. Further establishing a NAD⁺ cofactor recycling system by expressing a NADH oxidase（llnox）from Lactococcus lactis subsp. cremoris MG1363 D-glucaric acid yield was improved to 115.65 mg/L. Finally reducing the carbon flux to glycolysis and pentose phosphate pathway enabled the highest D-glucaric acid production of 313.65 mg/L, which lays the foundation for efficient production of glucaric acid and the corresponding downstream chemicals by microorganisms.

Key words: glucaric acid, Aspergillus niger, cofactor recycling system, RNA interference

郭宇飞, 闫荣媚, 张小茹, 曹威, 刘浩. 代谢工程改造黑曲霉生产葡萄糖二酸[J]. 生物技术通报, 2022, 38(11): 227-237.

GUO Yu-fei, YAN Rong-mei, ZHANG Xiao-ru, CAO Wei, LIU Hao. Metabolic Engineering Modification of Aspergillus niger for the Production of D-glucaric Acid[J]. Biotechnology Bulletin, 2022, 38(11): 227-237.

图/表 11

参考文献 33

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年份Year	菌株Strain	基因改造Genes madified	碳源Carbon source	产量Yield/（g·L^-1）	引用文献Reference
2009	E. coli BL21 Star（DE3）	ino1，MIOX，udh	Glucose	1.13	[12]
2010	E. coli BL21 Star（DE3）	ino1，MIOX，udh	Glucose	2.50	[13]
2014	E. coli MG1655	ino1，SUMO-MIOX，udh	Myo-inositol	4.85	[17]
2016	S. cerevisiae CEN. PK2-1D	ino1，inm，MIOX，udh	Glucose	1.60	[14]
2017	E. coli L19S	ino1，MIOX，udh	Glucose	0.80	[18]
2017	S. cerevisiae CEN. PK2-1C	MIOX，udh，ino1，pfk1	Glucose	0.23	[15]
2018	E. coli BL21 Star（DE3）	cscB，cscA，cscK，ino1， MIOX，udh，suhB	Sucrose	1.42	[19]
2018	Pichia pastoris GS115	MIOX，udh	Glucose	2.60	[16]
2018	S. cerevisiae BY4741	ino1，MIOX，udh	Glucose，myo-inositol	6.00	[20]
2020	E. coli BL21 Star（DE3）	ino1，MIOX，udh，suhB	Glucose	5.35	[21]
2020	S. cerevisiae	ino1，inm，MIOX，udh，vgb	Glucose	6.38	[22]

年份Year	菌株Strain	基因改造Genes madified	碳源Carbon source	产量Yield/（g·L^-1）	引用文献Reference
2009	E. coli BL21 Star（DE3）	ino1，MIOX，udh	Glucose	1.13	[12]
2010	E. coli BL21 Star（DE3）	ino1，MIOX，udh	Glucose	2.50	[13]
2014	E. coli MG1655	ino1，SUMO-MIOX，udh	Myo-inositol	4.85	[17]
2016	S. cerevisiae CEN. PK2-1D	ino1，inm，MIOX，udh	Glucose	1.60	[14]
2017	E. coli L19S	ino1，MIOX，udh	Glucose	0.80	[18]
2017	S. cerevisiae CEN. PK2-1C	MIOX，udh，ino1，pfk1	Glucose	0.23	[15]
2018	E. coli BL21 Star（DE3）	cscB，cscA，cscK，ino1， MIOX，udh，suhB	Sucrose	1.42	[19]
2018	Pichia pastoris GS115	MIOX，udh	Glucose	2.60	[16]
2018	S. cerevisiae BY4741	ino1，MIOX，udh	Glucose，myo-inositol	6.00	[20]
2020	E. coli BL21 Star（DE3）	ino1，MIOX，udh，suhB	Glucose	5.35	[21]
2020	S. cerevisiae	ino1，inm，MIOX，udh，vgb	Glucose	6.38	[22]

菌株Strain	特征描述Characteristics	来源Source
Escherichia coli JM109	-	本实验室保存
Agrobacterium tumerfaciens AGL-1	-	本实验室保存
S834	A.niger ATCC1015，Tet-on：：cre，ΔoahA，ΔcexA	本实验室保存
S985	ΔoahA，ΔcexA，OEppudh	本研究
S1223	ΔoahA，ΔcexA，OEppudh，OEanmioxA	本研究
S1877	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1	本研究
S2086	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA	本研究
S2390	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA，OEllnox	本研究
S2603	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA，OEllnox，RNAipfkA	本研究
S2635	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA，OEllnox，RNAipfkA，RNAizwf	本研究

菌株Strain	特征描述Characteristics	来源Source
Escherichia coli JM109	-	本实验室保存
Agrobacterium tumerfaciens AGL-1	-	本实验室保存
S834	A.niger ATCC1015，Tet-on：：cre，ΔoahA，ΔcexA	本实验室保存
S985	ΔoahA，ΔcexA，OEppudh	本研究
S1223	ΔoahA，ΔcexA，OEppudh，OEanmioxA	本研究
S1877	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1	本研究
S2086	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA	本研究
S2390	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA，OEllnox	本研究
S2603	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA，OEllnox，RNAipfkA	本研究
S2635	ΔoahA，ΔcexA，OEppudh，OEanmioxA，OEscJEN1，OEaninoA，OEllnox，RNAipfkA，RNAizwf	本研究

质粒Plasmid	特征描述Characteristics	来源Source
pLH454	loxP-hph-loxP，gpdA promoter，trpC terminator，hyg^r，kan^r	本实验室保存
pLH509	loxP-hph-loxP，pkiA promoter，trpC terminator，hyg^r，kan^r	本实验室保存
pLH756	loxP-hph-loxP，mbfA promoter，trpC terminator，hyg^r，kan^r	本实验室保存
pLH1418	loxP-hph-loxP，glaA promoter，gfp loop，trpC terminator，hyg^r，kan^r	本实验室保存
pLH1453	loxP-hph-loxP，pkiA promoter，gfp loop，trpC terminator，hyg^r，kan^r	本实验室保存
pLH690	loxP-hph-loxP，gpdA promoter，ppudh gene，trpC terminator，hyg^r，kan^r	本研究
pLH737	loxP-hph-loxP，pkiA promoter，anmioxA gene，trpC terminator，hyg^r，kan^r	本研究
pLH782	loxP-hph-loxP，gpdA promoter，scJEN1 gene，trpC terminator，hyg^r，kan^r	本研究
pLH1020	loxP-hph-loxP，gpdA promoter，aninoA gene，trpC terminator，hyg^r，kaⁿr	本研究
pLH1317	loxP-hph-loxP，mbfA promoter，llnox gene，trpC terminator，hyg^r，kan^r	本研究
pLH1421	loxP-hph-loxP，glaA promoter，sense sequence of zwf gene，gfp loop，antisense sequence of zwf gene，trpC terminator，hyg^r，kan^r	本研究
pLH1454	loxP-hph-loxP，pkiA promoter，sense sequence of pfkA gene，gfp loop，antisense sequence of pfkA gene，trpC terminator，hyg^r，kan^r	本研究