Biosensor-assisted transcriptional regulator engineering for   Methylobacterium extorquens AM1 to improve mevalonate synthesis by   increasing the acetyl-CoA supply

doi:10.1016/j.ymben.2016.11.010

CORC > 北京大学 > 化学与分子工程学院

	Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply
	Liang, Wei-Fan ; Cui, Lan-Yu ; Cui, Jin-Yu ; Yu, Kai-Wen ; Yang, Song ; Wang, Tian-Min ; Guan, Chang-Ge ; Zhang, Chong ; Xing, Xin-Hui
刊名	METABOLIC ENGINEERING
	2017
关键词	Methylobacterium extorquens AM1 QscR regulation Biosensor Cyclic pathway Acetyl-CoA Mevalonate SERINE CYCLE GENES ESCHERICHIA-COLI C1 COMPOUNDS METHANOL PATHWAY FAMILY PROTEIN METABOLISM ACTIVATION EXPRESSION
DOI	10.1016/j.ymben.2016.11.010
英文摘要	Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal a-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. C-13 labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67 g/L, which was equivalent to an overall yield of 0.055 mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.; National Natural Science Foundation of China [NSFC 21376137, NSFC 21627812]; Tsinghua University Initiative Scientific Research Program [20131089238]; SCI(E); ARTICLE; 159-168; 39
语种	英语
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/476422]
专题	化学与分子工程学院
推荐引用方式 GB/T 7714	Liang, Wei-Fan,Cui, Lan-Yu,Cui, Jin-Yu,et al. Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply[J]. METABOLIC ENGINEERING,2017.
APA	Liang, Wei-Fan.,Cui, Lan-Yu.,Cui, Jin-Yu.,Yu, Kai-Wen.,Yang, Song.,...&Xing, Xin-Hui.(2017).Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply.METABOLIC ENGINEERING.
MLA	Liang, Wei-Fan,et al."Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply".METABOLIC ENGINEERING (2017).