Spatial modulation and cofactor engineering of key pathway enzymes for fumarate production in Candida glabrata.

Fumarate is a naturally occurring organic acid that is an intermediate of the TCA cycle, and has numerous applications in food, pharmaceutical, and chemical industries. However, microbial fumarate production from renewable feedstock is limited by the intrinsic inefficiency of its synthetic pathway caused by week metabolites transportation and cofactor imbalance. In this study, spatial modulation and cofactor engineering of key pathway enzymes in the reductive TCA pathway were performed for the development of a Candida glabrata strain capable of efficiently producing fumarate. Specifically, DNA-guided scaffold system was firstly constructed and optimized to modulate pyruvate carboxylase, malate dehydrogenase, and fumarase, increasing the fumarate titer from 0.18 g/L to 11.3 g/L. Then, combinatorially tuning cofactor balance by controlling the expression strengths of ADP-dependent PEP carboxykinase and NAD+ -dependent formate dehydrogenase led to a large increase in fumarate production up to 18.5 g/L. Finally, the engineered strain T.G-4G-S(1:1:2) -P(M) -F(H) was able to produce 21.6 g/L fumarate in a 5-L batch bioreactor. This strategy described here paves the way to develop efficient cell factories for the production of the other industrially useful chemicals. This article is protected by copyright. All rights reserved.