Automatic Redirection of Carbon Flux between Glycolysis and Pentose Phosphate Pathway Using an Oxygen-Responsive Metabolic Switch in Corynebacterium glutamicum

Abstract

Controlling the carbon flux into a desired pathway is important for improving product yield in metabolic engineering. After entering a cell, glucose is channeled into glycolysis and the pentose phosphate pathway (PPP), which decreases the yield of target products whose synthesis relies on NADPH as a cofactor. Here, we demonstrate redirection of carbon flux into PPP under aerobic conditions in Corynebacterium glutamicum, achieved by replacing the promoter of glucose 6-phosphate isomerase gene (pgi) with an anaerobic-specific promoter of the lactate dehydrogenase gene (ldhA). The promoter replacement increased the split ratio of carbon flux into PPP from 39 to 83% under aerobic conditions. The titer, yield, and production rate of 1,5-diaminopentane, whose synthesis requires NADPH as a cofactor, were increased by 4.6-, 4.4-, and 2.6-fold, respectively. This is the largest improvement in the production of 1,5-diaminopentane or its precursor, lysine, reported to date. After aerobic cell growth, pgi expression was automatically induced under anaerobic conditions, altering the carbon flux from PPP to glycolysis, to produce succinate in a single metabolically engineered strain. Such an automatic redirection of metabolic pathway using an oxygen-responsive switch enables two-stage fermentation for efficient production of two different compounds by a single strain, potentially reducing the production costs and time for practical applications.

Bifunctional optogenetic switch for improving shikimic acid production in E. coli

“…Here, we present a strategy to circumvent the humps in mining new regulatory elements; specifically, two orthogonal proteins, TetR and TEVp, were designed and optimized to control the transcriptional and protein level, respectively. To date, the regulatory switches available have been exploited for upregulation [ 38 ], downregulation, or bifunctional regulation [ 39 ]. However, most switched designs operate on one regulatory level, such as transcriptional level [ 33 , 40 , 41 ] or protein level [ 42 ].…”

Improving succinate production by engineering oxygen-dependent dynamic pathway regulation in Escherichia coli

“…The OBS can regulate two different protein targets under different regulation levels. By recruiting anaerobic-inducible promoters (P ldhA [42], P nar [43], P adhE [44], P hmp [45] and P 8vgb [46]) or regulatory protein (FNR [38,47]), several oxygen-responsive biosensors have been developed for building efficient microbial cell factories. For example, an anaerobic activation system was recently designed to enhance the metabolic flux towards the reductive TCA pathway under anaerobic conditions for glutaric acid production [47].…”

Green chemical and biological synthesis of cadaverine: recent development and challenges

“…55 Though the yield of cadaverine has been improved by metabolic engineering, secretion transporter, strong promoter, and efficient heterologous lysine decarboxylase, the carbon originating from glucose can pass through different pathways, such as glycolysis and pentose phosphate pathway depending on either aerobic or anaerobic conditions. 36 Control of the split ratio of carbon ux could be another possibility to increase the cadaverine production. Recently, a new strategy has been developed for improving the production of cadaverine by automatic redirection of carbon ux between glycolysis and PPP under aerobic conditions in C. glutamicum YT14.…”

“…This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.promoter of the lactate dehydrogenase gene (ldhA). The new process can increase the yield of cadaverine by 4.4 times 36,37.…”