Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production

Abstract

L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH r ). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22±1.41) μmol g CDM 1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH r improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C 1 units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.

Characterization, modification, and overexpression of 3-phosphoglycerate dehydrogenase in Corynebacterium glutamicum for enhancing l-serine production

“…However, the growth rate decreased, which is consistent with the findings of previous reports indicating that deletion of sdaA decreased the rate of L-serine co-metabolism with glucose by 47 %, but still results in degradation of L-serine to pyruvate (Netzer et al 2004). Furthermore, it has been reported that attenuation of the conversion of L-serine to pyruvate and glycine, in combination with the co-expression of phosphoglycerate kinase and feedback-resistant PGDH, could dramatically improve Lserine production (Lai et al 2012). Therefore, it can be concluded that enhancement of L-serine production not only requires overexpression of the C-terminal truncated PGDH in the L-serine biosynthesis pathway, but also reduction of the L-serine degradation pathway in C. glutamicum SYPS-062.…”

A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum

“…Organic acids were determined by high performance liquid chromatography equipped with an SB-Aq column (4.6 × 250 mm; Agilent Technologies, USA) at 210 nm. Mobile phase A (20 mM KH 2 PO 4 , pH 2.3) and mobile phase B (acetonitrile) were at a ratio of 95:5 [42]. To measure the enzyme activity, the cells were resuspended in the indicated buffer (100 mM Tris-HCl pH 7.5 and 10% glycerol) and disrupted by ultrasonic treatment at 4°C.…”

Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes

“…For the gene clusters (nrps1, nrps3, pks6, pks-nrps2, lanti) that were expressed in one of the three media tested, gene knock-out combined with the comparative metabolic profile between mutants and wild type can be used to identify their corresponding products. For the silent gene clusters (pks2, pks8, pks-nrps1) that were not expressed in any of the three media, various methods could be used to activate them [27], and subsequently to dissect the metabolic pathway and flux [28].…”