Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

Abstract

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7: [182][183][184][185][186][187][188][189][190][191][192][193][194][195][196] 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

Arabitol Metabolism of Corynebacterium glutamicum and Its Regulation by AtlR

“…The mtlD gene product shows a striking level of identity (up to 68%) with bacterial ManDHs, which, at least in some cases, have also been shown to catalyze arabitol oxidation with NAD ϩ (e.g., see references 14, 15, and 72). The xylB gene product shows up to 65% identity with XylK genes from other bacteria, and the functionality of the xylB gene product as XylK was previously shown by Kawaguchi et al (39). All genes for the enzymes of D-arabitol uptake and metabolism in C. glutamicum are organized into two operons, i.e., the atlR-xylB operon and the rbtT-mtlD-sixA operon (Fig.…”

The LacI-Type Transcriptional Regulator AraR Acts as an l -Arabinose-Responsive Repressor of l -Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831

“…Cellulosic biomass contains significant amounts of pentose sugars, such as D-xylose and L-arabinose, that are often difficult for conventional bioprocesses to exploit. Recent advances in metabolic engineering of C. glutamicum have helped to open up new possibilities for efficient utilization of substrates containing mixtures of D-glucose, D-xylose, and L-arabinose (8)(9)(10)(11)). An understanding of the transcriptional regulation of sugar metabolism genes in C. glutamicum is an attractive avenue toward optimization of utilization of such sugar mixtures.…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…These sugars, namely xylose and arabinose, display significant fractions in agricultural residues and other lignocellulosic biomass recently receiving increasing interest as a cheap and most abundant raw material for biobased production [161]. Through heterologous expression of the E. coli genes xylA and xylB C. glutamicum was able to consume xylose as sole source of carbon [162]. In substrate mixtures, glucose-mediated regulation still exerts a measurable influence on xylose consumption kinetics.…”