[Engineering of an L-arabinose metabolic pathway in Ralstonia eutropha W50]

To broaden the substrate spectrum including L-arabinose, Ralstonia eutropha W50, a mutant strain with high yield of poly-beta-hydroxybutyrate (PHB), was metabolically engineered by expressing the genes encoding L-arabinose catabolic enzymes and high-affinity L-arabinose transporter from Escherichia coli.

The promoter fragment of PHB synthase gene phaC1 (P(pha C1)) from R. eutropha H16 and the araBAD genes from E. coli W3110 were cloned by PCR and inserted into expression vector pBBR1 MCS. The resulting recombinant plasmid was transformed into W50 to generate W50-1. The araFGH gene from E. coli W3110 was introduced into W50-1 by plasmid system or homologous recombination, yielding W50-2 and W50-3 respectively. The fermentation characteristics of the three engineered strains were investigated.

The flask fermentation experiments of the engineered strains show that W50-1 carrying the arabinose catabolic genes under the control of P(pha C1) could grow in the fermentation medium containing 0.1 mol/L arabinose as the sole carbon source, but could not utilize low concentration arabinose (0.01 mol/L). However, W50-2 and W50-3 containing the gene of high-affinity arabinose transporter were able to utilize low concentration arabinose. In the fermentation medium containing 0.1 mol/L arabinose, the biomass of W50-3 was 2.5 fold higher than that of W50-1, and the PHB accumulation amount of W50-3 accounted for 38.6% of the cell dry weight.

R. eutropha W50 was able to metabolize L-arabinose by the expression of araBAD genes, and the simultaneous expression of araFGH genes could further improve its ability of L-arabinose utilization. By using L-arabinose as the sole carbon source, the recombinant strain W50-3 can accumulate a noticeable level of PHB.