Engineering of a D-xylose metabolic pathway in Ralstonia eutropha W50

Abstract:

Abstract:［Objective］This study aimed to broaden the substrate spectrum of Ralstonia eutropha W50 to use D-xylose，which can produce poly-β-hydroxybutyrates(PHB) at a high level.［Methods］The D-xylose transporter gene xylE from Escherichia coli K-12 W3110 was cloned by PCR technique and integrated into the R．eutropha W50 chromosome．The recombinant strain W50-E was obtained．The D-xylose catabolic genes xylAB from E. coli K-12 W3110 and the promotor of PHA synthase gene phaC1 from R．eutropha H16 were cloned into pBBR1MCS to construct a recombinant plasmid．The plasmid was transformed into R．eutropha W50 and W50-E to generate the recombinant strains W50-AB and W50-EAB respectively．The characteristics of D-xylose utilization by W50-AB and W50-EAB were investigated.［Results］The expression of xylA and xylB genes in R．eutropha W50 was confirmed by enzyme assay．The recombinant strain W50-AB could grow on 0.1 mol/L D-xylose with the maximum specific growth rate of 0.025 h－1，but no growth and D-xylose consumption were observed when cultivated on 0.01 mol/L D-xylose．The recombinant strain W50-EAB exhibited a faster growth than W50-AB on 0.1 mol/L D-xylose，with the maximum specific growth rate of 0.035 h－1．Furthermore，it exhibited a slow but defined growth and D-xylose consumption on 0.01 mol/L D-xylose．The PHB content assay showed that both recombinant strains accumulated a small amount of PHB，with a proportion of 15.07±1.01% and 15.07±1.64% on the basis of dry cell weight respectively，by using D-xylose(0.1 mol/L) as substrate．And their final D-xylose-PHB conversion rates were 0.0920 g·g－1 and 0.0838 g·g－1 respectively，which were much lower than their glucose-PHB conversion rates(＞0.22 g·g－1)．However，the recombinant strains W50-AB and W50-EAB exhibited better fermentation performance and more PHB accumulation when using glucose(0.01 mol/L) and D-xylose(0.09 mol/L) mixed sugars as fermentative substrate.［Conclusion］The recombinant strain W50-AB can metabolize D-xylose by the expression of xylAB genes，and the further expression of xylE gene is able to improve its D-xylose consumption rate．Meanwhile，the two recombinant strains can accumulate a small amount of PHB by using D-xylose as the sole carbon source．