Limiting metabolic steps in the utilization of D-xylose by recombinant Ralstonia eutropha W50-EAB

Abstract:

Abstract:［Objective］ To further improve the efficiency of xylose fermentation by modifying the pentose phosphate pathway (PPP) and the aldehyde reductase gene h16_A3186 in Ralstonia eutropha W50-EAB．［Methods］The transketolase (tktA，cbbT2) and transaldolase (tal) gene were cloned from R. eutropha chromosome by PCR and inserted into expressing vector pBBR1MCS-3．The resulting recombinant plasmids were transformed into W50-EAB to generate W50-KAB，W50-CAB and W50-TAB，respectively．The aldehyde reductase gene h16_A3186 was shortened from 834 bp to 135 bp by in-frame deletion from strain W50-E in which the xylE gene coding for xylose transporter was chromosomally integrated to construct recombinant strain W50'-E．Then the xylAB gene coding for xylose isomerase and xylulokinase from Escherichia coli were expressed in W50'-E to generate recombinant strain W50'-EAB．Recombinant plasmid pWL1-TAL was transformed into W50'-EAB to construct the strain W50'-TAB．The fermentation characteristics of the engineered strains were investigated． ［Results］The expression of tktA，cbbT2 and tal genes in R.eutropha W50-EAB was confirmed by enzyme assay．The deletion of h16 _A3186 gene was confirmed by PCR analysis and enzyme assay．Amplification of transketolase activity in R.eutropha W50-EAB showed negative effect on cell growth and D-xylose consumption．The recombinant strain W50-TAB and W50'-EAB exhibited a faster growth than W50-EAB with the maximum specific growth rate of 0.039 h-1 and 0.040 h－1，respectively，when cultivated on 0.1 mol/L D-xylose．And the PHB accumulation of W50-TAB and W50'-EAB reached 16.2±1.01% and 19.8±1.05% on the basis of cell dry weight，respectively．Furthermore，recombinant strain W50'-TAB exhibited better fermentation performance with the maximum specific growth rate of 0.042 h－1 and PHB content of 27.9±0.47%，respectively．Meanwhile，the recombinant strains W50-TAB，W50'-EAB and W50'-TAB showed higher biomass and more PHB accumulation when using glucose (0.01 mol/L) and Dxylose (0.09mol/L) mixed sugars as fermentative substrate.［Conclusion］Overexpression of the tal gene resulted in incressed D-xylose consumption．Deficiency of the aldehyde reductase relieved inhibition to D-xylose metabolism．Combination of the two strategies contributed to a higher efficiency of D-xylose utilisation and more PHB accumulation of the engineered R.eutropha strain.