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

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 D-xylose (0.09 mol/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.