The Construction And Preliminary Fermentation Evaluation Of Xylose-utilizing Recombinant Z. Mobilis Strain

Making full use of all components in lignocellulosic hydrolysate is the key point of producing fuel ethanol from lignocellulose. As xylose is one of the principal constituents of lignocellulosic hydrolysate and the second abundant monosaccharide in nature, researchers have paid much attention in developing microbial strains for manufacturing ethanol from glucose and xylose. Z. molilis is a bacterium that efficiently metabolites glucose for ethanol production, but it does not make use of xylose. The purpose of present study is to construct recombinant strains of Z. mobilis CP4 capable of converting xylose to ethanol.Firstly, DNA fragments Peno-talB and tktA were prepared by PCR and inserted into vector pUC19, resulting in the formation of plasmid pUC19-Peno-talB/tktA. Secondly, pZB21-Pgap-xylA/xylB-Peno-talB/tktA, a plasmid composed of four E. coli genes related to xylose metabolism, was constructed by ligating Peno-talB/tktA with a shuttle plasmid pZB21-Pgap-xylA/xylB. The recombinant strains of Z. mobilis CP4(pZB21-Pgap-xylA/xylB-Peno-talB/tktA) were selected from T plate after electroporation. The enzyme assays of cell-free extracts revealed that the cloned four genes encoding xylose metabolic enzymes had been co-expressed in recombinant strains. The enzyme activity levels of recombinant stain 1# were shown as follows,XI: 0.0449 U/min/mg proteins, XK: 0.337 U/min/mg proteins, TAL: 0.047 U/min/mg proteins, TKT: 0.291 U/min/mg protein.Cell culture experiments verified that the recombinant strains were not capable growing in xylose medium. Besides, a little ethanol was produced in xylose-fermenting medium.In the experiment of fermentation performance of the xylose-fermenting strain, the result showed that the recombinant strain 1# was found to be capable of utilizing about 35.4% xylose in a 6% xylose-fermenting medium in 168 hours with a ethanol yield about 52.94% of the theoretical value. On the contrary, the xylose conversion of control strain Z. mobilis CP4(pZB21) was zero and there was no production of ethanol. These results provided evidence for the potential xylose-fermenting ability of the recombinant Z. mobilis CP4(pZB21-Pgap-xylA/xylB-Peno-talB/tktA) strain.