Construction Of Two Targeting Vectors For Deleting PyrG And LadA Gene From Aspergillus Oryzae

As an important GRAS industrial fungus, Aspergillus oryzae possessing relatedenzymes for hemicellulose and xylose catabolism, is able to integrate enzymeproduction, saccharification and fermentation into a one step process for efficientxylitol production. With knocking-out xdh gene to prevent xylitol’s transformation toD-xylulose, the yield of xylitol had been increased by10times than the parent strain.Nevertheless, there existed problems to be solved:1) The pyrG-parental strain wasscreened by UV mutagenesis. As targeting vector carries a pyrG gene, homologousrecombination tends to occur at the mutated pyrG gene rather than the target genelocus. Besides, ability to produce extracellular glucoamylase of the parental strain isought to be improved for higher productivity of xylitol;2) Loss of the xylitoldehydrogenase step can be partially compensated for some dehydrogenase likeL-arabinitol dehydrogenase encoded by ladA gene that should be knocked-out toblock up catabolism of xylitol further. Hence, an A.oryzae strain owing highlyproductivity of extracellular glucoamylase was chosen as our parental strain for alltransformation, and then the targeting vector pDispyrG and pBSK-pyrG-ladABCwere constructed for knocking-out the pyrG gene and for deleting the ladA generespectively.1. Using genome of A.oryzae as a template, pyrG gene was cloned. Then pyrGupstream fragment PA and downstream fragment PB were amplified and fused intoone by PCR. The fusion product was ligated to pMD-19T plasmid and transformedinto E.coil, reaching the goal of constructing the vector pDispyrG.2. With genome of A.oryzae as a template, ladA upstream segments LA and LB anddown stream segment were cloned. A single enzyme cutting sites KpnI and Bsp1407Iwere found by primer preimer5.0, which divided these segments into three sections—imtermediate fragments1,2and3. By normal PCR and fusion PCR, fragments1,2and3were amplified. Afer restriction digestion, intermediate fragments1,2, and3were ligated to pBluescript II SK+plasmid. Thus the targeting vector was constucted sucessfully.Plasmid pDispyrG is constructed for a satble pyrG auxotroph transformationsystem with high productivity of extracellular glucoamylase, which lays thefoundation for subsequent mutiple genetic manipulation. And plasmidpBSK-pyrG-ladABC is used to marker-freely delete ladA gene, recycling the markerpyrG and blocking up catabolism of xylitol futher as expected.