Development And Preliminary Application Of FLP/FRT Recombination System In Candida Amazonensis

Lignocellulose represents the largest source of renewable carbohydrate on earth,and the efficient utilization of xylose is one of the current bottleneck problems to achieve the industrial application of lignocellulose raw material.At present,the selection of microorganism which can ferment xylose effectively has become the key to solve the above problems,and the auxiliary means of genetic engineering technology plays important role in development and selection of new strains.Candida amazonensis,a novel yeast of the Scheffersomyces clade,is expected to be a new xylose-fermenting model strain,because of its outstanding capacity to ferment xylose and its good resistance to environmental stress.However,few available molecular tools limits the metabolic engineering and industrial application on this yeast strain.In view of the above problems,we used C.amazonensis C BS 12363 as the starting strain,and four promoters(SpXYLp,Sp MAL6 p,SpMAL1p,SpGAL1p)from Spathaspora passalidarum and ScGAL1 p promoter from Saccharomyces cerevisiae were amplified and fused to the reporter gene of green fluorescent protein(gfpm)to study the regulation performance in C.amazonensis under corresponding inducible conditions,respectively.The results showed that SpMAL1p(induced by maltose)and SpGAL1p(induced by galactose)were strictly inducible promoters in C.amazonensis.SpGAL1p was selected to control the expression of the C.amazonensis-adapted FLP gene(caFLP),encoding the site-specific recombinase FLP.The promoter-caFLP fusion fragment was used to ligated with the hphm marker gene that conferred resistance to Hygromycin B,and the ligation product was flanked by direct repeats of the FLP recognition target(FRT).Then with the addition of the homologous arms,we consructed the PDC deletion cassette(PRFgHRP).The cassette was transformed into C.amazonensis successfully.After selection of Hyg B-resistant transformant(designated as C.amazonensis PDC01)in which the deletion cassette was inserted into the PDC target gene,FLP expression was induced by growth of the transformant in galactose-containing medium,and Hyg B-sensitive transformant in which hphm and caFLP flippers were excised from the genome was obtained,designated as C.amazonensis PDC02.This is the first time to apply the FLP/FRT recombinant system in C.amazonensis.To further validate the effectiveness of the FLP/FRT recombinant system,and study the xylose fermentation ability of C.amazonensis,the recombinant expression vector pRMFH-ldh D was constructed by combining the D-lactate dehydrogenase gene(ldh D)derived from Lactobacillus plantarum with the FLP/FRT recombination system described above.After the linearized vector was transformed into C.amazonensis,the expression of FLP recombinase was accomplished by transferring the positive transformants into maltose-containing media,and then we obtained a ldhD recombinant strain(Ca-DM)without resistant marker gene.The fermentation results of the Ca-DM strain showed that the metabolic flow of xylose and glucose was mostly in the direction of D-lactic acid,and when the xylose was used as the carbon source,the concentration and yield of D-lactic acid was up to 117 g·L-1(99.9% optical purity or higher)and 0.9 g·g-1 xylose,respectively,illustrating the excellent ability to produce D-lactic acid on xylose.In addition,fermentation by the Ca-DM strain was carried out with corncob hemicellulose hydrolyzate as the substrate,and the production of D-lactic acid was achieved in hydrolyzate-containing media.