Study On Genetic Transformation System Construction Of Rhizopus Oryzae And Modification Of Xylose Metabolism

Rhizopus oryzae is an important producer of organic acids including L-lactic acid. It possesses high potential for industrial applications because of its advantage of simple culture conditions and fermentation process, as well as the high optical purity of lactic acid product. However, the xylose metabolism rate and intensity of pentose-hexose co-fermentation are low, which hinder its efficient use of lignocellulosic biomass. In this paper, with Rhizopus oryzae strain AS 3.819 as the research object, genetic engineering technology was used to construct an integrating expression vector and the methods of transformation and screening for Rhizopus oryzae, and xylose reductase was genetically engineered, with the goal of enhancing the pentose-hexose co-fermentation capacity of Rhizopus oryzae. The main research work and achievements accomplished are summarized as follows:1) Xylose metabolism and pentose-hexose co-fermentation of Rhizopus oryzae. Induction and inhibition effect of carbon substrates on activities of key enzymes in xylose metabolism was analyzed. Analysis of Rhizopus oryzae xylose fermentation and glucose-xylose co-fermentation conditions and comparing of xylose and xylitol fermentation suggested that the limiting key enzyme of xylose metabolism was xylose reductase. The fermentation results with the existence of surfactant confirmed that glucose repression on xylose metabolism is the main reason for the limitation of pentose-hexose co-fermentation of Rhizopus oryzae.2) Cloning of Rhizopus oryzae promoter fragment and study of the promoting activities. A promoter probe vector pUKMR was constructed and used in Escherichia coli to analyze and compare the functions and activities of gene promoter fragments from Rhizopus oryzae. The substrate induction of promoter fragments was studied through comparing the promoting activities under different carbon substrates. The effect of lactate dehydrogenase promoter length on promoting activity was studied.3) Genetic transformation method of Rhizopus oryzae based on hygromycin B resistance as selectable marker. Gene splicing by overlap extension PCR (SOE-PCR) technology was used to construct an integrating eukaryotic expression vector with hygromycin B resistance as selectable marker. Rhizopus oryzae was transformed using this vector and transformants were achieved. The effects of transformation conditions on transformation frequencies were studied and optimal transformation conditions were obtained. Quantitative PCR was used to study the effect of integrated plasm id copy number on antagonistic genetic stability of transformants.4) Cloning of Rhizopus oryzae xylose reductase gene and its heterologous expression. The cDNA fragment of xylose reductase gene was obtained by using reverse transcription PCR. The fragment was expressed with Pichia pastoris heterologous protein expression system, and recombinant protein with xylose reductase activity was obtained. The enzymatic properties, substrate and coenzyme preference of recombinant protein were studied after it was isolated and purified. Application of site-directed mutagenesis analysis suggested that amino acid sites Thr226 and Val274 in the recombinant protein had effect on its coenzyme preference.5) Genetic modification of Rhizopus oryzae xylose reductase gene. Rhizopus oryzae was genetic transformed with xylose reductase gene as object. Rhizopus oryzae transformants with multiple copies of the xylose reductase gene was achieved. The multiple copies of the xylose reductase genes were promoted as constitutive expressing gene, which resulted in the depletion of glucose repression. The results of xylose fermentation and glucose-xylose co-fermentation suggested that the xylose metabolism capacity of transformants had been improved. However, xylose metabolism was still subject to repression in the case of co-fermentation. The increase of cell permeability of the transformants by using surfactant resulted in a low rate of xylose utilization in co-fermentation.