Genome Shuffling Improves L-lactic Acid Production From Xylose By Rhizopus Oryzae

In recent years,as the food crisis strengthening day by day, the development ofrenewable resource has been put on the agenda.As a result,L-lactic acidfermentation using lignocellulosic biomass has become a new hotspot. Xylose is amajor component of hemicellulose,accounting for up to30%of lignocellulosichydrolysates.Besides glucose,xylose is one of the most abundant monosaccharidefound in nature. Generally,wild Rhizopus oryzae can’t convert xylose intoL-lactic acid efficiently.Thus screening of Rhizopus oryzae with high L-lactic acidproduction from xylose is meaningful to the bioconversion of lignocellulosicbiomass. In this thesis,genome shuffling was used to improve L-lactic acidproduction of Rhizopus oryzae from xylose.The fermentation characteristic and keyenzyme activities of genome shuffled strain,mutant and the original strain werestudied.Comparative proteomics analysis of proteins from different strains werecarried out by two-dimensional electrophoresis.The main conclusions are asfollows:（1）Rhizopus oryzae As3.819was mutagenized by using⁶⁰Coγ-irradiation.Considering the influence of irradiation dose on the lethal rate andpositive mutant rate,400Gy was choosen as the best irradiation dose.After repeatedmutagenesis treatment under the best irradiation dose,the mutant strains mut-33andmut-43,with high L-lactic acid production and stable genetic characteristic,wereobtained by selection.The L-lactic acid production of mut-33and mut-43was18.1%and15.3%higher than that of original strain respectively.（2）The electrofusion conditions of protoplasts from Rhizopus oryzae werestudied.0.6mol/L sorbitol was used as osmotic stabilizer. A selective system bybiparental inactivation was used to isolate fusants, and the inactivation conditions formut-33and mut-43were ultraviolet irradiation for180seconds and heating treatment at50℃for6minutes respectively.The electrofusion parameters of protoplasts were optimizedby unifactor experiment.The optimal parameters were as follows: direct current（DC） pulsevoltage2⁶⁰ V,pulse duration40μs,number of DC pulse2，and the maximum fusionfrequency reached7.29%.（3）Mutants mut-33and mut-43were used as parent strains,and F2-17was obtainedafter the second round of shuffling.The L-lactic acid production of F2-17achieved 58.12g/L after108hours’fermentation.Compare to the original strain in the samefermentation batch,F2-17’s fermentation period was reduced by24hours,and itsL-lacic acid production was increased by44.87%.The L-lactic acid yield of F2-17was28.80%higher than that of the original strain.The XR,XDH and LDH activitiesof original strain As3.819,mutant mut-33and shuffled strain F1-19,F2-17wereassayed in vitro analysis,the results showed that the XR,XDH and LDH activities ofthe shuffled strains were much higher than that of other strains.（4）Methods to the prepatation of Rhizopus oryzae mycelium protein have beendetermined.Two-dimensional electrophoresis maps of original strainAs3.819,mutant mut-33and shuffled strain F1-19,F2-17were obtained.21differentially expressed protein spots were found after image analysis,14spots ofthose were identified by using MALDI-TOF-MS analysis and databasesearching.These identified proteins could be divided into six categories accordingto their functions:①pyruvate cytoplasmic metabolic enzymes,②citric acid cycleenzymes,③glycolysis-related enzyme,④heatshock protein,⑤cytoskeletal proteins,⑥protein synthesis-related enzymes.（5）A speculation about the mechanism leading to the improved phenotypes ofshuffled strains was made.There was an increased ATP synthase expression in theshuffled strain.More ATP was synthesized,and a larged number of NAD+wasgenerated at the same time.Thus,XDH could get enough coenzyme,and the abilityof the shuffled strain to consume xylose was increased.Glycolytic rate of theshuffled strain was increased to a certain extent,and the generation of pyruvate waspromoted.As a result,the formation rate of L-lactic acid,a downstream product,wasincreased.Pyruvate flow in other branches was reduced,and more pyruvate flowshifted into L-lactic acid synthesis pathway.Thus,the L-lactic acid production wasincreased.