Breeding Of Rhizopus Oryzae Producting L-Lactic Acid Based On SR Soft X-Ray Radiation

L-lactic acid is an important organic acid and widely applied in food,pharmaceutical and other industries. There is an increased interest inappling L-lactic acid as a starting material for the synthesis ofpolylacticacid, a biodegradable and good biocompatibility plastic.Rhizopus oryzae could be a high-performance strain to produce highoptical purity L-lactic acid, because it does not require complicatenutrition in fermentation. For all that, the primary limitation of theRhizopus fermentation for L-lactic acid is in the relatively poor yield oflactate per mole of glucose consumed and low performance in conditionsof higher temperature or lower pH. Radiation mutagenesis is an importanttechnology for microbiology to produce high-performance strains.Because synchronous radiation(SR) soft X-ray coveres K-absorption edgewavelength of fundamental life elements such as C, N and O, which hasstronger radiation biological effect, the radiobiological studies of it hadbeen payed more attentions in recent years.Based on SR soft X-ray radiation, breeding of Rhizopus oryzae hasbeen developed towards the goal of increasing L-lactic acid yield, highvolumetric productivity, and high performance in conditions of low pH.And mutation effects were carefully investigated on metalbolism, cDNAand proteomics level. Summary and main conclusions as follows:(1) The soft X-ray mutagenesis was carried out in SynchronousRadiation Laboratory (SNRL) at University of Science and Technology ofChina (USTC). The soft X-ray radiation conditions for Rhizopus oryzaemutagenesis were established after investigated several main factors suchas sample holder for spores and sample carriers. The lethal curves of Ck(4.4nm), Nk (3.02nm), and Ok (2.3nm) of soft X-ray on the spores ofRhizopus oryzae were developed, respectively. Using lethal curves andenergy deposition of short wavelength radition analysis, Nk wavelengthsoft-X ray with moderate energy deposition was used for Rhizopus oryzaemutagenesis. Optimum radiation dose range,0.4-0.8kGy, for Rhizopusoryzae mutagenization was concluded by analysising relation betweenlethal rate and positive mutation rate of weakened alcohol dehydrogenase（ADH） activity mutation by Nk wavelength soft X-ray radiation.(2) A mutant ADH-21, with high L-lactic acid yield and weakenedADH activity, was screened with yeast peptone dextrose（YPD） platecontaining allyl alcohol from Rhizopus oryzae mutants which weremutagenized by soft X-ray. L-lactic acid yield of ADH-21and paraentstrain were94.01g/L and82.49g/L, respectively. Compared with that ofparaental strain, L-lactic acid yield of ADH-21increased by13.97%,while ethanol yield decreased by70.99%. Furthermore, compared withthat of parental strain, lactate dehydrogenase(LDH) activity of ADH-21 increased by34.7％, while ADH activity of ADH-21decreased by79.85％. These results indicate that inhibition of the activity of ADH is aneffective way to reduce the production of ethanol while improve theproduction of L-lactic acid. ADH of Rhizopus oryzae AS3.819andADH-21were purified for further chartacterization. Effects of pH,concentration of Zn2+, and Mg2+on Rhizopus oryzae AS3.819andADH-21were similar, while optimum reaction temperature of ADH frommutant were lower than that from parental strain. In addition, cDNAsequence of adh gene from parant strain was compared with mutant.(3) For increasing the glycolytic flux of Rhizopus oryzae by defectingoxidative phosphorylation, a mutant F0F1-17, with high L-lactic acid yieldand65%of F0F1-ATPase activity decrease, was screened with PDA platecontaining neomycin from Rhizopus oryzae mutants which weremutagenized by soft X-ray. As a consequence, the glycolytic pathway keyenzyme phosphofructokinase(PFK), pyrutate kinase(PK), glyceraldehydesphosphate dehydrogenase (GAPDH) activity in the mutant increased by58.5%,24.8%and19.8%, which resulted that rate of glucose consumed,average specific glucose consumption rate, L-lactic acid productivity andaverage specific L-lactic acid production rate of the mutant increased by17.5%,85.9%,24.1%and61.5%than those of the paraental strain,respectively. The maximum L-lactic acid yield (91.54g/L) by mutant wasobtained within about48h of fermentation which was about6h shorter than that by the paraental.(4) The specific activity of hexokinase increased with the increase ofMg2+concentration in fermentation medium. Effects of positivlyregulating Mg2+concentration on the fermentation performance of F0F1-17and AS3.819were resulted that a slightly increase of the hexokinaseactivity resulted in a slightly increase of performances of the L-lactic acidfermentation, especially in more harsh effects on the low-energy chargeF0F1-17strains than those on the Rhizopus oryzae AS3.819. Consequently,for increasing the glycolytic flux of Rhizopus oryzae by increasing HKactivity, a mutant HK-3, with high L-lactic acid yield and85.3%ofhexokinase(HK) activity increase, was screened with medium containing2-DG as a sole carbon source from Rhizopus oryzae mutants which weremutagensised by soft X-ray. Compared with paraental strain, rate ofglucose consumed, average specific glucose consumption rate, L-lacticacid productivity and average specific L-lactic acid production rate of themutant increased by29.8%,27.9%,71.8%and41%, respectively.Compared with AS3.819, rate of glucose consumed, average specificglucose consumption rate, L-lactic acid productivity and average specificL-lactic acid production rate of the mutant increased by141.4%,50.3%,177.5%and75%, respectively. The maximum L-lactic acid yield (97.53g/L) by mutant was obtained within~36h of fermentation. It is provedthat a Rhizopus oryzae mutant with lower F0F1-ATPase activity and higher hexokinase activity could be a good performance industry strain.(5) For increasing fermentation performance of Rhizopus oryzae inacid-resistant conditions, a mutant acid-18, with26.53%higher L-lacticacid yield (83.5g/L), was screened with accumunation in mediumcontaining L-lactic acid from Rhizopus oryzae mutants which weremutagenized by soft X-ray. Consistently, compared with that of parentalstrain, LDH activity of mutant increased by42.2％, and H+-ATPaseactivity increased. All proteins from mutant and parental strain wereseparated by two-dimensional electrophoresis. PDQuest software analysisto electrophoresis map showed that there were475proteins with differentabundance between the acid-resistant mutant and parental strain, and12points with significantly different abundance whose molecular weight,isoelectric point and other properties were analysised and5proteins of12were identified by Maldi-toff mass spectrometry and database search.This thesis results have laid the experimental foundation for SR softX-ray applications to Rhizopus oryzae mutagenization. On the basis ofstudy to radiation mutagenesis conditions by SR soft X-ray with Rhizopusoryzae as a model object, methods aimed at increasing fermentationperformance, including breeding mutant with weakened ADH activity,breeding mutant with weakened F0F1-ATPase activity and increased HKactivity, breeding mutant with increased acid-resistant, were studied by SRsoft X-ray radiation mutagenesis. And mutation effects of above mutants were analysised in metabolism, key enzymes activity, proteome and cDNAlevels. This study presents underlying data for breeding a goodfermentation performance Rhizopus oryzae suitable for industryprocessing.