Screening Of 2,3-butanediol-producing Saccharomyces Cerevisiae And The Effect Of Prdc Gene Knockout On Yield

2,3-Butanediol(2,3-BD)is an important platform chemical with widely application in fields of chemical industry,food,medicine and aerospace.The demand for 2,3-BD production is tremendous while the main way of 2,3-BD production is fermented by microorganisms due to the shortage of petroleum.However,most of the microorganisms used to produce 2,3-BD are pathogenic that is disaccord with the security principles of industrial production.Thus it's a hot topic to produce 2,3-BD by utilization of safety strains without pathogenesis.Saccharomyces cerevisiae can be a 2,3-BD-producing strain which has great advantage because the cultivation of this strain is pretty simple and rapid without pathogenic and too much by-products.Furthermore,the strain is considered as eukaryotic model organisms and has been widely applied in traditional and modern biotechnology fields.Nevertheless,during the process of yielding 2,3-BD fermented by S.cerevisiae,most of the glucose will be consumed to produce ethanol which is adverse for the accumulation of 2,3-BD.To improve the production of 2,3-BD,ethanol pathway in S.cerevisiae must be interdicted.Thus the paper aims at interrupt the ethanol pathway in S.cerevisiae by knocking out pyruvate decarboxylase genes?and?(pdc5and pdc1)through the method of homologous recombination which is helpful to convert more of the glucose into 2,3-BD.Thus 2,3-BD production will be enhanced.First of all,the original strain was selected and its specialties were analyzed.By application of shake-flask fermentation,the alcohol production performance of three candidate strains was detected.The S.cerevisiae W5 was finally selected as the original strain according to the conversion ratio of ethanol,growth,yielding of intermediate products and key enzyme activities.Under the best fermentation conditions that is 30?with a shaking speed at 200 r/min and glucose concentration of 80g/L,the most conversion ratio of ethanol was 0.397±0.02 g/g,10.0%and 8.3%less than that of S.cerevisiae WBG3(0.441±0.02)and S.cerevisiae RHZ7(0.433±0.01a).Furthermore,ethanol productions of the 3 strains improved by 16.80%?-7.72%and 10.41%with an enhancement of productivities that were 43.58%?10.20%and 11.09%respectively when 0.2 g Ca CO₃ was added in 12 h of the fermentation.While ethanol productions decreased by 8.59%?8.49%and 16.93%with an reduction of productivities that were6.30%?8.84%and 12.01%less than the control when 0.5%acetic acid was added initial process of the fermentation.The intermediate products of 2,3-BD pathway were also determined and the results showed that the concentrations of pyruvate,acetoin and?-acetolactic acid of S.cerevisiae W5 were the most that were 0.189±0.006 g/L?0.276±0.008 g/L and 3.475±0.035 g/L respectively,1.99 times and 2.45 times,2.85times and 1.42 times,1.03 times and 1.04 times more than that of S.cerevisiae WBG3and RHZ7.Meanwhile,fructose-6-phosphate kinase,pyruvate kinase,pyruvate decarboxylase,ethanol dehydrogenase and lactate dehydrogenase were detected by kits and the maximal enzyme activities of them were 8.417±0.190 U/mg(24 h)?98.410±2.952 U/mg(24 h)?0.258±0.003 U/mg(24 h)?0.379±0.011 U/mg(24 h)?46.89±0.60 U/mg(48 h)respectively,demonstrating that the key enzyme activities of W5 were the most at 24h.Second,homelogens recombinant technology(SFH-PCR)was applied to interrupt the ethanol pathway.With plasmid p EGFP-C3 and p CAMBIA1300 genomic DNA as a template,40 bp homologous linear segments the same as pdc5-EGFP(949 bp)and pdc1-hy B(1109 bp)were cloned in each side pdc5 and pdc1 contained in S.cerevisiae W5 where the green fluorescent protein(EGFP)and hygromycin(hy B)were uased as screening markers respectively.The homologous linear segments were transformed into S.cerevisiae W5 by acetic acid lithium,which homologous recombinate with pdc5 and pdc1 genes respectively.The properties of the recombinant strain were determined by PCR,q RT-PCR,SDS-PAGE,pyruvate decarboxylase enzymes activity assay and2,3-butanediol production.The results showed that the relative expressions of pdc5contained in recombinant strains S.cerevisiae W5-01 and pdc1 in S.cerevisiae W5-02were 49%,30%and 53%,55%lower than that of the original strain at 12h and 48h respectively while the the relative expressions of bdh1 were 2.50,33.59 and 1.67,89.88times more than that of the original strain at 12h and 48h respectively.The gray value of proteins encoded by pdc5 in S.cerevisiae W5-01 and pdc1 in S.cerevisiae W5-02 were21.93%and 30.1%lower than the original strain at 48h respectively.The pyruvate decarboxylase enzymes activities of S.cerevisiae W5-01(0.39±0.1 U/mg)and S.cerevisiae W5-02(0.307±0.1 U/mg)were 59.75%and 60.13%less than the original strain(0.77±0.09 U/mg)respectively while the 2,3-butanediol production of them were1.20±0.1 g/L and 0.47±0.1 g/L,263.64%and 67.86%more than that of the original strain(0.28±0.07 g/L).In this paper,more carbon source flows to generate 2,3-butanediol path by koncking out the pdc5 and pdc1genes that is helpful to amplify the choice of strain for industrial production of 2,3-butanediol and lay the foundation of 2,3-butanediol production by microbial fermentation in some degree.