Study Of The Efficient Synthesis Of Fuel Ethanol From Saccharomyces Cerevisiae Using Pentacharide Sugar

Industrial Saccharomyces cerevisiae C5 has good fermentation properties,can efficiently use glucose and xylose in the lignocellulose digestion,synthetic fuel ethanol,for industrial production.In order to increase the production of fuel ethanol in industrial Saccharomyces cerevisiae and respond to the national"carbon neutrality"goal,relevant experiments were conducted to explore the scheme of heterotrophic microorganisms using CO₂to synthesize ethanol.Existing literature reports and research progress show that it is feasible to convert heterotrophic microorganisms into autotrophic microorganisms,so an attempt was made to transfer the Rubisco enzyme gene rbc from the Calvin cycle into industrial Saccharomyces cerevisiae C5 to compare the ethanol yield of recombinant yeast and C5.The main findings are as follows:（1）Construct heterologous carbon sequestration vector K4-r DNA-HYB-FBA1p-rbc-CYC1t-ENO2p-prk-SLM5t-TEF2p-ca-TYS1t and chaperone protein gro E expression vector K2-d1g-zeocin-TEF1p-gro E-PGI1t,using lithium acetate conversion method,sequentially transformed into the C5 genome of industrial Saccharomyces cerevisiae,to obtain recombinant carbon sequestration strains C5-rpc,C5-rpcy,C5-rpcy-gro E.（2）The transcription level,growth status and fermentation performance of the recombinant strain were verified and compared,and the absorbance value of OD600in the fermentation process of the strain was determined,and it was found that the growth of recombinant Saccharomyces cerevisiae was relatively slow in the early stage,and after multi-generation domestication,it could grow normally,and its growth rate could reach a similar level of the starting strain C5.The results of the expression verification of recombinant Saccharomyces cerevisiae showed that after 6the transformation and integration of the codon-optimized carbon sequestration gene into the industrial Saccharomyces cerevisiae genome,due to its low codon adaptability,the carbon sequestration gene could be transcribed normally m RNA,but could not express the corresponding protein,and after codon optimization,the optimized strain was constructed in the same way,and the chaperone protein gro E system was transformed and integrated.The carbon sequestration pathway can be expressed normally with the assistance of gro E chaperone protein and the sugar alcohol conversion rate is increased by 4.07%compared with the C5 strain.The results of the study show that it is feasible for industrial Saccharomyces cerevisiae to produce fuel ethanol,but it is necessary to continue strain optimization and adaptive evolution under industrial culture conditions to improve the stability of recombinant strains.