Construction And Optimization Of Fermentation Conditions For High-yielding Engineered Nervonic Acid Bacteria

Nervonic acid(C24:1)is a very-long chain monounsaturated fatty acid that restores nerve fiber activity and promotes nerve cell regeneration,promotes brain development and maintains normal brain function,and has significant efficacy in many neurological diseases.At present,nervonic acid is mainly extracted from a very small number of animals and plants,the cost is high and expensive,the main obstacle to the industrial production of nervonic acid is the lack of high-yielding engineering bacteria.Based on the construction of a nervonic acid synthesis pathway,it is proposed to increase the yield of nervonic acid by replacing the expressed host and optimizing the metabolic regulation and fermentation conditions of nervonic acid production in Saccharomyces cerevisiae.1.Production of nervonic acid using Rhodotorula glutinisas as an expression host.The plasmid pYES-PTEF1-KCS-TCYC-PPGK-FAE1-TCYC with the key rate-limiting enzyme gene was transferred to Rhodotorula glutinisas for overexpression.Fermentation results showed that KCS-FAE1 expression did not have much effect on the fatty acid composition of Rhodotorula glutinisas,nor did it produce nervonic acid.Although Rhodotorula glutinisas is rich in oleic acid,little research has been done on its fatty acid elongation system and regulatory mechanisms,so Rhodotorula glutinisas is temporarily unable to serve as a host bacterium for nervonic acid production.2.Metabolic regulation of nervonic acid production by Saccharomyces cerevisiae.Based on the construction of the pYES-PTEF1-KCS-TCYC-PPGK-FAE1-TCYC plasmid,firstly,overexpression of the transcription factor Mga2 to promote the synthesis of the intracellular substrate oleic acid,and secondly,an attempt to replace the screening marker with G418 resistance to allow the engineered yeast to grow on the more nutrient-rich YPD medium;then the yeast’s own fatty acid elongase ELO2 was knocked down to reduce the competitive role of metabolic bypass;finally,the exogenous KCS was integrated into the genome of Saccharomyces cerevisiae to increase the stability and copy number of KCS genes.The results showed that replacement of the G418 screen marker resulted in significantly higher biomass but slightly lower nervonic acid production in engineered yeast;overexpression of Mga2 increased C16:1 and C20:1 production but reduced C24:1 production;knockdown of Elo2 increased nervonic acid production in engineered yeast by 60.3%;and integration of KCS into the genome resulted in slightly lower nervonic acid production.3.Optimization of nervonic acid producing culture conditions for engineered yeast.With increasing C/N,there was a decreasing trend in nervonic acid yield,with a maximum C24:1 yield of 0.959%at a C/N molar ratio of 4.4.Low-temperature fermentation favored the monounsaturated fatty acid content of the engineered yeast,which increased nervonic acid production by 24.32%at 18℃ compared to 22℃.Raising the initial pH of the fermentation broth facilitated the increase in Saccharomyces cerevisiae biomass,and the accumulation of nervonic acid was most beneficial at an initial pH of 6.0.Fermentation to day 12 yielded a maximum of 0.55%of nervonic acid,followed by 0.48%of nervonic acid on day 6,and the rotation speed had less effect on the nervonic acid yield.Exogenous addition of 0.5%Tween 80 and 1 g/L Asn increased the nervonic acid yield by 81.9%and 48%,respectively.The response surface optimization method was used to investigate the interaction of Tween 80,Asn and fermentation time on the nervonic acid production of engineering bacteria,and the software predicted that the optimal culture conditions were 1.47 mM oleic acid,1.97 g/L Asn,and 8.57 days of fermentation,with a maximum nervonic acid production of 1.37%for engineering bacteria.