Construction Of An Engineered Yarrowia Lipolytica For The Efficient Conversion Of Acetic Acid For Squalene Synthesis

Squalene is a natural linear triterpene with various applications in food,medical and cosmetic fields.Currently,the traditional production methods of squalene include plant extraction and chemical synthesis.Plant extraction is resource-intensive and difficult to separate terpene complexes with similar properties to squalene;chemical synthesis is a complex synthesis route with high environmental pollution.Compared with the traditional production methods,the microbial synthesis method of biosynthesizing squalene by constructing cell-engineered bacteria with renewable biomass as raw material has the advantages of low cost,low environmental pollution and sustainability,and is a potential choice for achieving efficient squalene production in the future.Acetic acid is one of the main electrocatalytic or biocatalytic products of one-carbon compounds such as CH₄,CO₂,and CO,and is also a major by-product of industrial waste as well as in the fermentation industry.The utilization of acetic acid is not only a prerequisite for the high-value conversion of syngas such as CO₂,but also an effective way to solve the problems of environmental pollution and resource waste caused by waste acetate.The Yarrowia lipolytica is a typical oil-producing yeast,rich in intracellular acetyl coenzyme A and high-throughput MVA pathway,and it has the ability to utilize a variety of substrates,making it an advantageous host for the conversion of acetic acid to synthesize squalene.Therefore,in this project,we have enhanced the squalene synthesis pathway,metabolic regulation and bypass-assisted regulation of engineering yeast for the efficient utilization of acetic acid,and applied the acetic acid-glucose co-utilization system for acetic acid with cytostatic property,and the specific research includes:（1）The squalene synthesis pathway was enhanced using Yarrowia lipolytica as the starting bacterium.Highly efficient squalene production potential strains were obtained by overexpressing endogenous squalene synthase SQS,endogenous HMG.By comparing the fermentation of different carbon sources,it was found that the metabolic pathway of acetic acid conversion to acetyl coenzyme A was shorter compared to glucose.The loss of carbon flow is smaller,which is a metabolic shortcut for the synthesis of squalene and has certain production advantages.The yield of squalene reached110.54 mg/L with acetic acid as raw material.（2）A multi-modular regulation of the biosynthetic pathway for the conversion of acetic acid to squalene was carried out.The metabolic pathway of acetic acid was enhanced by overexpression screening of endogenous acetyl coenzyme A synthase（ACS）,Salmonella enterica mutant Se ACS;the MVA pathway was enhanced by adding different copies of squalene synthase（SQS）,endogenous HMG,and overexpression of MVA pathway rate-limiting enzymes yl ERG10,ato B,yl ERG12 and yl IDI,and the final squalene production was increased to 184.24 mg/L,a 66%enhancement in yield.（3）Weakening the branched pathway of acetyl coenzyme A metabolism and enhancing neutral carbon flow utilization.The synthesis of fatty acids and citric acid is the main intracellular acetyl coenzyme A metabolic branched pathway in Yarrowia lipolytica.In order to weaken the consumption of acetyl coenzyme A,this study constructed a g RNA-CRISPRi system targeting fatty acid synthase and citrate synthase by using CRISPR interference,and guided d CAS9 to the target site after transcription by g RNA to achieve the weakening of citrate synthase and Fatty acid synthase was weakened.After the weakening,a squalene yield of 379.078 mg/L was obtained,which is the highest yield of squalene production from acetic acid by Yarrowia lipolytica reported so far.（4）A glucose-acetic acid co-utilization system was explored to alleviate the inhibition of cell growth by acetic acid.In order to relieve the inhibition of cell growth by acetic acid,this study optimized the co-utilization system of glucose and acetic acid,and found that the best effect was achieved when 10g/L of glucose and 30 g/L of acetic acid were used as carbon sources,and the cell biomass reached 30 OD₆₀₀,which was two times higher than that of acetic acid alone.It showed that the acetic acid and glucose co-utilization system effectively increased the cell biomass and alleviated the inhibition of cell growth by acetic acid.In this study,by optimizing and regulating the metabolic pathway of acetic acid conversion to squalene and weakening the branched pathway of acetyl coenzyme A metabolism,we successfully constructed a Yarrowia lipolytica engineering bacterium that can efficiently use acetic acid to convert squalene,explored the glucose-acetic acid co-utilization system,alleviated the inhibition of acetic acid on cell growth,and laid the foundation for the research to realize the efficient conversion of acetic acid to produce terpenoids.