| Crotonic acid is an organic carboxylic acid containing four carbon atoms and a double bond.Its chemical nature is more active due to its carbon-carbon double bond structure in its molecule.The application of crotonic acid is very extensive,and it is of great significance in the fields of medicine and chemical industry.At present,the production of crotonic acid mainly relies on chemical synthesis,which takes ethylene as one of the raw materials produced by petroleum cracking,and finally converted from crotonaldehyde to crotonic acid through a series of chemical reactions.With the increase of the international energy crisis and the deterioration of the environment,it is clear that the industry depending on fossil fuels will be affected to a certain extent in terms of cost and sustainable development.Therefore,human beings have to assist or replace the industry in other ways.With the development of genetic engineering methods,humans have gradually succeeded in obtaining a series of substances from microorganisms.The first model organisms also the most well-researched organisms so far are the representative E.coli of prokaryotes.Although most of the current research is carried out in this microorganism,the certain pathogenicity within E.coli and some biological characteristics of the prokaryotic expression system have limited its large-scale application in industrial production.The yeast system that was born with it is considered to be an ideal expression system model organism in industrial production.Compared with prokaryotes,eukaryotes make the eukaryotic expression system in the industrial fermentation industry better than the large intestine due to the integrity of its own expression system.The expression system of bacilli is more advantageous.Yarrowia lipolytica is widely accepted as a novel yeast expression system.Due to its high safety(GRAS),the products produced by this yeast metabolism are widely used in food,medicine and other fields.At the same time,Yarrowia lipolytica itself has a fatty acid metabolism pathway and is more resistant to extreme environments such as low pH.In summary,the use of Yarrowia lipolytica as a carrier is one of the mainstream directions of current microbial fermentation research.In this study,at the beginning of the design of the crotonic acid metabolic pathway,there are relative path and key enzymes of synthetic crotonaldehyde in the path of its own metabolic production of butanol within Clostridium acetobutylicum,and the excavation was continued based on this.A synthetic pathway for the synthesis of crotonic acid was successfully constructed in Yarrowia lipolytica.The medium contains crotonic acid as determined by gas chromatography mass spectrometry.The yield of crotonic acid was then determined by high performance liquid chromatography to be 62.24 mg/L.Subsequently,the path of the construction was further optimized,and the key enzymes in the overexpression pathway were used to achieve excessive accumulation of crotonic acid precursors for high yield of crotonic acid.The yield of the liquid was determined to be 123.49 mg/L,which has nearly doubled.Then the path was optimized continually,the pyruvate to acetyl-CoA to achieve one-step synthesis,reducing the metabolic consumption of each step that would otherwise need three steps to synthesize,so that more precursors flow to the downstream of the synthesis of crotonic acid,and finally In the liquid phase,the yield of crotonic acid was increased to 220.00 mg/L,and the overall yield was increased by a factor of two.Compared with the chemical synthesis method,this study realized the process of converting sugar into crotonic acid in vivo,which has a relatively low requirement on substrate and production conditions,and the product has no strong harm to the environment;compared with Escherichia coli In terms of production mode,the strains used in this study are suitable for high-density culture,and are relatively resistant to low-pH environments,meeting industrial production requirements. |
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