Study On The Construction Of A Synthesis Pathway For 1,3-butanediol By Saccharomyces Cerevisiae

1,3-butanediol is an important raw material of organic and fine chemical industry,widely used.At present,the industrial production of 1,3-butanediol mainly uses the acetaldehyde condensation hydrogenation process,and the raw materials come from the petrochemical industry.With the depletion of global fossil energy,it has become imperative finding renewable alternative resources.It is a significant work of studying biosynthesis 1,3-butanediol.In this work,Saccharomyces cerevisiae was used as the research object.And this is the first time to use Saccharomyces cerevisiae as the expression system to construct a non-natural synthesis pathway of 1,3-butanediol.Based on the inhere metabolic pathways and native genes,design artificially the synthesis route of 1,3-butanediol in Saccharomyces cerevisiae cells,starting from acety-CoA,then acetoacetyl-CoA reduced to 3-hydroxybutyrl-CoA,which deacetylated to form 3-hydroxybutyraldehyde,and finally reduced to 1,3-butanediol by alcohol dehydrogenase.Using the yeast strain,a safe and reliable edible strain,as the initial strain,CRISPR/Cpf1 technology was used to break up the URA3 gene of yeast,resulting the loss of target gene function,and obtain the auxotrophic host strain Saq lacking URA3 function successfully.As for the enzymes required for the catalysis 3-hydroxybutyryl-CoA to 3-hydroxybutanal in the synthetic route,two kinds of exogenous genes,Aldh and BphJ with a wider catalytic range of acetyl-based substrates,were selected.Then using the CRISPR/Cas9 technology,the expression cassettes of Aldh,BphJ and EGFP were integrated into the ? sites of yeast genome,obtaining the engineering strain Saq-Fab with the 1,3-butadediol pathway.Both the phenotype and mocular experiments confirmed the exogenous genes had been intergrated into genome and stably genetically expressed.However,the target product 1,3-butanediol was not detected in the metabolites,with the non-natrual pathway completed theoretically.In order to produce the target production,further studies had been conducted through regulating the metabolic flow and reducing the production of ethanol.The CRISPR/Cas9 technology was used to site-directed mutation of the ADH1 gene to make it lose its function,successfully reducing the ethanol production,accumulating intermediate metabolites upstream of the ethanol pathway.Then exogenous gene thlA was introduced into cells for promoting the amout of acetoacetyl-CoA.In theory,there had sufficient precursors for synthesizing 1,3-butanediol.However,the target production,1,3-butanediol,had not yet been detected.It is speculated that the enzymes,Aldh and BphJ,are not effective in catalyzing the conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde,resulting the metabolic flow cannot be pulled to 1,3-butanediol pathway.