The Study Of Interaction Between Genome-encoding Phosphatases And Lycopene Biosynthesis

Lycopene,as a representative of carotenoids,is a useful natural product that holds great commercial value and has been widely used in food,medicine and other fields.Currently,engineering of microbial cell factory for its synthesis has become one of the spotlight fields to realize the production of this compound via green biomanufacturing technology.The traditional metabolic engineering strategy focuses on relatively simple heterogeneous pathway integration and competition pathway knockout.However,due to the complex interactions between different pathways in metabolic network,there still exists a large gap between the actually realized and theoretical yield of engineering strains constructed based on conventional strategies.This suggests that it is necessary to reconstruct the metabolic network of microbes at the genome level.More specifically,it is necessary to mine the interactions of the gene loci in host genome and lycopene synthesis pathway,reconstruct the microbial metabolic network and achieve higher yield via the editing of these sites.In recent years,a few studies have indicated that many endogenous enzymes possess substrate promiscuity.These enzymes carry activities upon a variety of different substrates.Phosphate hydrolase,or phosphatase is a representative of such promiscuous enzyme family with the most extensive studies.Moreover,there are a series of phosphorylated intermediates in lycopene synthesis pathway,which can presumably be consumed by the endogenous promiscuous phosphatases.Therefore,we made a hypothesis that there are a series of phosphorylated intermediates in lycopene biosynthesis pathway,which can presumably be consumed by the endogenous promiscuous phosphatases,reduce the carbon flow of the pathway,leading to the impairment of target product accumulation.Therefore,the basic strategy of this study is to screen the potential promiscuous phosphatase pool by gene expression,thus restore the carbon flow and enhance lycopene production.In this thesis,we chose Escherichia coli DH1 as host strain.Starting from the previously constructed lycopene-producing kickoff strain,we integrated one additional copy of crt EIB cluster,the key genes in the lycopene biosynthesis pathway at the smf site.This modification led to 34% increase in lycopene yield compared with the parent strain.We thus named this engineered strain after lyc011 and used it as the host for the following phosphatase screen.Secondly,endogenous promiscuous phosphatases probably enabling the hydrolysis of the intermediates in lycopene pathway were predicted by bioinformatics and system biology approaches,giving rise to 57 candidate genes.We applied CRISPR interference(CRISPRi)based gene repression to screen the impact of these genes on lycopene accumulation.To this end,57 plasmids containing double single guide RNAs(sg RNA)targeting these genes were successfully constructed based on.The pd Cas9 plasmid and sg RNA expression plasmid were introduced into the lyc011 strain and the lycopene yield was assessed for each construct.Finally,15 genes encoding phosphatases(yjj G,aph A,nag D,yqa B,yid A,pph A,bac A,yah A,glp Q,pgp B,spo T,cpd B,nud J,dgt,pho Q)were identified with positive effects on lycopene production.As a proof-of-concept,this study could help us more comprehensively understand the effect of the enzyme promiscuity on the metabolic system,especially the incorporated heterogeneous pathway.By releasing the interaction between the heterologous pathway and endogenous metabolism,similar strategy is expected to be applied as a general method in metabolic engineering.