Metabolic Engineering Of Escherichia Coli For Efficient Production Of Carotenoids

Astaxanthin and?-carotene both are important carotenoids,and are widely used in aquaculture,animal husbandry,and medical care due to the variety of biological functions.The chemical synthetic products have poor safety,and low yields and high cost limits the direct extraction from organisms.With the development of biotechnology,several microorganisms were engineered to synthetize astaxanthin and?-carotene,especially Escherichia coli.Here,engineered E.coli strains were genetically modified for efficient production astaxanthin and?-carotene.Firstly,nine new operons containing?-carotene hydroxylase genes crt Z and?-carotene ketolase genes crt W from different sources were constructed and,respectively,introduced into the strain.Among the nine resulting strains,five accumulated detectable amounts of astaxanthin ranging from 0.49 to 8.07 mg/L.Subsequently,the protein fusion Crt Z to Crt W were constructed based on operons,and strains expressing fusion proteins with Crt Z rather than Crt W attached to the N-terminus accumulated much more astaxanthin.The introduction of peptide linkers into fusion proteins further validated the phenomenon and increased the production of astaxanthin.The astaxanthin production of the best strain ZF237T/Crt Z_As-(GS)₁-W_Bswas 127.6%and 40.2%higher than that of strains ZF237T/crt Z_AsW_Bs and ZF237T/crt Z_BsW_Ps,respectively.The production produced by the best strain reached 26.16mg/L after fermentation optimization.Secondly,deletion of the zwf gene increased the resultant strain's?-carotene production and content by 5.1 and 32.5%,respectively,relative to the values of strain ZF431gdh A,but decreased the biomass by 26.2%.Subsequently,the deletion of pts HIcrr operon in strain ECW1 further increased the?-carotene production titer from122.0 to 197.4 mg/L,but the provitamin content was decreased.The determination of intracellular cofactors indicated that lower NADPH contributed the decreased content in strain ECW2.Therefore,several strategies to increase the NADPH supply were evaluated for enhancement of the?-carotene content.The best strain,ECW4/p5C-nad K,containing the combination of yjg B gene deletion and nad K overexpression,produced 2,579.1 mg/L of?-carotene in a 5-L bioreactor.Comparative transcriptomic analysis of strains ECW1 and ECW2 was carried out to explore the transcriptional responses of the E.coli strains to the zwf and pts HIcrr deletions.The deletion of zwf significantly depressed the expression levels of genes participating in the de novo synthesis of purine and formation of the large subunit of ribosome in strain ECW1.In addition,the deletion downregulated the expression levels of the most EMP pathway and PPP genes,while upregulated TCA cycle and anaplerotic pathways supplying adequate acetyl-Co A and oxaloacetate for the strengthened TCA cycle.These resulted in lower biomass of strain ECW1.On the other hand,the expression levels of ppc and pck,genes involving NADPH and?-carotene heterologous were upregulated,and these were beneficial for higher intracellular content in strain ECW1.As for strain ECW2,the double deletions significantly downregulated central carbon metabolisms,energy and,factor metabolisms.This depression might achieve a new balance for strain ECW2 in global transcriptional profile,and allowed for better coordination of the carbon flux distribution.Whereas,the expression levels of genes involving NADPH,MEP and heterologous pathways were significantly depressed and decreased?-carotene intracellular content in strain ECW2.Finally,the results of RT-q PCR indicated that zwf deletion upregulated the relative transcriptional levels of genes pts G and gal P involving glucose transport in strain ECW1.Then,over-expressions of the two genes were exerted via promoter replacement in the genome of strain ECW1.The?-carotene production and intracellular content of resulting strains were increased to different degrees after fermentation adjustment.Afterwards,the over-expressions were introduced into strain ZF43?gdh A,the?-carotene production and intracellular content were increased when pts G was overexpressed.Lastly,strains over-expressing pts G produced the highest titer of linalool.Here,the production of carotenoids was significantly increased through metabolic engineering,the factors influencing production and genetic response to modifications of strains were explored through appropriate methods,and a new target was found to beneficial for terpenoids production in E.coli.These discoveries present important references for other natural products biosynthesis,and the best strains have industrial application potentials.