Studies On Lycopene Production By Recombinant Escherichia Coli And Recombinant Corynebacterium Glutamicum

Lycopene is a kind of carotenoid which has many biological and pharmaceutical activities.including anti-cancer,anti-inflammatory,and anti-oxidative activities.Consequently,lycopene is widely used as a supplement in functional foods,animal feed,nutraceuticals,and pharmaceuticals and as an additive in cosmetics.In recent years,researchers paid more attentions to produce lycopene by microrganism,and using Escherichia coli as host bacteria is a research focus for the ma jority of researchers.In this work,we studied a lycopene production strain E.coli K12f-pACLYC preserved in our lab which produced lycopene in high performance only when using fructose as carbohydrate resource.Adding to the medium is control,adding 6 g/L fructose to the medium is exprement.Lycopene yield of the strain when cultivated in the medium with 6 g/L glucose reached 45 mg/gDCW,and the yield of the strain when cultivated in the medium with 6 g/L glucose reached 177.4 mg/gDCW.Gene transcription pattern of the strain in middle and late period of logarithm when grown on fructose and glucose respectively was studied by using transcriptome sequencing and quantitative PCR analysis.385 differentially expressed genes(DEGs)with fold>4 were identified,which mainly exist in EMP.TCA and oxidative phosphorylation pathway.The high lycopene production of the strain when using fructose as carbon source was explained by analyzing the expression pattern of the genes in related pathways:Firstly,the expression pattern of the genes in EMP pathway suggested the accumulation and metabolic balance of glyceraldehyde-3-phosphate(G-3P)and pyruvate;Second,TCA cycle and oxidative phosphorylation pathway were very active,which provided enough NADH and ATP to synthesize lycopene;Thirdly,the gene pntA which convert NADH to NADPH was up-regulation obviously,which provided enough NADPH for synthesizing lycopene.We selected 11 key genes and analyzed their expression levels by quantitative real-time PCR analysis.The results verified the reliability of the transcriptome sequencing.Besides,we reconstructed a recombinant Corynebacterium glutamicum strain to produce lycopene by using C.glutamicum ATCC13032 as host.A recombinant expression vector pEC-XK99E-crtBI which includes crtB and crtI genes coming from Ervinia herbicola was built,and then transformed to C.glutamicum by electrotransformation.However,lycopene could not be detected in the culture.Considering the expression of gene Cgl0626 and Cgl2172 from C.glutamicum which encodes geranylgeranyl pyrophosphate synthase is relatively weak,the crtE gene which comes from E.herbicola was also introduced to pEC-XK99E to form recombinant expression vector pEC-XK99E-crtEBI.Lycopene was successfully detected in the culture with the maximum concentration of 31.43 mg/L,when the strain was induced at 25 ?for 8 h with 1mM IPTG as inducer.It is the first study to use C.glutamicum as a host strain to produce lycopene.