Construction Of Escherichia Coli For The Lycopene Production And Its Metabolic Pathways Optimization

Lycopene is a fat-soluble carotenoids with C40 and 11 conjugated double bonds,which is not only healthy,but also has strong antioxidant properties.It is widely used in food,medicine and other industries.At present,the price of lycopene products?5%?is about 1000 RMB/kg.lycopene is known as"plant gold"due to the high price.Current commercially available lycopene is usually extracted by multi-step chemical synthesis or natural products from.That the complexity of the synthetic and the high cost of natural product extraction limits its industrialized production.Thanks to the development of synthetic biology and metabolic engineering,the production of lycopene by microorganism fermentation has been widely concerned.In this paper,the engineered Escherichia coli strains with high-level production of lycopene was constructed by the method of metabolic engineering including overexpressing some genes of metabolic pathways,choosing suitable expression vector,selecting the high-expression promoters,integrant expression and performing fed-batch fermention.In order to obtain more relevant functional gene resources of Isopentenyl diphosphate isomerase?IDI?and 1-deoxyxylulose-5-phosphate synthase?DXS?,four idi and dxs genes were cloned from Escherichia coli,Bacillus subtilis,Myxococcus stipitatus and Deinococcus gobiensis,and were expressed in Escherichia coli with pTRC99aEBI.As results,Myxoidi and Myxodxs were the most efficient species for lycopene production and the yield of lycopene reached to 10.86 mg/g DCW and 7.94 mg/g DCW respectively.Lycopene yield reached to 15.26 mg/g DCW by co-expression of the codon optimized IDI?DXS.This study described new genetic resources for lycopene production by comparing the different IDI and DXS.The results will provide new idea for further metabolic engineering.According to the preference of codon bias of E.coli,the CrtE?CrtB and CrtI genes of lycopene were optimized and the CrtEBI gene was synthesized.Four different types of expression plasmid were constructed using induced plasmid pTRC99a and pCDF plasmid.As results,the lycopene production of plasmid pCDF-EBI and plasmid pCDF-IEB were stronger than pTRC99a-EBI and pTRC99a-IEB.In order to simplify the production technology of recombinant Escherichia coli for lycopene and reduce the production cost,we selected 17 promoters that regulated by transcription factor in E.coli.By cloning its promoter sequences and ligating with report gene CrtEBI,we constructed the Escherichia coli for the Lycopene.Then the best expression levels of promoters were evaluated by the ability to accumulate lycopene and measured by High Performance Liquid Chromatography?HPLC?.As results,the strain of expression lycopene with the promoter P_lpp?P_mglbglb was the highest.And the results revealed that the lycopene yield increased by 50%compared with the control strain pTRC99aEBI.Integrant expression has the advantages of stability and the production process without adding antibiotics.To increase the lycopene yield,the function gene CrtEBI and idi-dxs were integranted into the genomes with the genome-editing technique CRISPR-Cas9.And on the basis of shaking bottle experiment,the experiment of 5 L fermentation tank was performed.The fed-batch fermentation experiment results showed that the lycopene production were 118.75 mg/L with a yield of 7.5 mg/g DCW.