| Lycopene(C40H56,536.85Da)is one of the carotenoids produced by some plants,algae,fungi,and bacteria.There are 11 conjugated double bonds and 2 unconjugated double bonds in its molecular structure,which is a straight chain hydrocarbon.This unique conjugated polyene structure makes lycopene have strong antioxidant function.Many studies have shown that lycopene can inhibit the growth of prostate cancer,colorectal cancer,gastric cancer,pancreatic cancer,and lung cancer.Meanwhile,lycopene can protect the cardiovascular system from coronary heart disease.Therefore,there is a great application prospect for lycopene in the treatment and prevention of human diseases.High-level yield is an important prerequisite for the application of lycopene.In general,lycopene can be extracted directly from plants or obtained by chemical synthesis.However,extraction from plants such as tomatoes and carrots is costly because of unstable supply and low yield of plant.The chemical synthesis of lycopene often raises safety concerns because of chemical reagent residue.Microbial engineering,also known as fermentation engineering,is an important part of bioengineering,which operates microorganisms with specific metabolic pathways to catalize raw materials into products of interest under appropriate conditions.This method has several advantages,including low cost,high yield,short production cycle,and less pollution.There are two pathways for lycopene production by organisms,one is mevaleric acid（MVA）pathway and the other terms 2C-methyl-D-erythritol4-phosphate（MEP）pathway.Eukaryotes usually use MVA pathway to synthesize lycopene,while most bacteria use MEP pathway.Although MEP pathway exists naturally in Escherichia coli,it is a long pathway with many key enzymes,and the energy efficiency of MEP pathway is also low.In contrast,the alternative MVA pathway is more energy efficient in the production of lycopene.Chassis strains are the basis of microbial engineering.In contrast to chassis strains like E.coli and Saccharomycetes cerevisiae,Halomonas bluphagenesis has many unique advanages.H.bluphagenesis TD1.0,a mutant of TD that was isolated and screened from the high-saline soil of Aidin Lake in Xinjiang,is a kind of Gram-negative bacteria.The optimum culture condition of TD1.0 is in the media with salt concentration of 60 g/L and p H of 9.0.The culture medium of Halomonas can inhibit the reproduction of other miscellaneous bacteria,which enables the fermentation of H.bluphagenesis TD1.0 continuously without sterilization and non-contamination.Using Halomonas as chassis bacteria can save energy and fresh water and improve the economy of industrial fermentation process.In this study,H.bluphagenesis TD1.0 was used as a chassis strain,and the main contents and results are as following:1.Construction and characterization of the heterologous MVA synthesis pathway in H.bluphagenesis for the production of lycopene.Firstly,we divided the MVA pathway for lycopene production into two modules,the upstream modul included key enzyme genes to synthesize isopentenyl pyrophosphate（IPP）and dimethylallyl pyrophosphate（DMAPP）from acetyl-Co A,and the downstream module carried genes for lycopene synthesis from IPP and DMAPP.Then,the key genes of upstream molel,including Efmva E,Efmva S,Spmva K1,Spmva K2,Spmva D,and Ecidi,were cloned into one plasmid under the control of two inducible promoters(PMmp Iand Ptrc),and the resultant plasmid termed pTer7.The downstream module genes such as crt E,crt B,and crt I from Streptomyces avermitilis were constructed into plasmid pTer3.After that,the two plasmids were co-transformed into H.bluephagenesis TD1.0 to form a complete lycopene production pathway in the engineered TD1.0/pTer7-pTer3.Shake flask fermentation of TD1.0/pTer7-pTer3 was performed and the production of lycopene was induced with different concentrations of isopropyl-β-d-thiogalactoside（IPTG）.The expression product was characterized by high performance liquid chromatography（HPLC）with lycopene standard.The results showed that the maximum lycopene yield was 0.20 mg/g dried cell weight（DCW）of TD1.0/pTer7-pTer3 under the concentration of 5μM IPTG.2.Optimization of the downstream module to improve lycopene production.Many studies showed that lycopene production is variable usder the catalization of downstream crt E,crt B,and crt I genes from different species,indicating the replacement of downstream module genes may improve lycopene yield.Therefore,we changed the source of crt E,crt B,and crt I genes in the downstream moduel with those from Streptomyces lividans to generate pTer5 plasmid.Co-transformation of pTer7 and pTer5 into TD1.0 obtained TD1.0/pTer7-pTer5,which was fermented with induction of different concentrations of IPTG for 48 h.Results showed that the highest yield was0.70 mg/g（DCW）at 5μM IPTG,and this yield was 3.5 times higher than that of TD1.0/pTer7-pTer3.At the same time,the plasmids pTer3 and pTer5 were separately transformred into TD1.0.After fermentation,it was found that the H.bluephagenesis TD1.0 transformed pTer5 only could produce lycopene of 0.30 mg/g（DCW）,indicating that the production capacity of crt E,crt B and crt I genes of S.lividans was better than those of S.avermitilis in H.bluephagenesis TD1.0.3.Optimization of promoter for the upstream genes further to improve lycopene production.Promoter activity determines the expression levels of functional genes,which directly relate to the yield of target products.Firstly,we changed two promoters of the upstream moduel to PMmpⅠto construct plasmid pTer14.Through the characterization of TD1.0/pTer14-pTer5,it was found that the highest yield was 0.75mg/g（DCW）,which had no obvious change compared with TD1.0/pTer7-pTer5.We speculate that the metabolic disorder may be caused by the high intensity of PMmpⅠ,which is not suitable for the upstream pathway.Then,we changed promoters with two weak Ptrcpromoters.After characterization of TD1.0/pTer1-pTer5,it was found that when the concentration of IPTG was 5μM,the maximum yield reached 1.22 mg/g（DCW）,which was 6 times higher than that of TD1.0/pTer7-pTer3.In conclusion,we constructed an MVA pathway for lycopene production in H.bluephagenesis TD1.0 that did not naturally produce lycopene.Replacement of the host source of key genes crt E,crt B and crt I in the downstream module of lycopene production and optimization of gene expression promoters in the upstream module constructed in H.bluephagenesis TD1.0 could improve lycopene yield to some extent.The new Halomonas host for lycopene production provided in this study can not only lay down a solid foundation for further engineering operations,such as collateral pathway blocking and product degradation pathway elimination,but also provide useful way for the construction of other bacteria with microbial engineering strategies. |
