The Study Of Lycopene Biosynthesis In Mucor Circinelloides

Lycopene derived from isoprene units belongs to carotenoids. As one of the strongest antioxidants in nature, it is effective in anti-carcinogenesis and prevention of cardiovascular, and possesses some other important biological properties. To meet the requirement for medicinal and mutritional purposes, the lycopene needs to be biologically safe and high purity. Therefore biosynthesis of lycopene becomes the inevitable technology trends. Mucor circinelloides which belongs to filamentous fungus accumulates carotenoids as secondary metabolites. This study focused on both of biosynthesis and regulative pathways of carotenogenesis of the mutants of M.circinelloides which yiled more carotenoids than whild-type and raised the lycopene production by molecular biology methods and optimization of the culture medium. Main results were decribed as follows:(1) The carotenoids content was raised significantly by removing the negative regulator of carotenogenesis in M. circinelloides. Firstly, crgA gene was knocked out in MU218 and MU206, 5 !crgA strains were obtained. The !-carotene content of the !crgA strains were 4.8~47.6 folds of the parental strains. Compared to the crgA null derivatives of MU206, knocking out crgA in MU218 background increased the !-carotene more dramatically. MU606 which was derived from MU218, possessed the highest !-carotene content. The !-carotene production of it is 3.00 mg/g and 4.01 mg/g in the dark and in light, respectively. In addition, the additive effect of mutation in crgA and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA.(2) A lycopene producing strain was obtained by inactivating lycopene cyclase in MU606 to block the reaction from lycopene to !-carotene. Site-directed mutagenesis and random mutagenesis were performed and three red mutants were obtained subsequently. The sequencing result revealed that the gene encoding for lycopene cyclase was mutant in all three red mutants. The mutation resulted the partial loss of the activity of lycopene cyclase of MUt1 and MUt2, and blocked the activity of it in MUt3 completely. And the lycopene content of it is 1.90 mg/g and 3.64 mg/g in the dark and light respectively, 30.6 and 21.4 times of MU606. According to the carotene composition and mutation locations, it is speculated that the first R domain of lycopene cyclase is responsible for the catalization of "-carotene to !-carotene and the second repeat R domain is in charge of cataliztoin of lycopene to "-carotene, besides the fuction of the first repeat R domain depends on the second one.(3) The lycopene content rised due to the increasement of carbon flux by removing the bottleneck of lycopene biosynthesis pathway. According to the trnacriptome data, we speculated that HMG-CoA reductase was the rate-limiting enzyme in the lycopene synthesis pathway. Three copies of hmgr genes(hmgr1, hmgr2 and hmgr3) were overexpressed in MUt3. The subsequent results shown that overexpression hmgr2 and hmgr3 doubled the lycopene content approximately, implicating that HMGr encoded by them are rate-limiting enzyme of the lycooene biosynthesis pathway. In contrast, overexpression hmgr1 chould not improve the lycopene content any more, indicating that HMGr coded by hmgr1 might participate in other isoprene biosynthesis instead of lycopene. Among all the recombinant strains, MUhr3-1 possessed the highest lycopene production which is 3.75 and 7.16 mg/g in the dark and light respectively.(4) The lycopene content of MUhr3-1 was improved further by depression the competitive pathway(lipid biosynthesis pathway). Firstly, to understand the relationship between fatty acid and lycopene biosynthesis in M. circinelloides, nitrogen limited medium was applied in the research. And the result suggested that lycopene synthesis in M. circinelloides is also regulated by nitogen starvation, fatty acid and lycopene accumulated simultaneously and competed with each other for acetyl-CoA; the trnacriptome data exhibited lower expression level of FAS and ACC in high carotenoids-producing strains, which comfirmed the competive relationship between fatty acid and lycopene biosynthesis; at last FAS and ACC RNAi recombinant strains were constructed to reduce the expression level of FAS and ACC, the lycopene production displayed an increase of 14%~43% and 13%~46% respectively while the growth of the recombinants were affected; to avoid the inhibition of growth by the depression of FAS and ACC, the inhibitors(cerulenin, palm oil, safflower oil, olive oil) of them were added to the medium during stationary phase. The lycopene content was improved in different extent, and it was raised to 11.10 and 11.55 mg/g when cerulin and palm oil was added, while lycopene content was not improved by combination of both inhibitors.(5) The lycopene yield was raised significantly by optimization of carbon and nitrogen source. Effect of different concentrations of culture glucose and soybean meal on the growth and the lycopene production of MUhr3-1 was investigated, and the lycopene yield was 2.5 times more compared to it before optimization. Furthermore fermentation with the optimized culture medium in fermentor with palm oil as ACC inhibitor for 120 h, the final lycopene content is is 12.77 ± 1.79 mg/g which is 387 times of it in the starting strain MU218. And the yield is up to 261.28 mg/L, 5 times of the highest production of lycopene in Mucor cinelloides, fulfilling the perpose of increasing lycopene production in M. circinelloides.