Cloning And Functional Expression Of Key Genes Of Carotenoids Biosynthesis Pathway In Schizochytrium Limacinum

Schizochytrium is known as a marine fungus,which has attracted much attention due to its high value-added product(DHA).Studies have shown that the metabolism of fatty acids is accompanied by many non-lipid metabolites such as squalene,sterols and carotenoids.In recent years,the growth and metabolism of Schizochytrium mainly focus on the content of lipid,especially DHA,while few people study its carotenoids accumulation.In this paper,the carotenoids synthesis pathways,especially astaxanthin synthesis pathway,are mainly studied,which will enhance astaxanthin content significantly.In order to explain this phenomenon,transcriptome was used in this study.Cloning and overexpression of key genes in astaxanthin biosynthesis pathway was also studied to investigate the astaxanthin biosynthesis pathway of Schizochytrium.It was found that the colour gradually deepened from yellow-white to reddish-brown when Schizochytrium treated with stressed,which has been proved to produce a large number of carotenoids in its cells.Through high performance liquid chromatography(HPLC)analysis,the main carotenoids were β-carotenoids,canthaxanthin,astaxanthin and lycopene in Schizochytrium limacinum B4D1.S.limacinum B4D1 was used in this paper.Firstly,the best inducer(methanol)and the best medium(fermentation medium A)for Schizochytrium growth and metabolism were screened by shaking flask experiment.And then,the effects of methanol on the growth,lipid synthesis and carotenoids accumulation with the optimum conditions were investigated.Results showed that there was no significant change in biomass,lipid content and carotenoids content of S.limacinum B4D1 under low methanol concentrations cultivating(≤3.2%).However,when treated with high methanol concentrations(≥4.8%),the biomass and lipid content decreased sharply,while carotenoids content increased significantly.Especially in 5.6% methanol group,the carotenoids accumulation reached at maximum(3529.05 ug/g DCW),of which astaxanthin content was 3388.92 ug/g,accounting for 96.03% of the total carotenoids.Secondly,in 5 L fermentor,the best concentration(5.6%)of methanol and the best fermentation medium were used to cultivate S.limacinum B4D1 to explore its lipids and astaxanthin accumulation.Results showed that in the early stage of fermentation,bacteria grow vigorously and had higher lipid content,however,the astaxanthin synthesis was at low expression level.After the middle stages,especially after the second addition of methanol,the lipid accumulation rate decreased significantly,while the astaxanthin synthesis rate increased significantly.At 132 h,the astaxanthin content reached the maximum of 3086.53 ug/g.It was also found that with the increase of astaxanthin content,the DHA content(%)increased gradually,which enhanced the antioxidant capacity in S.limacinum B4D1 cells,likewise,indirectly demonstrated the super antioxidant capacity of astaxanthin.In order to explore the phenomenon of astaxanthin accumulation under methanol-induced conditions,transcription group analysis(0%,3.2% and 5.6%)was performed.Results showed that the expression of most key genes was down-regulated in the central carbon cycle pathway,especially in the EMP pathway,three key enzyme genes were significantly down-regulated(the FPKM of hexokinase(HK),6-phosphofructokinase(pfk A)and pyruvate kinase(pyk)were 13.86%,30.53% and7.69% of the control,respectively),resulting in a significant decline in biomass.Among the two competitive pathways of astaxanthin synthesis(fatty acid synthesis pathway and squalene/sterol synthesis pathway),the down-regulation of key enzymes(the FPKM of acetyl-Co A carboxylase(ACACA),farnesyl diphosphate synthase(FDPS)and farnesyl-diphosphate farnesyltransferase(FDFT1)were 0.98%,19.95%and 19.95% of the control,respectively,resulting in a decrease of the synthesis of two competitive pathways,and the up-regulation of key genes in carotenoids biosynthesis pathway(geranylgeranyl diphosphate synthase,type III(GGPS),lycopene beta-cyclase(LCYB)and beta-carotene 3-hydroxylase(crt Z)increased by 6.38,1.19 and 2.43 times,respectively,compared with the control),which resulting in a significant increase in astaxanthin content in Schizochytrium.Finally,in this study,we constructed a basic plasmid p UCm-T-HM suitable for homologous expression of S.limacinum B4D1.Through transcriptome analysis,the sequence of key genes in carotenoids biosynthesis pathway was obtained.The target genes HMGCR,GGPS,crt B and crt O/crt W were obtained by gene cloning.Zeocin was used in overexpression plasmids(p UCm-T-HM-HMGCR,p UCm-T-HM-GGPS,p UCm-T-HM-crt B and p UCm-T-HM-crt O/crt W)as resistance screening.After being transformed into S.limacinum B4D1 by electrotransformation,HMGCR,GGPS,crt B and crt O/crt W overexpression strains were finally obtained.Quantitative Real-time PCR(q-RT-PCR)was used to analyze the related-genes expression in overexpressed strains.Results showed that the relative expression of HMGCR gene was up-regulated by 238.15 times and GGPS by 178.53 times(p ≤ 0.05).This study deepens our scientific understanding of astaxanthin biosynthesis in Schizochytrium and provides an important new functional genomics information resource.These results will facilitate future genetic studies on the molecular mechanisms and cell factories construction of astaxanthin biosynthesis in Schizochytrium.