Study On DO-stat Fed-batch Fermentation To Improve β-carotene Yield By Engineering Yarrowia Lipolytica-C11

β-carotene is one of the carotenoids,in addition to transforming into vitamin A in human body,β-carotene has the function of anti-cancer,anti-oxidation,and promotes intercellular communication.It is widely used in food,medicine,cosmetics and other industries.Therefore the market demand of β-carotene is huge.Yarrowia lipolytica is recognized as a safe strain.There are many advantages of Y.lipolytica such as simple genetic manipulation,easy cultivation,and the presence of lipid droplets in the cell,which is good for the storage of β-carotene.At present,using engineering Y.lipolytica to produce β-carotene has increased more attention.However,most of the fermentation of Y.lipolytica is at the level of shake flask fermentation or simple fed-batch fermentation,and its fermentation process has not been systematically optimized.Based on this,this research optimized the fermentation process of Engineering Y.lipolytica YL-C11 at 4L fermenter,explored the energy and gene changes during the fermentation process,and established fermentation kinetic models.The main conclusions obtained are as follows:(1)Fed-batch fermentation of YL-C11.Fed-batch fermentation was used to control the glucose concentration at about 5 g/L,ammonia water was used to control the pH maintain at 5.5.After 120 hours of fermentation,a total of 1200g glucose was consumed,73.5g/L of dry cell weight and 1.6 g/L of β-carotene were obtained.The ATP content in the cells showed a downward trend and the NADP+/NADPH content showed an upward trend during fed-batch fermentation.Kinetic studies show that the Logist equation and Luedeking-Piret equation fit well the cell growth process and product generation process of YL-C11.The kinetic parameters μm(0.113),Xm(71.18),α(0.0216),β(1.52 × 10-5)were obtained and the values of Yx/s(0.306)and Yp/s(0.007)were calculated.The results of fed-batch fermentation indicates that insufficient supply of dissolved oxygen is main factor,which limited the Y.lipolytica YL-C11 to further growth.(2)DO-stat fed-batch fermentation of YL-C11.In order to solve the insufficient oxygen supply in fed-batch fermentation,DO-stat fed-batch fermentation was established to maintain the dissolved oxygen at 10%-20%,and pH was maintained at 5.5.After 120 hours of fermentation,a total of 1350 g glucose was consumed,94 g/L of dry cell weight and 2.01 g/L of β-carotene were obtained.The dry cell weight andβ-carotene content is 1.3-fold higher than that in fed-batch fermentation.The ATP content in the cells showed a downward trend and the NADP+/NADPH content showed an upward trend during DO-stat fed-batch fermentation.The ATP and NADP+/NADPH content are higher than that in fed-batch and more glucose was consumed at log period.Kinetic studies show that the Logist equation and Luedeking-Piret equation fit well the cell growth,glucose consumption and product generation process of engineering Y.lipolytica YL-C11.The kinetic parameters μm(0.527),Xm(94.73),α(-0.5876),β(0.2673),Yx/s(0.353)and Yp/s(0.158)were obtained.The kinetic results showed that the engineering Y.lipolytica YL-C11 had higher growth rate and glucose utilization rate in the DO-stat fed-batch fermentation.The transcription levels of four key genes(tHMG,GGS1,carRA,and carB)on theβ-carotene synthesis pathway revealed that higher expression levels were obtained when compared to fed-batch fermentation.(3)Study on transcriptomics based on RNA-seq.The Illumina Hi SeqTM 2000 platform was used to carry out high-throughput sequencing for the engineering Y.lipolytica YL-C11 of 24h(F-24,D-24)and 48h(F-48,D-48)during fed-batch fermentation and DO-stat fed-batch fermentation.The results of sequencing quality showed that the sequencing quality can meet the research requirements.854 genes were up-regulated and 732 genes were down-regulated in the F-24 and F-48,748 genes were up-regulated and 237 genes were up-regulated in the D-24 and D-48,64 genes were up-regulated and 95 genes were up-regulated in the F-24 and D-24 samples.The results of GO enrichment analysis showed that "oiological process" and"molecular function" had the most enrichment items.The most genes enriched to"metabolic process" in the "biological process".Among the "molecular functions",the genes enriched to "catalytic activity" were the most.In the "cell function",the gene enriched to the "membrane" were the most.The results of GO enrichment analysis of ATP and NADP+/NADPH-related genes showed that the expression of ATP and NADP+/NADPH-related genes was inhibited,which suggest that the cells produced more energy in DO-stat fed-batch fermentation than that in fed-batch fermentation.The results of KEGG enrichment analysis showed that the genes enriched to"metabolic" pathway were the most,followed by "organic system".In metabolism,the genes enriched to "carbohydrate metabolism" are the most,followed by "amino acid metabolism".There are also different degrees of enrichment in other metabolism.The results of KEGG enrichment analysis of different genes related to the TCA cycle in F-24,F-48 and D-24,D-48 showed that the expression of genes related to the TCA cycle was inhibited in the fed-batch fermentation.Indicating that the TCA pathway was strengthened in the DO-stat fed-batch fermentation when compared to fed-batch fermentation.Strengthened TCA pathway in the DO-stat fed-batch fermentation produced more energy and higher glucose conversion.The KEGG enrichment analysis of "carbon metabolism" in F-24 and D-24 showed that the gene expression of"carbon metabolism" related pathway was enhanced in DO-stat fed-batch fermentation when compared with the fed_batch fermentation.Which also indicated that higher consumption rate and utilization rate of glucose in the DO-stat fed-batch fermentation were obtained.To sum up,this study used the engineering Y lipolytica YL-C11 to produceβ-carotene,and established the DO-stat fed-batch fermentation strategy,which provide certain reference for the industrial production of beta-carotene.