Metabolic Engineering Of Yeast For Heterologous Biosynthesis Of Vitamin A(retinoids)

Vitamin A is a class of lipophilic compounds,which participates in physiological activities such as vision,immune system,gene transcription and skin cell differentiation,and plays an important role in the normal function of body metabolism,and thus is widely used in food,feed,pharmaceutical and cosmetics industries.Most of the commercially available commercial vitamin A is produced by chemical synthesis,which suffers from the critical problem of low yield of the all-trans isomer,which has the highest bioactivity.Taking the two forms of vitamin A,all-trans retinol and all-trans retinoic acid as the target products,enzyme mining,pathway assembly,metabolic balance reconstruction,and fermentation optimization were conducted to obtain engineered yeast strains producing retinol and retinoic acid,respectively.Referring to the natural synthesis pathway of vitamin A,codon-optimizedβ-carotene 15,15’-oxygenase(Blh)from uncultured marine bacteria MED66A03 was introduced into a Saccharomyces cerevisiae strain containing the β-carotene synthesis pathway.The expected product retinal(23.65 mg/L)was detected together with a small amount of retinol(6.89 mg/L)formed by unknown endogenous alcohol dehydrogenases.Through the application of bi-phasic culture system,the addition of Fe2+and the optimization of culture conditions,99.67%of total retinoids were extracted extracellularly,and its titer(200.21 mg/L)increased by 4.21 times.Then,by increasing the copy number of the upstream precursor GGPP synthesis gene(CrtE03M)and overexpressing the truncated NADH kinase(tPos5)to promote the cytoplasmic NADPH supply,the total retinoids titer was further increased to 264.03 mg/L,which contained 221.37 mg/L of retinal and 42.66 mg/L of retinol.To achieve selective biosynthesis of retinol,the heterologous alcohol dehydrogenase/aldehyde reductase ybbO from Escherichia coli was first introduced into the retinoids-producing yeast strain to verify its catalytic activity.and then the endogenous Env9 protein from S.cerevisiae was mined based on ybbO sequence homology search.The reduction activity of Env9 protein on retinal was verified both in vitro and in vivo for the first time.The co-expression of ybbO and Env9 in strain Y03-342,which had the highest total retinoids titer,resulted in strain Y03-43 with a retinol synthesis titer of 122.03 mg/L.Considering that retinoids,especially retinols,are easy to be degraded due to oxidation,antioxidants were introduced in the culture,leading to a retinol titer of 401.65 mg/L in strain Y03-43,accounting for 99.86%of the total retinoids.In high-density bi-phase fermentation,2542.68 mg/L retinoids(62.32 mg/g DCW)was successfully produced,of which 97.51%was retinol(2479.34 mg/L)and 2.49%was retinol(63.34 mg/L).To construct a S.cerevisiae strain for efficient retinoic acid production,the retinoids detection method was first adjusted to isolate retinol,retinal and retinoic acid.The new detection method revealed existence of aldehyde dehydrogenases in yeast endogenous to produce retinoic acid with retinal as substrate,which had been neglected in the earlier work.By comparing the catalytic activity of several heterogenous aldehyde dehydrogenases,the retinaldehyde dehydrogenase from Mus musculus(MmAldh)was selected as the query sequence for homologous protein search,and eight candidate endogenous aldehyde dehydrogenases were found,of which only overexpression of Hfd1 resulted in obvious increase in retinoic acid accumulation.The oxidative activity of Hfdl to retinal was further confirmed by gene knockout and in vitro enzyme assay.Subsequently,by continuously increasing the copy number of the gene encoding Hfdl and adjusting its integration site,strain Y03-784 with retinoic acid as the main product was obtained,and its shake flask titer could reach 99.71 mg/L.In high-density fermentation,the retinoic acid titer reached a maximum of 545.28 mg/L,accounting for 84.19%of the total retinoids.In order to explore the potential of Yarrowia lipolytica,the most productive host reported for β-carotene,as a platform for retinoids production,the procedures of the"YaliBricks" toolkit for rapid gene assembly and the 26s rDNA site-based integration plasmid prDNAloxP were first Optimized.Then a series of sentinel integration plasmids were derived by modifying the prDNAloxP plasmid,which achieved a one-time sentinel integration of 4 genes(14 kb).The kit and the modified integration plasmids were applied to successfully construct a β-carotene-producing Y.lipolytica strain,which laid the foundation for the subsequent investigation of retinoids synthesis.Retinoids synthesis was achieved by introducing the YpBLH gene,however,expression of the heterologous ybbO protein,Env9 protein and Hfdl protein was unable to have an effect on the ratio of the target product.So,an attempt was made to mine the endogenous dehydrogenases/oxidases and only the YALI0C07414p protein with alcohol dehydrogenation function was found.Finally,a modified prPOX41oxPLEU plasmid was applied in Yp-14 strain to introduce two YpBLH genes to construct Yp-32 strain,which could reach a retinol titer of 346.14 mg/L in bi-phase culture without the addition of antioxidants,laterally reflecting the natural advantage of Y.lipolytica as a platform for retinol production.In summary,this study successfully constructed vitamin A-producing yeast cell factories based on S.cerevisiae and Y.lipolytica respectively,laying foundation for fermentative production of vitamin A,especially all-trans retinol and all-trans retinoic acid.