Metabolic Engineering Of Yarrowia Lipolytica For Carotenoids Production

Carotenoids are fat-soluble isoprenoid pigments responsible for different colors such as yellow,orange and red in plants,fungi,algae and some bacteria.There are more than 600 various carotenoids in nature,but only a few of them are used industrially such as lycopene,β-carotene,canthaxanthin and astaxatnthin.They have received considerable attentions in food industry,medicine and cosmetics because of their beneficial effects on human health.Carotenoids are mostly obtained by chemical synthesis,but the growing demand of natural additive has stimulated scientists to increase carotenoid production in carotenogenic sources and even produce them in non-carotenogenic strain such as Escherichia coli and Saccharomyces cerevisiae by metabolic engineering approaches.Yarrowia lipolytica has emerged as preferred non-conventional yeast for metabolic engineering,especially for the production of chemicals derived from acetyl-CoA,fatty acids and lipids.It is considered to be a generally recognized as safe(GRAS)organism and has already been used commercially to produce many value-added biochemicals.In this study,Y.lipolytica was used as a platform to efficiently produce carotenoids through synthetic biology,metabolic engineering,and fermentation optimization,and pave the way for industrial production of carotenoids.1.In the research of constructing carotenoids production platform in Y.lipolytica;lycopene was chosen as the first target product.To construct the lycopene synthetic pathway,the phytoene synthase gene crtB,the phytoene dehydrogenase gene crtI and GGPP synthase gene crtE from Pantoea agglomerans were introduced into Y.lipolytica strain Polf and the initial yield of 8.5 mg/L was obtained.Furthermore,the effects of copy numbers of lycopene synthesis genes and a variety of key central metabolic genes on lycopene production in Y.lipolytica were investigated.The results indicated that monocopy integration of the crtE and crtB genes and multicopy integration of the crtI gene led to the highest lycopene production(124 mg/L).Overexpression the AMPD gene encoding for AMP deaminase resulted in approximately three-fold increase in lycopene production(371 mg/L).The engineered strain with the best lycopene production performance was therefore cultivated in a 5 L bioreactor with intermittent supplementation of glucose,and 745 mg/L lycopene was obtained at day 4 of the cultivation,together with the highest lycopene content(46.2 mg/g DCW)ever reported in Y.lipolytica.2.In constructing of the strains for lycopene production,the integrative plasmids were employed to express exogenous genes.Further metabolic engineering in Y.lipolytica forβ-carotene and astaxanthin production based on the lycopene-producing strains constructed above was time-consuming due to repetitive works for plasmid construction and yeast transformation,and the restricted number of genes integration.Recently,the CRISPR/Cas9 system has been successfully adapted to generate genetically modified Y.lipolytica,and five sites were found as integration targets.However,for carotenoids production,the sites were still not enough.To further increase gene targets for CRISPR/Cas9 system,twenty genomic sites that accepting gene integrations without impacting cell growth were screened,the sgRNA and donor plasmids were constructed for each site.The set of sites included nine sites that are involved in lipid accumulation(POX1,POX2,POX3,POX4,POX5,POX6,GUT2,PEX10,LIP1),eight sites with nonfuctional pseudogenes due to frameshift(E1,A1,B1,A2,F1,E2,F2,C1)and threes sites that are involved in xylose metabolism(XLK,XYR,XDH).3.CRISPR/Cas9 integration strategy was used for β-carotene and astaxanthin production in Y.lipolytica strain Polf.To construct the β-carotene synthetic pathway,the codon-optimized phytoene synthase/lycopene cyclase gene carRP and the phytoene dehydrogenase gene carB from Mucor circinelloides were introduced into Y.lipolytica and the initial yield of 17.1 mg/L was obtained.Then,the activities of GGPP synthase gene GGS1 and HMG-CoA reductase gene HMG were optimized through overexpression and obtained 57.3 mg/L β-carotene.In order to further facilitateβ-carotene production in Y.lipolytica,the enhancement of the MVA pathway,acetyl-CoA availability through overexpression of AMP deaminase-encoding gene AMPD,and increasing in copy numbers of carRP and carB were performed in shaking flask cultures.The results showed that the multicopy expression of carRP and carB,and overexpression of ERG13 gene which encodes HMG-CoA synthase boosted β-carotene production up to 168.1 mg/L.Additional downregulation of the competing squalene synthase gene SQS was also explored,however only adverse effect on β-carotene production was observed.Finally,the optimized strain contained the overexpression of a total of eight genes including three copies of carRP,two copies of carB,and single copies of GGS1,HMG and ERG13.To achieve a high titer of β-carotene,high biomass concentration is necessary.However,the strain constructed above retained both the ura3 and leu2 auxotrophies that were present in the starting Y.lipolytica Polf strain thus not suitable for high-cell density fermentation.Recovering uracil and leucine auxotrophies resulted in higher overall cell mass and a corresponding increase inβ-carotene production(407.7 mg/L).The engineered strain with the best β-carotene production performance was therefore cultivated in a 5 L bioreactor with continuous supplementation of glucose,4.5 g/L β-carotene was obtained between day 6 and day 7 of the cultivation.4.Based on the efforts of β-carotene production in Y.lipolytica,the obtained strain was further engineered to confer a novel biosynthetic pathway for the production of astaxanthin.The catalytic ability of β-carotene hydroxylase from different sources was investigated based on astaxanthin accumulation.Coexpression the PspcrtW gene from Paracoccus sp.N81106 along with the HpcrtZ gene from Haematococcus pluviali led to astaxanthin production of 61.3 mg/L and by far the highest astaxanthin concentration in Y.lipolytica.In this study,Y.lipolytica was engineered to be an attractive platform cell factory to produce carotenoids efficiently.With rational gene manipulation and cultivation optimization,the original strains were effectively engineered for producing lycopene,β-carotene and astaxanthin at high titers.This systematic strains engineering presented here,provides both biological insights into carotenoid production and a roadmap for future terpenoid engineering studies in Y.lipolytica.