Heterologous Synthesis Of Lycopene In Yarrowia Lipolytica

Lycopene has anti-oxidant,anti-aging,cardiovascular disease prevention and anticancer effects.It can be used as food colorants,feed supplements,nutritional products as well as cosmetics and drugs.At present,the main production methods of lycopene are plant extraction,chemical synthesis and biosynthesis.Plant extraction is limited by the growth cycle of the plant and is prone to oxidation during the extraction process.There are many by-products in the chemical synthesis process,which easily cause environmental pollution.The by-products of biosynthesis are few,the cost is low and it is not affected by factors such as time and geographical conditions.In this study,Yarrowia lipolytica was used as a chassis cell and a lycopene synthetic pathway was introduced to produce lycopene in the cytoplasm and peroxisome organelles.Established lycopene-producing cell factory and subcellular factory provide new ideas for lycopene biosynthesis.In the present study,a lycopene synthesis pathway was introduced into the cytoplasm and peroxisome of Y.lipolytica through the construction of a 2A peptide multi-gene coexpression vector strategy.Y.lipolytica can be used as a chassis,and gene editing tools can be used to increase yield of lycopene.The main research work is as follows:(1)At first,the 2A peptide multi-gene co-expression vector construction strategy was used to connect two fluorescent protein genes to transform into yeast,and it was found that two fluorescent proteins can be expressed simultaneously,thereby verifying the functionality of the 2A peptide sequence.The lycopene synthesis pathway genes,Crt E,Crt B,Crt I,HMG1 and GGS1 were linked together to construct a lycopene expression body using a 2A sequence,and then transformed into Y.lipolytica to construct lycopene synthesis pathway.The strain with the highest lycopene production in the cytoplasm was obtained,reaching 29.42 mg/L.(2)The validity of the peroxisome resident signal SKL was verified.The SKL tag was linked to the e GFP reporter gene.It was found that the SKL signal tag could carry the target gene into the peroxisome organelle.Then,the peroxisome resident signal SKL was fused with the lycopene synthesis pathway gene to locate the lycopene synthesis pathway in the peroxisome.The yield of lycopene was 47.26 mg/L.The results showed that the final yield of lycopene-engineered engineering yeast was higher than that in the cytoplasm and subcellular compartmentation could increase lycopene production.(3)The yield of lycopene was further increased by increasing oil content in Y.lipolytica.During lipogenesis pathway,the GUT2 gene encodes a glycerol 3-phosphate dehydrogenase gene.The absence of GUT2 reduces the process of glycerol 3-phosphate into gluconeogenesis,thereby lipid accumulation is improved.CRISPR-CAS9 technology was used to knock out GUT2,lycopene production reached 59.09 mg/L.During lipid metabolism,the MFE1 gene,the peroxisome hydroxyacyl-Co A dehydrogenase gene,is related to lipid ?-oxidation,and disrupting ?-oxidation can also increase the content of oil.CRISPR-CAS9 technology was used again to continue to knock out MFE1,lycopene production increased to 66.18 mg/L.In addition,the lycopene peroxisome synthesis pathway and cytoplasmic synthesis pathway are combined to produce lycopene simultaneously in the peroxisome and cytoplasm,which improves the final yield of lycopene,reaching 70.52 mg/L.