Metabolic Engineering Of The Oleaginous Yeast Yarrowia Lipolytica For Microbial Synthesis Of Bisabolene

Bisabolene is an origin natural active substance of plant.It belongs to monocyclic sesquiterpenes and has three isomers:?-bisabolene,?-bisabolene,and y-bisabolene.Yarrowia lipolytica is a typical representative unconventional yeast,is also an excellent microbial production host,because it is an oleaginous yeast with a large amount of intracellular acetyl-CoA,it has potential to be reattached to produce excess farnesyl pyrophosphate(FPP)in order to be used as a precursor for bioconversion to bisabenene,and Y.lipolytica can grow on a variety of cheap hydrophobic and hydrophilic carbon sources,including alkanes,alkenes,fats,alcohols,and organic acids,so it can be used as an effective whole-cell biocatalyst to repair the polluted environment.In this paper,Y.lipolytica was successfully engineered and optimized to produce bisabolene from WCO.We envision that this work will provide strategic insights for the development and use of Y.lipolytica as a microbial cell factory to sustainably and economically produce valuable chemical substances from waste oily raw materials.The main research contents are as follows:(1)The ?-bisabolene synthase gene(?BS)from Abies grandis and the ?-bisabolene synthase gene(?BS)from Zingiber officinale and the?-bisabolene synthase gene(?BS)from Helianthus.Integrate the recombinant plasmid into the Y.lipolytica Polg ?KU70 genome.The engineering strains Polg K?BS,Polg K?BS and Polg KyBS and the original strain Po1g ?KU70 obtained by cultivating and fermenting in YPD medium were analyzed and detected by gas chromatography-mass spectrometry(GC-MS).These strains will be used as the basic strains for the production of bisabolene for further engineering design and optimization.(2)Then,the 10 functional genes in the MVA pathway were overexpressed with a single gene to realize their effect on the overproduction of bisabolene,so as to determine the rate-limiting enzyme gene for the biosynthesis of bisabolene in the MVA pathway.The above plasmids were recombined into the Y.lipolytica genome,and 30 recombinant yeasts were obtained.The strains Po1g KaHR,Po1g K?HR,and Po1g KyHR are the targets of the next step of further improvement to increase the production of bisabolene.(3)In order to further increase the yield of bisabolene and avoid the burden of yeast growth and reduce the toxicity of the product,the heterogeneous expression of AcrB pump from Escherichia coli and ABC-G1 pump from Grosmania clavigera in yeast was integrated into the chromosomes of Po1g KaHR,Po1g K?HR,and Po1g K?HR strains,and six recombinant yeasts were obtained.(4)In order to prove the potential use of the engineered Y.lipolytica strains to produce bisabolene using WCO as the carbon source,the six engineered strains were cultured in a medium using WCO instead of glucose as the carbon source.The fermentation results showed that using WCO as the sole carbon source successfully produced ?-bisabolene,?-bisabolene,and ?-bisabolene.In the case of having the same number of carbon units,when 1.18%(w/v)WCO is added as the carbon source.This indicates that the Y.lipolytica strains engineered to produce bisabolene with WCO as the sole carbon source can grow very efficiently and steadily,and it can potentially be used for the sustainable and economic platform to produce bisabolene.(5)Research shows that magnesium ions and other metal ions have certain activation effect on lipase of oleaginous yeast.By adding certain magnesium ions to the culture medium to increase the activity of the enzyme and increase the conversion efficiency of fatty acids in the waste oil,thereby providing more precursor substances for the production of bisabolene and further increasing the production of bisabolene.Six engineering strains were cultured in an optimized medium with WCO as the carbon source.The Y.lipolytica engineering strains Polg KaBS-ABCG1,Polg K?BS-ABCG1 and K?BS-ABCG1 can produce ?-bisabolene,?-bisabolene and y-bisabolene was increased to 973.03mg/L,68.17 mg/L,20.22 mg/L,respectively.In summary,this study investigated the ability of Y.lipolytica heterologous synthesis of bisabolene through a metabolic engineering strategy,and successfully realized the conversion of WCO to bisabolene.This study also laid the foundation for the transformation of Y.lipolytica to use kitchen waste oil to synthesize more valuable industrial products.