Metabolic Engineering Of Kluyveromyces Lactis For Microbial Synthesis Of Bisabolene

Bisabolene is an important natural active substance of plant origin,which has extremely important applications in various fields such as food,cosmetics and medicine,and has great market demand.However,the production of bisabolene is still low,and there are few synthetic methods,and the synthesis methods are complex and pollute the environment.With the increase of synthetic biology elements and the development of metabolic engineering transformation,more and more natural active substances turn to microbial cell factory production.Microbial synthesis method has a wide range of application because of high transformation efficiency,strong environmental adaptability,low cost and green sustainable development,and has very broad application prospects.Kluyveromyces lactis is typical unconventional yeast that has extremely important applications in basic microbial research and applied research.The range of available carbon sources of K.lactis is extremely wide,and the yeast can be grown with lactose as the sole carbon source and energy source due to the fact that the yeast can produceβ-galactosidase and lactose permease to decompose and utilize lactose.There are many similarities between K.lactis and Saccharomyces cerevisiae in terms of metabolism and cellular properties.In recent years,a series of basic and applied studies in synthetic biology with K.lactis as the biotechnological chassis have developed rapidly.In this paper,a series of studies were carried out in the production of bisabolene using K.lactis as the biotechnological chassis,mainly including the following aspects:（1）First of all,theα-bisabolene synthase from Abies grandis,theβ-bisabolene synthase from Zingiber officinale and theγ-bisabolene synthase from Helianthus were introduced in K.lactis.Because of the nature MVA pathway,the production pathways ofα-bisabolene andβ-bisabolene were successfully constructed in yeast,with yields of 3.9 mg/L and 5.1 mg/L,respectively.Theβ-bisabolene was selected as the target product for subsequent production.Subsequently,there are five fermentation methods were selected to ferment the engineered strains containingβ-bisabolene synthase.According to the yield,the method of adding lactose on the basis of YPD medium was selected as the optimal fermentation method.（2）Use different promoters to overexpress HMGR genes.The commonly used endogenous promoters P_{Kl PDC1},P_{Kl ADH3},P_{Kl GAL7} and the synthetic promoter P_hp4d from Yarrowia lipolytica were selected to overexpress the HMGR gene.By comparing the yields,the engineered strain which the HMGR gene was overexpressed by P_{Kl GAL7} has highest yield which is 37.0 mg/L.Therefore,P_{Kl GAL7} was chosen as the subsequent expression promoter.（3）Increase the metabolism of MVA pathway in yeast.a.The endogenous gene of MVA pathway,truncated S.cerevisiae HMGR gene and truncated endogenous HMGR gene were overexpression in K.lactis,respectively.The results show that the overexpressed IDI gene strain has the highest yield,reaching 45.1 mg/L.b.Combined overexpression of the IDI,HMGR and GGPPS genes which are the better genes in single gene overexpression assays.These genes were overexpression with a combination of dual gene or triple gene,but the yield does not rise but falls.The three-gene combination overexpressed strain had the highest yield at 32.5 mg/L.（4）The transporter was used to accelerate the transport of bisabolene out of cells,and the Acr B transporter from Escherichia coli,the YALI0F19492p transporter from Y.lipolytica and the ABC-G1 transporter from Grosmania clavigera were introduced in K.lactis.After fermentation,GC-MS was used to detect the production of bisabolene of the engineered strains.The results show that the YALI0F19492p transporter gene strain has the highest yield,reaching 34.6 mg/L.（5）Use whey as carbon source to product bisabolene in K.lactis.Using dairy by-product whey as the sole carbon source,the yield of engineered strains which were cultured by medium with different concentrations of whey was compared.The strain of overexpressing IDI gene was used as the experimental strain,and the optimal whey addition concentration was finally determined to be 10%,the yield of the strain is 95.5 mg/L.In summary,this paper constructs an engineered strain of K.lactis that produces high-value plant natural products,and realizes the effectively use of whey,providing an effective solution for the construction of efficient green microbial cell factories.