Engineering Yarrowia Lipolytica For Limonene Synthesis

Limonene,as an important monocyclic monoterpene,is widely used in food,medicine and cosmetics because of its antioxidant,antibacterial and aromatic properties.With the development of synthetic biology and metabolic engineering,the use of microorganisms to synthesize limonene has a good potential.At present,although microorganisms such as bacteria or yeast have been used as a chassis for limonene synthesis,due to the limited synthesis of the precursor geranyl diphosphate(GPP),the synthesis efficiency of limonene is low.Yarrowia lipolytica,as an unconventional oleaginous yeast,is a chassis for the synthesis of terpenoids due to its high acetyl coA synthesis flux and good robustness.In this study,a high level of limonene synthesis was achieved by introducing orthogonal limonene synthesis pathway and enhancing precursor supply.The results are as follows:(1)Construction of synthesis pathway of orthogonal limonene:Taking AHH12,a strain of mevalonic acid(MVA)produced in the laboratory,as the starting strain,limonene synthase(CltLS1)from Citrus Limon was screened and identified.On this basis,synthesis of neryl diphosphate(NPP)by introducing neryl diphosphate synthase(SltNPPS)from Solanum lycopersicum,which is orthogonal to the endogenous GPP synthesis pathway.The titer of limonene was 35.94 mg/L and the synthesis efficiency of limonene was higher than that of GPP and 13.8-folds that of CltLS1 alone.In order to increase the selectivity and activity of limonene synthase,CltLS1H570Y was co-expressed with SltNPPS to get strain LN008.The titer of limonene was 38.53 mg/L,which was 11.9%higher than the wild CltLS1 co-expressed with SltNPPS.The preliminary construction of limonene synthesis strains was realized effectively by engineering Yarrowia lipolytica,which laid a foundation for the subsequent optimization.(2)Enhancement of MVA pathway:In order to increase the accumulation of precursors of limonene synthesis,ERG20F88W-N119W was further overexpressed to obtain 54.56 mg/L limonene.Phosphomevalonate kinase(ERG8)and mevalonate diphosphate decarboxylase(ERG 19)downstream of the MVA pathway were overexpressed and the strain LN010 was constructed.The titer of limonene was 248.23 mg/L,6.44-folds that of strain LN008.In addition,in order to balance the ratio of intracellular precursors isopentyl diphosphate(IPP)to dimethylallyl diphosphate(DMAPP),mutating isopentenyl diphosphate isomerasel(IDI1)into IDI1L132H/Y182F/W239C,IDI1L132H/Y182F and IDI1L132H.The effects of IDI1 and its three variants on the synthesis of limonene were analyzed.The results showed that the mutated IDI1 resulted in a significant decrease in the titer and content of limonene,and only the overexpression of IDI1,which was derived from Yarrowia lipolytica,improved the titer of limonene by 14.9%.After further overexpression of mevalonate kinase(ERG 12),the titer of limonene was increased to 300.08 mg/L and the content was 13.32 mg/g DCW.(3)Introduction of isopentenol utilization pathway(IUP):In order to increase the accumulation of IPP/DMAPP,artificially designed IUP is introduced.IPP or DMAPP was two-step catalytically synthesized by expressing isopentenyl phosphate kinase from Arabidopsis thaliana(AtIPK)and choline kinase from Saccharomyces cerevisiae(ScCK).After AtIPK and ScCK expression,the addition of high concentration isoprenol(prenol)resulted in impaired cell growth,while the addition of low concentration isoprenol(prenol)resulted in a slight increase in limonene production.The addition of 2.5 mM isoprenol resulted in a 13.36%increase in the titer of limonene.(4)Synthesis of limonene by peroxisomal compartmentalization:cytoplasmic-peroxisomal dual expression of partial enzymes of orthogonal limonene synthesis pathway using enhanced peroxisome localization signal peptide(ePTSl)failed to effectively promote the synthesis of limonene.In this study,through the introduction of orthogonal limonene biosynthesis pathway,Yarrowia lipolytica strain was constructed for the limonene production,and the titer of limonene was increased to 300.08 mg/L through the enhancement of mevalonic acid pathway in shaking flask.In addition,the introduction of IUP and the ability of peroxisomal compartment synthesis into limonene were also explored,which established a basis for further improving the production of limonene and the synthesis of downstream derivatives.