Metabolic Engineering Of Yarrowia Lipolytica For ?-ionone Biosynthesis

Terpenoids and their derivatives have attracted great interest for their commercial application as biofuels,flavoring ingredients,fragrances,antiseptics and pharmaceuticals.With the development of synthetic biology tools,many terpenoids have been produced from microorganisms by the pathway reconstruction,which showing greatly potentiality to meet the need of market.Morever,the use of generally recognized as safe(GRAS)microorganisms to convert natural raw materials into products leads to aroma compounds that can be described as natural according to the United States and the European Union flavor regulations.However,since natural products anabolism involves long pathways and complex regulation,the construction of an efficient microbial cell factory is still a cumbersome work,and often resulting in low production performance.The GRAS,unconventional yeast Yarrowia lipolytica(Y.lipolytica)has rapidly emerged as a valuable cell factory for the produce of terpenoids due to its high production capacity for the biosynthesis of acetyl Co A derivatives.In this work,an efficient ?-ionone producing Y.lipolytica platform was constructed by the metabolic pathway optimizations and fermentation conditions optimizations.In this study,the ku70 and ku80 genes of Y.lipolytica were knockout to generate strain YLBI0003,to improve the homologous recombination(HR)efficiency,resulting in a 53% HR efficiency for 14.5 kb fragment integration.The car B and car RP genes that from Mucor circunelloides and CCD1 from Petunia hybrid were assembled into ?-ionone synthesis cassette and was then integrated into the r DNA locus of strain YLBI0003 to generate the initial strain YLBI0004,resulted in 3.5 mg/L of ?-ionone titer.The overall pathway from glucose to ?-ionone was organized into the following three modules,considering the two most important intermediates: acetyl-Co A and geranylgeranyl diphosphate(GGPP).Module 1,the acetyl-Co A supply module;Module 2,the GGPP supply module;Module 3,the ?-ionone synthesis module.We first engineered the strain YLBI0004 by the optimization of GGPP supply module to improve the ?-ionone production.Nine endogenous pathway genes were integred into strain YLBI0004 to geneate strain YLBI3017,resulted in a 43.7-fold increase of ?-ionone titer compared with initial strain.Subsequently,the acetyl-Co A supply module was optimized by the introduction of PK-PTA pathway that consisted of the phosphoketolase from Bifidobacterium breve and phosphotransacetylase from Bacillus subtilis into strain YLBI3017 to geneate strain YLBI3117,yield 202.2 mg/L ?-ionone.Finally,another copy of ?-ionone synthesis module was integrated into strain YLBI3117 to obtain strain YLBI3118.The strain YLBI3118 had yielded 220.7 mg/L(18.1 mg/g DCW).Then the Ph CCD1 enzyme from ?-ionone synthesis module was modified to improve the membrane affinity by directed mutation or the amino-terminal addition of membrane destination peptides,which had been reported to improve the Ph CCD1 membrane affinity and then promote the catalysis of ?-carotene.The ?-ionone production from two well strains YLBI3120 and YLBI3121 have up to 360.9 mg/L(15.9 mg/g DCW)and 354 mg/L(16.5 mg/g DCW),respectively.Furthermore,the optimization of fermentation conditions was performed to improve the ?-ionone production.The results showed that glucose and peptone yield the highest titer,which up to 358.4 mg/L(19.2 mg/g DCW).And the nitrogen limitation fermentation led to a 154% decrease of ?-ionone titer.In the subsequent 3-L bioreactor fermentation,the dissolved oxygen was found to be a very important factor for the biosynthesis of ?-ionone.This is related to the last metabolic step of ?-ionone biosynthesis,which is catalyzed by carotenoid cleavage dioxygenase and requieds two oxygen molecules to produce two ?-ionone molecules.With the optimization of bioreactor fermentation conditions,the ?-ionone titer improved 280-fold compared to the initial strain,up to 1 g/L,to the best of our knowledge,is the highest yield of ?-ionone obtained in cell factories to date.In this work,the PK-PTA pathway was found to be an effective strategy to improve ?-ionone titer and the dissoved oxygen was found to be a key factor for the biosynthesis of ?-ionone.This work should advance the potential of Y.lipolytica as a cell factory platform for the high-level production of terpenoids.