Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction

Abstract

Background: Limonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene.

Results: To construct the limonene synthetic pathway in Yarrowia lipolytica, two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica. Furthermore, to maximize limonene production, several genes involved in the MVA pathway were overexpressed, either in different copies of the same gene or in combination. Finally with the optimized pyruvic acid and dodecane concentration in flask culture, a maximum limonene titer and content of 23.56 mg/L and 1.36 mg/g DCW were achieved in the final engineered strain Po1f-LN-051, showing approximately 226-fold increase compared with the initial yield 0.006 mg/g DCW.

Conclusions: This is the first report on limonene biosynthesis in oleaginous yeast Y. lipolytica by heterologous expression of codon-optimized tLS and tNDPS1 genes. To our knowledge, the limonene production 23.56 mg/L, is the highest limonene production level reported in yeast. In short, we demonstrate that Y. lipolytica provides a compelling platform for the overproduction of limonene derivatives, and even other monoterpenes.

Keywords: Limonene; Limonene synthase; Neryl diphosphate synthase 1; Yarrowia lipolytica.

Fig. 1

Biosynthesis pathway for limonene production in Y. lipolytica. IPP and DMAPP are converted to NPP by neryl diphosphate synthase 1 (NDPS1) and then NPP is further converted to limonene by limonene synthase (LS). Blue arrows represent that the pathways were exogenously integrated in Y. lipolytica, while red arrows represent that the pathways were overexpressed in Y. lipolytica. Single arrows represent the one-step conversions, while triple arrows represent multiple steps

Fig. 2

GC–MS analysis of limonene from the dodecane phase of the cultures in engineered Y. lipolytica. The strain was cultivated in YPD medium for 72 h. a Limonene standard; b Mass spectrum of limonene standard; c Limonene obtained in YPD medium containing 4 g/L pyruvic acid of Po1f-LN-051; d Mass spectrum of limonene obtained in YPD medium containing 4 g/L pyruvic acid of Po1f-LN-051

Fig. 3

Quantitative analysis of limonene production in engineered Y. lipolytica. Limonene productions in the engineered strains Po1f-LN-000, Po1f-LN-001, Po1f-LN-002, Po1f-LN-003, Po1f-LN-004, Po1f-LN-005, Po1f-LN-006, Po1f-LN-007, Po1f-LN-008, Po1f-LN-011, Po1f-LN-021, Po1f-LN-031, Po1f-LN-041, Po1f-LN-051, Po1f-LN-061, Po1f-LN-071. All strains were cultured in YPD medium for 3 days. Three repeats were performed for each strain, and error bars represent standard deviations

Fig. 4

a The OD600 values of strains Po1f, Po1f-LN-000, Po1f-LN-004, and Po1f-LN-051 cultured in YPD medium, measured at 0, 8, 14, 24, 32, 48, 72, and 96 h. b Squalene production in strains Po1f, Po1f-LN-000, Po1f-LN-004, Po1f-LN-051 cultured in YPD medium for 5 days

Fig. 5

Effects of different combinations of glucose and glycerol as substrate and heterologous addition of pyruvic acid and dodecane on the production of limonene. a Glucose of 2, 1.5, 1, 0.5, 0 % were respectively combined with glycerol of 0, 0.5, 1, 1.5, 2 % as substrate in the YP medium of strain Po1f-LN-051. b Pyruvic acid was added to the YPD medium of strain Po1f-LN-051 to a final concentration of 0, 2 , 4, or 8 g/L. c Dodecane was added to the YPD medium of strain Po1f-LN-051 with 4 g/L pyruvic acid as the auxiliary carbon source and the proportion of dodecane was from 2 to 10 %. Three repeats were performed for each medium, and error bars represent standard deviations. t tests were conducted to evaluate statistical significance at p < 0.05. Particularly, for (a) the asterisk shows statistical significance between 2 % glucose and other combinations of glucose and glycerol, for (b) the asterisk shows statistical significance between YPD medium containing 0 g/L pyruvic acid and other concentrations of pyruvic acid, and for (c) the asterisk shows statistical significance between YPD medium containing 2 % dodecane and other proportions of dodecane

Fig. 6

Inhibitory effects of d-limonene on Y. lipolytica. A fixed volume of serially diluted d-limonene in ethanol ranging from 100 to 3000 mg/L was added to YPD medium which contained 0.5 % Tween 80 (v v-1), YPD medium contained 0.5 % Tween 80 (v v-1) and ethanol was regarded as control. Po1f was used as the trial strain