Host and Pathway Engineering for Enhanced Lycopene Biosynthesis in Yarrowia lipolytica

Abstract

Carotenoids are a class of molecules with commercial value as food and feed additives with nutraceutical properties. Shifting carotenoid synthesis from petrochemical-based precursors to bioproduction from sugars and other biorenewable carbon sources promises to improve process sustainability and economics. In this work, we engineered the oleaginous yeast Yarrowia lipolytica to produce the carotenoid lycopene. To enhance lycopene production, we tested a series of strategies to modify host cell physiology and metabolism, the most successful of which were mevalonate pathway overexpression and alleviating auxotrophies previously engineered into the PO1f strain of Y. lipolytica. The beneficial engineering strategies were combined into a single strain, which was then cultured in a 1-L bioreactor to produce 21.1 mg/g DCW. The optimized strain overexpressed a total of eight genes including two copies of HMG1, two copies of CrtI, and single copies of MVD1, EGR8, CrtB, and CrtE. Recovering leucine and uracil biosynthetic capacity also produced significant enhancement in lycopene titer. The successful engineering strategies characterized in this work represent a significant increase in understanding carotenoid biosynthesis in Y. lipolytica, not only increasing lycopene titer but also informing future studies on carotenoid biosynthesis.

Keywords: HMG1; carotenoids; lipid metabolism; metabolic engineering; mevalonate pathway; synthetic biology.

FIGURE 1

Engineering lycopene biosynthesis in Y. lipolytica. (A) Schematic of the lycopene biosynthetic pathway from acetyl-CoA. For simplicity, cofactors and ATP consumption is not shown. (B) Summary of strategies attempted in this work. Items shown in green enhanced lycopene yield, while items shown in red reduced lycopene yield.

FIGURE 2

Effect of leucine and uracil auxotrophies on lycopene production. (A) Lycopene titer for strains with and without leucine and uracil auxotrophies. (B) Specific lycopene content (mg lycopene/g DCW) for strains with and without leucine and uracil auxotrophies. Strains were grown for 4 days in 25 mL of YPD10 at 30°C in baffled shake flasks. Dry cell weight produced by each strain: HEBI-L-U 2.4 ± 0.4 g/L, HEBI-U 2.8 ± 0.2 g/L, HEBI-L 2.0 ± 0.4 g/L, and HEBI 4.3 ± 0.8 g/L. Statistical significance from the HEBI-L-U strain is indicated by “^∗”. HEBI indicates the following overexpressions in the PO1f background: H = HMG1, E = CrtE, B = CrtB, I = CrtI.

FIGURE 3

The effect of β-oxidation on lycopene production in Y. lipolytica PO1f. Relative specific lycopene content of strains with and without intact β-oxidation at 4 and 10 days of culture time. β-oxidation was disrupted by knockout of MFE1. Strains were grown for 4 and 10 days in 25 mL of YPD10 at 30°C in baffled shake flasks. Bars and error bars represent the mean and standard deviation, respectively, of biological triplicates. Statistical significance from the HEBI strain is indicated by “^∗”. At 4 days, the HEBI strain produced 2.8 ± 0.3 mg lycopene/g DCW.

FIGURE 4

The effect of PAH1 knockout on lycopene production in Y. lipolytica PO1f. Relative specific lycopene content of strains with and without disruption of PAH1. Strains were grown for 4 days in 25 mL of YPD10 at 30°C in baffled shake flasks. Bars and error bars represent the mean and standard deviation, respectively, of biological triplicates. Statistical significance from the HEBI strain is indicated by “^∗”. No statistically significant difference was detected. The HEBI strain produced 5.3 ± 0.3 mg lycopene/g DCW.

FIGURE 5

The effect of disrupting glycogen biosynthesis on lycopene production in Y. lipolytica PO1f. Relative specific lycopene content of strains with and without intact glycogen biosynthesis. Strains were grown for 4 days in 25 mL of YPD10 at 30°C in baffled shake flasks. Bars and error bars represent the mean and standard deviation, respectively, of biological triplicates. Statistical significance from the HEBI strain is indicated by “^∗”. The HEBI strain produced 3.4 ± 0.2 mg lycopene/g DCW.

FIGURE 6

Engineering mevalonate biosynthesis pathway. (A) Schematic representation of the mevalonate pathway. (B) Relative specific lycopene production from strains with a single gene in the mevalonate pathway overexpressed. Strains were grown for 4 days in 25 mL of YPD10 at 30°C in baffled shake flasks. Bars and error bars represent the mean and standard deviation, respectively, of biological triplicates. Statistical significance from the HEBI strain is indicated by “^∗”. The HEBI strain produced 3.2 ± 0.9 mg lycopene/g DCW.

FIGURE 7

Engineering the lycopene biosynthetic pathway. (A) Schematic representation of the lycopene biosynthetic pathway. (B) Relative specific lycopene production from strains with a single gene in the lycopene biosynthetic pathway overexpressed. Strains were grown for 4 days in 25 mL of YPD10 at 30°C in baffled shake flasks. Bars and error bars represent the mean and standard deviation, respectively, of biological triplicates. Statistical significance from the HEBI strain is indicated by “^∗”. The HEBI strain produced 3.2 ± 0.9 mg lycopene/g DCW.

FIGURE 8

Combing successful overexpressions to optimize lycopene production. Relative specific lycopene of strains combining successful engineering strategies to maximize lycopene production in 4-day shake flask cultures. Strains were grown for 4 days in 25 mL of YPD10 at 30°C in baffled shake flasks. Bars and error bars represent the mean and standard deviation, respectively, of biological triplicates. Statistical significance from the HEBI strain is indicated by “^∗”. The HEBI strain produced 2.0 ± 0.5 mg lycopene/g DCW. HV8I indicates the following overexpressions in the HEBI background: H = HMG1, V = MVD1, 8 = ERG8, I = CrtI. Names with multiple copies of a given letter indicate multiple overexpressions of the indicated gene.

FIGURE 9

Lycopene production in fed-batch shake flask cultures. Strains were grown for 12 days in 25 mL of YPD10 at 30°C in baffled shake flasks with glucose feeding as described. Bars and error bars represent the mean and standard deviation, respectively, of biological triplicates.

FIGURE 10

Lycopene production in fed-batch bioreactor. (A) Cell pellets taken from each day of bioreactor culture post-inoculation. Red coloration is due to lycopene production. (B) Specific lycopene production at each day of bioreactor culture post-inoculation. Data represents technical triplicates of a single bioreactor experiment.