Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway

Abstract

Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

Keywords: Yarrowia lipolytica; astaxanthin; metabolic engineering; submerged fermentation; β-carotene.

Figure 1

Effect of Synechococcus GGPPs7 (ST7434) and the 2^nd copy of crtE (ST7433) on β-carotene production. ST6899 is the parental strain to ST7434 and ST7433. (A) Yeast extract peptone dextrose (YPD) plate after 2 days cultivation. (B) Titers measured by HPLC. The error bars represent standard deviations calculated from biological triplicate experiments.

Figure 2

Carotenoid production of strains ST7906 (expressing multiple copies of PscrtW) and ST7972 (expressing multiple copies of HpBKT). All strains were cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. The error bars represent standard deviations calculated from triplicate experiments.

Figure 3

Carotenoid production by strains ST7925 and ST7926. Set of yeast transformants expressing PscrtW in combination with either PacrtZ or HpcrtZ. Positive control: ST7400; Parent strain: ST7906. All strains were cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. Three dots represent multiple integrations of genes and one dot represents single integration. The error bars represent standard deviations calculated from triplicate experiments (“iso” after each strain indicates the isolate number).

Figure 4

Carotenoid production by strains ST7973 and ST7974. Set of transformants expressing HpBKT in combination with either PacrtZ or HpcrtZ. Positive control: ST7400. Parent strain: ST7972. All strains were cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. Three dots represent multiple integrations of genes, and one dot represents single integration. The error bars represent standard deviations calculated from triplicate experiments (“iso” after each strain indicates the isolate number).

Figure 5

Carotenoid production by strains ST7927 and ST7928 transformed with combinations of PscrtW and PacrtZ/HpcrtZ genes. Molar ratios of DNA constructs are in brackets. Strains used as controls: ST7906 and ST7400. Three dots represent multiple integrations of genes. All strains were cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. The error bars represent standard deviations calculated from triplicate experiments (‘iso’ after each strain indicates the isolate number).

Figure 6

Carotenoid production by strains ST7975 and ST7976 transformed with combinations of HpBKT and PacrtZ/HpcrtZ genes. Molar ratios of DNA constructs are in brackets. Strain used as positive control: ST7400. Three dots represent multiple integrations of genes. All strains were cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. The error bars represent standard deviations calculated from triplicate experiments (“iso” after each strain indicates the isolate number).

Figure 7

Concentrations of dry cell weight (DCW), glucose, and carotenoids during the fed-batch cultivations of ST7976 (iso 3). The values are averages from three independent experiments; the error bars represent show standard deviations.

Figure 8

(A) Carotenoid concentrations during fed-batch cultivations (as in Figure 7) of ST7976 (iso 3). The values are averages from three independent experiments; the error bars represent show standard deviations. (B) Bioreactor at the end of the fed-batch fermentation of ST7976 (iso 3).

Figure 9

Engineered pathways. (A) Improvement of precursor supply. (B) Astaxanthin biosynthesis pathway. The white boxes indicate enzymes already expressed in the parental strain ST6899 [23]. The green boxes indicate enzymes additionally expressed in the parental strain in this study. IPP: Isopentenyl pyrophosphate; DMAPP: Dimethylallyl pyrophosphate; FPP: Farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate; ERG20: farnesyl pyrophosphate synthase; CrtE and GGPPs7: geranylgeranyl pyrophosphate synthase; CrtYB: phytoene synthase and lycopene cyclase; CrtI: phytoene desaturase; CrtW: β-ketolase from bacteria; BKT: β-ketolase from microalgae; CrtZ: β-hydroxylase.

Figure 10

Flowchart of the strains generated in this study.