Integrated pathway engineering and transcriptome analysis for improved astaxanthin biosynthesis in Yarrowia lipolytica

Abstract

Astaxanthin is a high value carotenoid with a broad range of commercial applications due to its superior antioxidant properties. In this study, β-carotene-producing Yarrowia lipolytica XK17 constructed in the lab was employed for astaxanthin biosynthesis. The catalytic effects of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ from various species were investigated. The PspCrtW from Paracoccus sp. and HpCrtZ^# from Haematococcus pluvialis were confirmed to be the best combination in converting β-carotene. Several key bottlenecks in biomass and astaxanthin biosynthesis were effectively eliminated by optimizing the expression of the above enzymes and restoring uracil/leucine biosynthesis. In addition, the effects of astaxanthin biosynthesis on cell metabolism were investigated by integrated analysis of pathway modification and transcriptome information. After further optimization, strain DN30 was able to synthesize up to 730.3 mg/L astaxanthin in laboratory 5-L fermenter. This study provides a good metabolic strategy and a sustainable development platform for high-value carotenoid production.

Keywords: Astaxanthin; Pathway engineering; Transcriptome analysis; Yarrowia lipolytica.

Graphical abstract

Fig. 1

Screening β-carotene ketolase (CrtW) and hydroxylase (CrtZ) from diverse organisms. (A) The biosynthetic pathway from β-carotene to astaxanthin. (B) Comparing the efficiency of different CrtWs in converting β-carotene to echinenone and canthaxanthin. (C) Comparing the efficiency of different CrtZs in converting β-carotene to β-cryptoxanthin and zeaxanthin (β-cryptoxanthin not detected). Evolutionary trees are constructed by maximum likelihood method, the percentage of trees in which the associated taxa clustered together is shown above the branches. Blue circle, bacteria. Green circle, algae. Error bar represents standard deviation of triplicate experiments.

Fig. 2

Combinatorial optimization of CrtW and CrtZ from diverse organisms. Carotenoids titers were determined in 13 strains expressing CrtW and CrtZ from various sources (other hydroxycarotenoids not detected). The different CrtW/CrtZ combinations are marked with asterisks under each group of bars. Error bar represents standard deviation of triplicate experiments.

Fig. 3

Effects of copy number of the PspCrtW, trCrCrtW and HpCrtZ^# genes on carotenoids biosynthesis (hydroxycarotenoid not detected). The copy number of the corresponding gene is indicated by the number of asterisks. Error bar represents standard deviation of triplicate experiments.

Fig. 4

Restoring uracil or/and leucine biosynthesis in strain DN21. (A) Two linear DNA fragments harboring the URA3 marker and the LEU2 marker, respectively. Bent arrow: a promoter; T-shape bar: a transcription terminator. (B) Biomass and carotenoids production of strains restoring uracil or/and leucine biosynthesis. The copy number of the corresponding genes is indicated by the number of asterisks. Error bar represents standard deviation of triplicate experiments.

Fig. 5

Transcriptome analysis of different carotenoid-producing strains. (A) Carotenoid production of strains DN25, DN26 and DN23. (B) β-carotene/astaxanthin production curves of strain DN25, strains DN23 and DN26. For each strain, we selected three samples (A, B, C) with different β-carotene/astaxanthin productivity for transcriptome analysis. Pink circle: early stage; Red circle: middle stage; Brown circle: final stage. (C) Expression pattern of genes involved in the MVA pathway and steroid biosynthesis pathway. Green or red arrows represent the down-regulation or up-regulation of gene transcriptions in Group B2 relative to Group A2. Values represent the corresponding fold changes. (D) Carotenoids production and biomass of strains overexpressing key genes based on transcriptome analysis.

Fig. 6

Construction of strain DN30 by NHEJ-mediated integration. (A) Two linear DNA fragments used to overexpress HpCrtZ^#, GND1 and HMG1 genes into strain DN21 by NHEJ-mediated integration. Bent arrow: a promoter; T-shape bar: a transcription terminator. (B) Astaxanthin production and biomass formation of strain DN30 in shake flasks.

Fig. 7

DO-stat fed-batch fermentation of the engineered strain DN30. (A) Final fermentation broth in 5-L fermenter. (B) DO-stat fed-batch fermentation curves of strain DN30. (C) Changes in the proportion of carotenoids during fermentation. Astaxanthin, canthaxanthin and echinenone were labeled in dark red, red and orange, respectively. Accumulations of β-carotene and other hydroxycarotenoids were not detected.