The enhancement of astaxanthin production in Phaffia rhodozyma through a synergistic melatonin treatment and zinc finger transcription factor gene overexpression

Abstract

Astaxanthin has multiple physiological functions and is applied widely. The yeast Phaffia rhodozyma is an ideal source of microbial astaxanthin. However, the stress conditions beneficial for astaxanthin synthesis often inhibit cell growth, leading to low productivity of astaxanthin in this yeast. In this study, 1 mg/L melatonin (MT) could increase the biomass, astaxanthin content, and yield in P. rhodozyma by 21.9, 93.9, and 139.1%, reaching 6.9 g/L, 0.3 mg/g DCW, and 2.2 mg/L, respectively. An RNA-seq-based transcriptomic analysis showed that MT could disturb the transcriptomic profile of P. rhodozyma cell. Furthermore, differentially expressed gene (DEG) analysis show that the genes induced or inhibited significantly by MT were mainly involved in astaxanthin synthesis, metabolite metabolism, substrate transportation, anti-stress, signal transduction, and transcription factor. A mechanism of MT regulating astaxanthin synthesis was proposed in this study. The mechanism is that MT entering the cell interacts with components of various signaling pathways or directly regulates their transcription levels. The altered signals are then transmitted to the transcription factors, which can regulate the expressions of a series of downstream genes as the DEGs. A zinc finger transcription factor gene (ZFTF), one of the most upregulated DEGs, induced by MT was selected to be overexpressed in P. rhodozyma. It was found that the biomass and astaxanthin synthesis of the transformant were further increased compared with those in MT-treatment condition. Combining MT-treatment and ZFTF overexpression in P. rhodozyma, the biomass, astaxanthin content, and yield were 8.6 g/L, 0.6 mg/g DCW, and 4.8 mg/L and increased by 52.1, 233.3, and 399.7% than those in the WT strain under MT-free condition. In this study, the synthesis and regulation theory of astaxanthin is deepened, and an efficient dual strategy for industrial production of microbial astaxanthin is proposed.

Keywords: Phaffia rhodozyma; RNA-seq; anti-stress; astaxanthin; melatonin; signal transduction; transcription factor engineering.

Figure 1

Effects of melatonin (MT) on biomass and astaxanthin accumulation in Phaffia rhodozyma. (A) Effects of different concentrations of MT on biomass, astaxanthin content, and yield in P. rhodozyma. (B) 96 h time-course effect of 1 mg/L MT on biomass in P. rhodozyma. (C) 96 h time-course effect of 1 mg/L MT on astaxanthin content in P. rhodozyma. (D) 96 h time-course effect of 1 mg/L MT on astaxanthin yield in P. rhodozyma. Data are given as means ± SD, n = 3. *p < 0.05 **p < 0.01.

Figure 2

The differentially expressed gene (DEG) number (A) and volcano plot (B) induced by melatonin treatment in P. rhodozyma.

Figure 3

GO annotation of the DEGs between the cells from MT treatment (M) and MT-free (C) conditions. (A) GO enrichment BarPlot; (B) GO enrichment ScatterPlot.

Figure 4

KEGG annotation of the DEGs between the cells from MT treatment (M) and MT-free (C) conditions. (A) KEGG enrichment BarPlot; (B) KEGG enrichment Scatter Plot.

Figure 5

Construction of a ZFTF gene overexpression vector and its validations. (A) Schematic diagram of vector construction through 2-step overlap PCR. Red parts: homologous oligonucleotide with up-stream or down-stream DNA fragments. 18sup, Pgdp, G418, Tgdp, Padh4, ZFTF, Tact, and 18sdown: upstream of 18SrDNA, gdp gene promoter, G418-resistence gene, gdp gene terminator, adh4 gene promoter, zinc finger transcription factor gene, act gene terminator, and downstream of 18SrDNA. (B) Electrophoretic profile of the constructed ZFTF overexpression vector of 6,785 bp. (M) marker; 1, the vector containing 18sup, Pgdp, G418, Tgdp, Padh4, ZFTF, Tact, 18sdown fragments with a length of 6,785 bp. (C) Confirmations of ZFTF’s integrations into the 18SrDNA location of P. rhodozyma genome by PCR. M, marker; lane 1–6, transformants ZFTF-1 to ZFTF-6 carrying the ZFTF-overexpression vector; lane 8, the wild strain (WT) D, The ZFTF gene expressions of WT and transformant ZFTF-3 at the MT-free and MT treatment conditions analyzed by RT-PCR. Data are given as means ± SD, n = 3. The significances without annotation indicate the samples from MT treatment versus MT-free, and ZFTF overexpression versus none ZFTF. *p < 0.05; **p < 0.01.

Figure 6

Biomass, astaxanthin content, and yield of P. rhodozyma transformant ZFTF-3 overexpressing a ZFTF gene under MT-free and MT treatment conditions. Data are given as means ± SD, n = 3. The significance without annotation indicate the samples from MT treatment versus MT-free and ZFTF overexpression versus none ZFTF. *p < 0.05; **p < 0.01.

Figure 7

Schematic diagram for the molecular mechanism of MT improving astaxanthin synthesis in P. rhodozyma at the global-cell level. Red circle, melatonin (MT); large arrows with different colors, various signal pathways; pentagrams, transcription factors (TFs) of different signal pathways with the same color as their related signal pathways; dashed box, the target genes or DEGs induced by MT. The mechanism of MT regulating astaxanthin biosynthesis is that (1) MT interact with the components of related signal pathways to alter signal transduction, or (2) MT as a signal molecule directly regulate the transcriptional levels of the target genes. The changed signal transduction transmitted to transcription factors (TFs), and TFs regulate the expressions of target genes at transcriptional level, leading to the quantity changes in related effector proteins. A part of effector proteins are components or TFs of related signal pathways, which reversely affect the signal pathways and regulate the expressions of target genes, while other parts of effector proteins regulate astaxanthin synthesis.