GATA transcription factor WC2 regulates the biosynthesis of astaxanthin in yeast Xanthophyllomyces dendrorhous

Abstract

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one-hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two-hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild-type strain did not largely modify the carotenoid profile, indicating translational and/or post-translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild-type strain and the XdWC2-disrupted strain apparently improved the production of monocyclic carotenoid 3-hydroxy-3', 4'-didehydro-β, ψ-carotene-4-one (HDCO) rather than β-carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over-producing strains.

FIGURE 1

The pathway of carotenoid biosynthesis in X. dendrorhous. MVA, mevalonate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; HDCO, 3‐hydroxy‐3′, 4′‐didehydro‐β, ψ‐carotene‐4‐one. IDI, IPP delta isomerase gene; CrtE, GGPP synthase gene; CrtI, phytoene desaturase gene; CrtYB, phytoene synthase gene or lycopene cyclase gene; CrtS, astaxanthin synthase gene; CrtR, cytochrome P450 reductase gene.

FIGURE 2

Identification of the GATA transcription factor. (A) The cell lethal rate of CBS 6938 in ARTP mutagenesis. (B) Flowchart for the ARTP mutagenesis of X. dendrorhous. M1 denotes a red mutant obtained in the first round of irradiation and M1‐3RC denotes the yellow mutant obtained in the second round of irradiation. (C) The production of astaxanthin and β‐carotene in the ARTP mutant M1‐3RC and the control CBS 6938. (D) The ‘TG’ insertion in the gene of the putative GATA transcription factor CDZ96439.1. LN483144.1 denotes the sequence Scaffold_54 in GenBank database. (E) Sequence analysis showing the typical PAS (Per‐Arnt‐Sim) domain and a C‐X₂‐C‐X₁₈‐C‐X₂‐C type GATA zinc‐finger domain in CDZ96439.1. The ‘TG’ insertion located in the GATA zinc‐finger domain.

FIGURE 3

Comparative sequence analysis of GATA transcription factors. (A) A phylogenetic tree of GATA transcription factors in fungi constructed by ClustalX 2.1 using neighbour‐joining method with 1000 bootstrapped replicates. (B) Comparative analysis of WC2 proteins by MEME suite exhibiting 7 conserved motifs. The sequences were derived from GenBank according to the accession numbers BAO20283 (Grifola frondose), KAG1754311 (Suillus lakei), KAG1891459 (Suillus subluteus), CUA69058 (Rhizoctonia solani), KAE9395386 (Gymnopus androsaceus JB14), KAB5596254 (Ceratobasidium theobromae), KAG9318635 (Chiua virens) and KAF9246368 (Melanogaster broomeanus).

FIGURE 4

Expression of XdWC2 influencing the biosynthesis of carotenoid. (A) The production of astaxanthin and β‐carotene detected by HPLC. Wildtype, the strain CBS 6938; Xd‐wc2, disrupting XdWC2 in CBS 6938; Xd‐Cwc2, restoring XdWC2 at the native locus in Xd‐wc2; Xd‐rDL‐WC2, overexpressing XdWC2 at rDNA loci in Xd‐wc2. (B) Expression of XdWC2 assayed by qRT‐PCR in the wild‐type strain CBS 6938, and the recombinant strains Xd‐wc2, Xd‐Cwc2 and Xd‐rDL‐WC2 when cultivated for 120 h.

FIGURE 5

Transcriptomic analysis of the XdWC2‐disrupted strain Xd‐wc2 and the XdWC2‐overexpressed strain Xd‐rDL‐WC2. (A) Comparison of the pigment in strains Xd‐rDL‐WC2 and Xd‐wc2 cultivated in 50 ml liquid YM medium for 24 and 28 h. (B) and (C) Volcano plots of gene expression of the cultures growing for 24 h (B) and 28 h (C). (D) Venn plot of down‐regulated and up‐regulated differentially expressed genes (DEGs). (E) Gene Ontology term enrichment of overlapping DEGs between the 24 h data set and 28 h data set. (F) KEGG pathway analysis of overlapping DEGs between the 24 h data set and 28 h data set. (G) Comparison between the transcriptomic data and the qRT‐PCR assay of the 12 selected genes. Clp1, Hypothetical protein; GH3, Glycoside hydrolase family 3 protein; ACP, Acid phosphatase; AbcT, ATP‐dependent transporter; Cry, Cryptochrome; Rho, Rhodopsin; BLUF, BLUF domain protein; CNH, Carbon‐nitrogen hydrolase; CryD, Cryptochrome DASH; AIF, Apoptosis‐inducing factor. (H) The growth phenotype of the gene‐disrupted strains cultivated on YM plates.

FIGURE 6

Detection of the interaction between XdWC2 and genes CrtI and CrtS. (A) Interactions between XdWC2 and the promoters of CrtI and CrtS detected by the Y1H assay. (B) The expression of CrtI and CrtS assayed by qRT‐PCR in the wild‐type strain CBS 6938, and the recombinant strains Xd‐Cwc2 and Xd‐rDL‐WC2 when cultivated for 120 h. (C) Gene expression of CrtI and CrtS assayed by qRT‐PCR in wild‐type strain CBS 6938, and the recombinant strains Xd‐wc2, Xd‐wc2‐CI, Xd‐wc2‐CS and Xd‐wc2‐CIS when cultivated for 120 h. (D) Gene expression of CrtI and CrtS assayed by qRT‐PCR in the wild‐type strain CBS 6938, and the recombinant strains Xd‐CI, Xd‐CS and Xd‐CIS when cultivated for 120 h. (E) Comparison of the carotenoid production between the wildtype strain CBS 6938 and the CrtI overexpressing strain Xd‐CI detected by HPLC. The peaks at 6.53 and 6.56 min denote the predicted HDCO. The content of HDCO in Xd‐CI is 2.75‐fold of that in CBS 6938. (F) Determination of HDCO by the TLC assay referring to a previous study (Chi et al., 2015). 1, astaxanthin; 2, HDCO; 3 and 4, keto derivatives; 5, β‐carotene. (G) Relative content of HDCO in the wild‐type strain, Xd‐wc2, Xd‐Cwc2 and Xd‐rDL‐WC2. The peak area per 10 mg dry cells weight (mAU·s/10 mg DCW) was used to denote the relative content of HDCO. (H) The content of β‐carotene, astaxanthin and HDCO detected by HPLC in the wild‐type strain CBS 6938, Xd‐CI, Xd‐CS and Xd‐CIS when cultivated for 120 h. (I) The content of β‐carotene, astaxanthin, and HDCO detected by HPLC in the wild‐type strain, Xd‐wc2, Xd‐wc2‐CI, Xd‐wc2‐CS and Xd‐wc2‐CIS when cultivated for 120 h.

FIGURE 7

Functional analysis of XdWC1. (A) Structure analysis of XdWC1 identifying functional domains. (B) Disruption of XdWC1 affects the production of astaxanthin, β‐carotene and HDCO. Wildtype, the CBS 6938 strain; Xd‐wc1, the XdWC1‐disrupted strain. (C) The expression of CrtYB, CrtI and CrtS assayed by qRT‐PCR in the wild‐type strain CBS 6938 and Xd‐wc1 when cultivated for 48 h. (D) Detection of the interaction between XdWC1 and XdWC2 by the Y2H assay. BD‐53 and AD‐T denote the positive control plasmids pGBKT7‐53 and pGADT7‐T, respectively. BD‐WC2/AD and BD/AD‐WC1 denote the negative controls. Cell suspension was diluted and dripped on the auxotrophic plates.