Functional characterization of the Xanthophyllomyces dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors

Abstract

The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.

Figure 1. Synthesis of isoprenoids in X. dendrorhous.

Metabolite abbreviations: IPP (isopentenyl pyrophosphate), DMAPP (dimethylallyl pyrophosphate), GPP (geranyl pyrophosphate), FPP (farnesyl pyrophosphate) and GGPP (geranylgeranyl pyrophosphate). The arrows represent the catalytic step with the respective enzyme-encoding gene. Genes controlling the steps shown in the figure are written in italics: idi [Genbank: DQ235686, IPP-isomerase], crtE [Genbank: DQ012943, GGPP-synthase], FPS [Genbank: KJ140284, FPP-synthase], crtYB [Genbank: DQ016503, Phytoene-β-carotene synthase), crtI [Genbank: Y15007, Phytoene desaturase], crtS [Genbank: EU713462, astaxanthin synthase] and crtR [Genbank: EU884133, Cytochrome P450 reductase]. The three proposed model systems to achieve the synthesis of GGPP are shown: 1) first system, a FPP- and a GGPP- synthase are involved and act sequentially, 2) second system, only one GGPP synthase is involved and 3) third system, participation of the first and second system simultaneously.

Figure 2. X. dendrorhous and C. neoformans FPS and crtE deduced amino acid sequence analysis.

Seven conserved regions have been previously reported in prenyl transferase enzymes; among these are the two aspartic acid-rich motifs, FARM and SARM, and the CLD chain length domain, which is responsible for determining the resulting isoprene chain length. The sequence alignment shows similarity between the compared proteins, particularly for the aforementioned motifs. However, the seventh reported conserved region shows little or no agreement between the crtE and the FPS translated sequences. This was consistent for geranylgeranyl pyrophosphate synthases from other related organisms. Amino acid sequences were deduced from: X. dendrorhous FPS [Genbank: KJ140284] and crtE [Genbank: DQ012943.1]; C. neoformans FPS [Genbank: XP_571137] and crtE [Genbank: XP_572774].

Figure 3. Colony pigmentation of the X. dendrorhous wild-type strain and the FPS and crtE deletion and over-expressing transformants over time.

Micro-drops of strains UCD 67–385 wild-type (1), 385-FPS^{(+/+, +1)} (2), 385-crtE^{(+/+, +1)} (3), 385-FPS^(+/−) (4) and 385-crtE^(+/−) (5), were seeded on YM-agar plates and incubated at 22°C. Pictures were taken after 1 (Panel A), 3 (Panel B) and 5 (Panel C) days of cultivation. Pigmentation is apparent earlier in both of the gene over-expressing strains (2 and 3). The deletion mutant strains (4 and 5) showed similar pigmentation as the wild-type (1) strain after 3 days of cultivation, but at day 5, the 385-crtE^(+/−) strain was paler than the wild-type strain.

Figure 4. Transcript level changes in the over-expressing and deletion mutant strains versus the parental wild-type strain.

The FPS (A), crtE (B), crtS (C) and crtR (D) genes expression in transformant and wild-type strains was determined by RT-qPCR and normalized to the actin gene expression after 72 h of cultivation. The respective gene transcript level in the control (wild-type strain) was considered to be 100%. Values are the mean ± standard error of the mean (SEM) of three independent experiments. (*p≤0,01, **p≤0,05; Student's t test).