Discovery of Geranylgeranyl Pyrophosphate Synthase (GGPPS) Paralogs from Haematococcus pluvialis Based on Iso-Seq Analysis and Their Function on Astaxanthin Biosynthesis

Abstract

Haematococcus pluvialis is widely distributed in the world and well known as the richest natural source of astaxanthin that is a strong antioxidant with excellent commercial value. The pathway of astaxanthin biosynthesis in H. pluvialis has been documented as an enzymatic reaction. Several enzymes have been reported, but their isoforms or homologs have not been investigated genome-wide. To better understand the astaxanthin biosynthesis pathway in H. pluvialis, eight candidates of the geranylgeranyl pyrophosphate synthase gene (HpGGPPS) predicted from Iso-seq data were isolated in this study. The length of coding region of these candidates varied from 960 bp to 1272 bp, composing of 7–9 exons. The putative amino acids of all candidates composed the signature domain of GGPPS gene. However, the motifs in the domain region are varied, indicating different bio-functions. Phylogenetic analysis revealed eight candidates can be clustered into three groups. Only two candidates in Group1 encode the synthase participating in the astaxanthin formation. The yield of astaxanthin from these two candidates, 7.1 mg/g (DW) and 6.5 mg/g (DW) respectively, is significant higher than that from CrtE (2.4 mg/g DW), a GGPPS gene from Pantoea ananatis. This study provides a potential productive pathway for astaxanthin synthesis.

Identification and Functional Analysis of Two Novel Genes—Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)—from Aurantiochytrium limacinum Significantly Enhance De Novo β-Carotene Biosynthesis in Escherichia coli

“…AlGGPPS functioned in β-carotene biosynthesis and performed better than the original CrtE gene of Pantoea ananatis with a higher β-carotene yield. This was consistent with the previously reported GGPPS gene of Haematococcus pluvialis with higher synthase activity, leading to higher carotenoid level [26]. It was proposed that high GGPPS activity may lead to a shortage of DMAPP and narrow the chance of carotenoid biosynthesis [24].…”

“…Thereby, IDI and GGPPS define the availability of IPP, DMAPP and GGPP, functioning as the key regulatory nodes by directing metabolic flux to carotenoid biosynthesis. The two key precursor synthases have been derived from different species [24][25][26][27][28][29], and it has proved to be an effective strategy to strengthen DMAPP and GGPP supply through overexpression of IDI or/and GGPPS, diverting the metabolite flow towards carotenoid biosynthesis [8,[29][30][31].…”

Transcriptomic and metabolomic analyses revealed regulation mechanism of mixotrophic Cylindrotheca sp. glycerol utilization and biomass promotion

“…Fucoxanthin was quantified with a HPLC (SHIMADZU, LC_2030C 3D, Japan) equipped with a reverse phase column (YMC, Carotenoid 5 µm, Japan) and a commercial standard (SolarBio, Fucoxanthin, China). Absorbance was recorded at 450 nm and elution program was previously reported by Huang et al with minor modifications [ 48 ]. In this study, organic solvent (methanol/isopropanol, 4:1) in 100% was maintained from 10 to 15 min and the whole procedure finished in 20 min.…”

The Histone Acetyltransferase HpGCN5 Involved in the Regulation of Abiotic Stress Responses and Astaxanthin Accumulation in Haematococcus pluvialis

“…The amplification was performed on the Bio-Rad T100 thermal cycler (Bio-Rad, United States) and the condition was as follows: initial denaturation at 98°C for 2 min; 35 cycles of 98°C for 10 s, 60°C for 10 s, 72°C for 1.5 min; and a final extension at 72°C for 5 min. Agarose gel electrophoresis, PCR products purification, T-A cloning including vector construction, E. coli transformation, colony PCR identification, and sequencing were performed as previously reported (Huang et al, 2019 ).…”