## 2 Result and Discussion
### Illustrating the Biosynthetic Pathway of Astaxanthin  
The candidate genes encoding the enzymes for the biosynthesis of astaxanthin were mined from the genome and transcriptome data. The mevalonate (MVA) pathway was found to be involved in the formation of isopentenyl pyrophosphate (IPP) (Figure 2A). The most genes involved in the MEP pathway were not found in transcriptomic data. Three enzymes were predicted to catalyze the formation of GGPP from IPP and DMAPP: Geranyl diphosphate synthase (GPPS), famesyl diphosphate synthase (FPPS), and geranylgeranyl diphosphate synthase (GGPPS). The genes encoding for the enzymes of phytoene synthase (CrtB), phytoene desaturase (CrtI), and lycopene cyclase (CrtY), which are involved in the biosynthesis of \(\beta\)-carotene from GGPP, were found to be consecutively fused into an open reading frame without stop codons (CrtIBY). Similar CrtIBY genes were also found in five other _Aurantiodotrium_ species, one of which was confirmed to catalyze \(\beta\)-carotene formation from GGPP [33]. The functional domains of CrtIBY of _Aurantiodotrium_ sp. SK4 and the alignment of different CrtIBY protein sequences were showed in Figure S6. A typical \(\beta\)-carotene hydroxylase (CrtZ) gene and three \(\beta\)-carotene ketolase (CrtO) genes were identified, which might be involved in converting \(\beta\)-carotene into astaxanthin. To reveal the regulation of astaxanthin biosynthesis at gene expression trends, the transcriptional patterns of five genes involved in the MVA pathway including 3-hydroxy-3-methylglutaryl coenzyme A synthase (_HMG-CoA syntthase_), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (_HMG-CoA reductase_), mevalonate kinase  
Figure 1: Overview of the fatty acid information in SK4. Fatty acid biosynthesis pathways (**A**), the expression trends of Type I fatty acid synthase and three subunits in the polyktetide synthase (PKS) pathway (**B**), relative fatty acid content (**C**) and percentage (**D**) of C14:0, C16:0, docosapentaenoic acid (DPA) and docosabexaenoic acid (DHA). \(\omega\)-3: \(\omega\)-3 desaturase; EL: elongase; KS: 3-ketoacyl synthase; KR: 3-ketoacyl-ACP reductase; ER: enoyl reductase; DH: dehydrase/isomerase. Data are shown as mean \(\pm\) SD, n = 3. (MVA kinase)_, phosphomevalonate kinase (_PMVA kinase_) and the _CrtlBY_ involved in the conversion of GGPP to \(\beta\)-carotene were detected at eight different time points by qRT-PCR (Figure 2B). All of the five genes reached expression peak at 36 h. The MVA pathway genes were upregulated at the early stages of cell development, but down-regulated when the cells were in the stationary phase. In contrast, _CrtlBY_ kept a relatively stable expression trend at stationary and decline phases. To further correlate the expression of the genes with the carotenoid composition, the dynamic evolution of carotenoids were analyzed. _Aurantiolchytrium_ sp. SK4 was found to consist of five main carotenoids: \(\beta\)-carotene, echienone, 3-OH echienone, canthaxanthin, adorinubin (phenicoxanthin), and astaxanthin (Figure 2C,D). \(\beta\)-Carotene and canthaxanthin were the major carotenoids, with the other three as minor ones. Since \(\beta\)-carotene was the predominant carotenoid (>60% of total carotenoids), the ketolation and hydroxylation of it should be the limiting steps for astaxanthin formation. The biosynthesis of astaxanthin in _Aurantiolchytrium_ sp. SK4 was proposed as shown in Figure 2A. Previously, overexpression of heterologous hemoglobin in _Aurantiolchytrium_ was found to enhance astaxanthin production [17], indicating that this microorganism could be manipulated to further enhance the production of astaxanthin and possibly other metabolites, such as DHA and carotenoids by the expression of some rate-limiting enzymes. Figure 2: The information on carotenoid biosynthesis in Aurantiochytrium sp. SK4.