## 3 Results
### C40 Carotenoid biosynthetic Pathways in the Antarctic Ocean as Elucidated by Metagenomic Analysis  
3.1 Assessment of the Prevalence of C40 Astaxanthin Biosynthesis Genes in Antarctic Microbial Communities  
Astaxanthin, a keto-carotenoid of significant biotechnological interest, is synthesized via the C40 carotenoid biosynthetic pathway [28]. Microbial inhabitants of the Antarctic Ocean utilize unique enzymatic processes to produce astaxanthin, which confers survival advantages under extreme conditions, such as high UV radiation and low temperatures [29]. Our metagenomic analysis focused on the genes associated with the astaxanthin biosynthetic pathway, which extends from the formation of phytoene to the final product, astaxanthin (Figure 4). Notable genes encoding \(\beta\)-carotene hydroxylase (CrtZ) and \(\beta\)-carotene ketolase (CrtW), which catalyzes the final step in the astaxanthin biosynthesis, were more abundant or uniquely present at 25 m.  
Figure 2: Carotenoid biosynthesis pathway genes identified from metagenomic data annotated with KEGG ID (ko00906). Carotenogenic genes found at 16 m are highlighted in red and those at 25 m in purple. The pathway diagram was sourced from the KEGG website. Figure 4: Astaxanthin biosynthesis from lycopene as revealed in metagenomic data. Each enzymatic step was annotated with its corresponding KEGG (Kyoto Encyclopedia of Genes and Genomes) identifier. Color coding was employed to signify enzymes with a higher gene abundance at specific depths. The absence of genes encoding enzymes was denoted by ’NA’, indicating a lack of detectable genes at the respective depths. The numbers enclosed in boxes reflect carotenoid gene abundance quantified by TPM (transcripts per million), offering a quantitative measure of gene abundance. Figure 3: Distribution of carotenoid biosynthesis pathway genes at depths of 16 and 25 m. (**A**) Profile of gene distribution related to the carotenoid biosynthesis pathway at 16 m. (**B**) Profile of carotenoid gene distribution at 25 m. (**C**) Genes showing differentially higher abundance at 16 m as determined by TPM. (**D**) Genes with differentially higher abundance at 25 m, also quantified using TPM. 3.2 Assessment of the Prevalence of C40 Myxol Biosynthesis Genes in the Microbiota of the Antarctic Ocean  
Myxol, a carotenoid derivative, undergoes synthesis via the C40 carotenoid biosynthetic pathway, involving distinct enzymatic steps that culminate in the formation of myxoxanthophyll--a glycosylated variant of myxol. This biosynthetic route is pivotal for microbial communities in the extreme environments of the Antarctic Ocean, aiding their adaptation and ecological prowess [30]. In our metagenomic study, we identified genes encoding key enzymes involved in converting typical carotenoid precursors into the structurally distinct myxol compounds (Figure 5). Carotenogenic genes such as genes encoding \(\beta\)-carotene hydroxylase (CrtR) and \(\beta\)-carotene/zeaxanthin 4-ketolase (CrtW or BKT) were prevalent at both depths. However, the gene encoding O-glycostransferase (CruG), crucial for sugar addition in myxol biosynthesis, was conspicuously absent at both depths. 3.3 Assessment of the Prevalence of C40 Okenone Biosynthesis Genes in the Antarctic Ocean Microbiomes  
Okenone, a ketocarotenoid with a unique pink-to-purple color, is synthesized through the C40 carotenoid biosynthetic pathway. This pathway plays a crucial role in the Antarctic Ocean's microbiomes, representing a complex biochemical adaptation essential for survival in harsh photic environments [31]. The synthesis of oxenone involves a sequence of enzymatic reactions starting from lycopene. These reactions include the specific introduction  
Figure 5: Biosynthesis of oxenone and myxol as revealed by metagenomic analysis. A color-coded scheme highlights carotenoid genes with increased abundance at specific depths. ’NA’ denotes the absence of detectable genes for certain enzymes at the respective depths.