Astaxanthin (AXT) is a red ketocarotenoid compound (3,3′-dihydroxy-β,β-carotene-4,4′-dione) with 40 carbon atoms, and is well known for its much higher antioxidant activity than coenzyme Q10 or some vitamins (such as C and E) (Zhou et al., 2024). Owing to the extreme antioxidant capacity, AXT plays critical roles in the antiaging, anti-inflammatory, anti-tumor and immune enhancement, etc., and currently wildly used in the aquaculture, cosmetic, food, and pharmaceutical industries (Elbahnaswy and Elshopakey, 2024; Van Doan et al., 2023). By now, chemical synthesis still dominates the current commercially available AXT, but such product has a lower antioxidant capacity (possibly because it is a racemic mixture, containing three isomers) than its natural counterpart and not been approved for human consumption (Zhang et al., 2020a). Hence, increasing studies have focused on the biological production of astaxanthin owing to the growing demand for natural products.

AXT is usually found in crabs, fish, or shrimps in our daily life. Nevertheless, these organisms cannot indigenously synthesize AXT and must assimilate it through diet (Wan et al., 2021). AXT is biochemically derived from oxidation of β-carotene and naturally produced by certain microalgae, yeast, and bacteria, whereas higher organisms do not possess the capacity to synthesize AXT de novo (Mussagy et al., 2023; Zhou et al., 2024). For mass-producing AXT, the microalgae Haematococcus pluvialis and the red-yeast Phaffia rhodozyma (synonym Xanthophyllomyces dendrorhous) have been used for decades (Fan et al., 2021; Mussagy et al., 2023; Zhang et al., 2020a; Zhou et al., 2024). Nevertheless, Haematococcus pluvialis has slow growth rate and suffers high risk to be contaminated, and both of the eukaryotic microorganisms have thick cell walls, resulting in increasing cost of downstream extraction efficiency and limited production capacity (Saini and Keum, 2018). With the development of synthetic biology, bacterial model microorganisms that are fast-growing with convenience for the downstream extraction are adopted as hosts to produce AXT (Wan et al., 2021). For example, the highest reported astaxanthin production by using the engineered Escherichia coli and Yarrowia lipolytica can achieve 1.18 g/L and 3.3 g/L, respectively (Zhou et al., 2024). Bacteria such as E. coli have weak cell wall, making extraction from this host more amenable (Zhang et al., 2020a). In addition, many improvements in AXT production has been made by the use of enzymes with high catalytic activities from various sources including bacteria (Mussagy et al., 2023; Zhang et al., 2020a; Zhou et al., 2024). However, AXT biosynthesis genes are rarely explored in bacteria.

So far, a few native bacterial AXT sources have been reported and they are usually halotolerant, such as Brevundimonas spp. (Asker, 2017; Asker et al., 2007b; Liu et al., 2020; Tao et al., 2006), Erythrobacter sp.(Asker et al., 2018), Halobacterium sp. (Calo et al., 1995), Paracoccus spp.(Asker et al., 2007b; Ide et al., 2012), Planococcus sp. (Sayed et al., 2023), Sphingomicrobium (S.) astaxanthinifaciens (Shahina et al., 2013a), and Sphingomonas astaxanthinifaciens (Asker et al., 2009; Asker et al., 2007b). In addition, AXT biosynthesis genes, i.e. crtW (coding beta-carotene/zeaxanthin 4-ketolase) and crtZ (beta-carotene 3-hydroxylase), were also identified in Altererythrobacter ishigakiensis (Shi et al., 2016) and other bacteria (Siddaramappa et al., 2018). It is notable that all these bacteria belong to the class Alphaproteobacteria except Planococcus sp. in Bacilli according to the List of Prokaryotic names with Standing in Nomenclature (LPSN, https://lpsn.dsmz.de/genus/sphingomicrobium) (Parte et al., 2020).

In this study, two marine bacteria, designated XHP0235T and XHP0239T, were isolated from coastal zone of China, which were identified as novel species in the genus Sphingomicrobium and able to synthetize AXT and other carotenoid compounds. Further genomic analysis revealed the crtW and crtZ were present in many taxonomic relatives of these two strains. Hence, polyphasic taxonomic study and comparative genomics analysis were performed to characterize strains XHP0235T and XHP0239T and to explore the diversity and evolution of the AXT biosynthesis gene in the family Sphingomonadaceae.