Ye, Yu-Qi

ABSTRACT Amino acid racemases are pivotal for d -amino acid (DAA) biosynthesis with wide-ranging biotechnological applications, yet their industrial deployment is hindered by narrow substrate specificity and instability. Here, we report the discovery of Halocola ammonii gen. nov., sp. nov. DA487 T , a novel taxon within the proposed family Halocolacceae fam. nov. (order Flavobacteriales ), isolated from hypersaline sediments. Genomic analysis revealed a robust DAA metabolic network, including a putative broad-specificity racemase RacX. Biochemical characterization demonstrated RacX’s exceptional catalytic efficiency ( k cat /K m = 151.2 s −1 mM −1 for l -Lys, k cat /K m = 17.8 s −1 mM −1 for d -Lys) and broad substrate spectrum (15/17 tested l -amino acids). Homology modeling and mutagenesis identified Ala79 and Cys193 as putative catalytic residues, based on structural conservation with EcL-DER. Remarkably, the A79C variant enhanced the reverse reaction efficiency ( d -Lys → l -Lys) by 44%, effectively shifting the enzyme’s catalytic bias and the resulting steady-state ratio of enzyme-bound species. Computational docking suggested that Asn80, Thr81, Asn121, and Thr124 may modulate substrate binding, though experimental structural validation is required. The thermostability-lability tradeoff ( T 1/2 55°C   =    70 min ) highlights targets for protein engineering. Our findings not only expand the phylogenetic diversity of microbial racemases but also identify a promising biocatalyst candidate for industrial DAA production. IMPORTANCE Microbial adaptations to extreme environments serve as a valuable source of novel biocatalysts with potential for sustainable industrial applications. In this study, we characterized Halocola ammonii DA487ᵀ, a halophilic bacterium representing the novel family Halocolaceae within the order Flavobacteriales , and identified a broad-specificity amino acid racemase, RacX. RacX demonstrates exceptional catalytic efficiency ( k cat /K m up to 151.2 s⁻¹ mM⁻¹ for l -Lys) across multiple amino acids and exhibits remarkable stability under neutral and alkaline conditions (pH 7.0–9.0)—properties intrinsically linked to its high-salt ecological niche. Unlike most known racemases from neutrophilic organisms, RacX originates from an understudied phylogenetic lineage and displays unique mechanistic features, including a strong innate bias toward d -amino acid (DAA) production that can be rationally reprogrammed via single-residue substitution (e.g., A79C). These functional and evolutionary insights, combined with its halotolerance and broad substrate scope, position RacX as a promising and engineerable biocatalyst for industrial processes requiring operation under high-salt or alkaline conditions, such as the synthesis of DAA precursors for antibiotics.

ABSTRACT The phylum Gemmatimonadota is widespread but rarely cultured and, in fact, there are only six described species isolated from soil, freshwater, and wastewater treatment. However, no isolates of Gemmatimonadota from marine environment have been described; thus, little is known about the physiology and metabolism of members of the marine lineages. In this study, four novel facultatively anaerobic bacterial strains belonging to Gemmatimonadota were isolated from marine sediments collected from Xiaoshi Island in Weihai, China, using an aerobic enrichment method. The integrated results of phylogenetic and phenotypic characteristics supported that these four strains represent one novel species in a novel genus, for which the name Gaopeijia maritima gen. nov., sp. nov. is proposed, as the first representative of novel taxa, Gaopeijiales ord. nov., Gaopeijiaceae fam. nov. in the class Longimicrobiia. Gaopeijiales was detected in 22,884 out of 95,549 amplicon data sets, mainly from soil. However, the highest mean relative abundances were in sponge (0.7%) and marine sediment (0.35%), showing salt-related character. Most of the Gaopeijiales subgroups potentially belong to the rare bacterial biosphere. The aerobic enrichment in this study could significantly increase the relative abundance of Gaopeijiales (from 0.37% to 2.6%). Furthermore, the metabolic capabilities inferred from high-quality representative Gaopeijiales genomes/MAGs suggest that this group primarily performs chemoorganoheterotrophic metabolism with facultatively anaerobic characteristics and possesses various secondary metabolite biosynthesis gene clusters (BGCs), mirroring those observed in the four novel strains. IMPORTANCE Despite rapid advances in molecular and sequencing technologies, obtaining pure cultures remains a crucial research goal in microbiology, as it is essential for a deeper understanding of microbial metabolism. Gemmatimonadota is a widespread but rarely cultured bacterial phylum. Currently, there are only six cultured strains of this interesting group, all isolated from non-marine environments. Little is known about the physiology and metabolism of members of the marine lineages. Here we isolated and characterized four novel marine strains, and proposed a new order Gaopeijiales within Gemmatimonadota . Furthermore, the global distribution, environmental preference, and metabolic potential of Gaopeijiales are analyzed using public data. Our work enriches the resources available for the under-represented phylum Gemmatimonadota and provides insights into the physiological and metabolic characteristics of the marine lineage ( Gaopeijiales ) through culturology and omics.

A novel slightly halophilic, aerobic, and Gram-stain-negative strain, designated as CH-27T, was isolated during a bacterial resource investigation of intertidal sediment collected from Xiaoshi Island in Weihai, PR China. Cells of strain CH-27T were rod-shaped with widths of 0.3–0.6 µm and lengths of 2.0–11.0 µm. Strain CH-27T grew optimally at 37 °C, pH 7.0 and with 2.0 % (w/v) NaCl. Catalase activity was weakly positive and oxidase activity was positive. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CH-27T was most related to Marinihelvus fidelis KCTC 92639T (93.6 %), followed by Wenzhouxiangella marina MCCC 1K00261T (92.0 %). Based on genome comparisons between strain CH-27T and M. fidelis KCTC 92639T, the average amino acid identity was 63.6 % and the percentage of conserved proteins was 48.3 %. The major cellular fatty acid of strain CH-27T (≥10 %) was iso-C15 : 0 and the sole respiratory quinone was quinone-8. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, and aminophospholipid. The DNA G+C content was 62.7 mol%. Based on comprehensive analysis of its phylogenetic, physiological, biochemical, and chemotaxonomic characteristics, strain CH-27T represents a novel species in a novel genus, for which the name Elongatibacter sediminis gen. nov., sp.nov. is proposed. The type strain is CH-27T (=MCCC 1H00480T=KCTC 8011T).