Expanding the biosynthesis spectrum of hydroxy fatty acids: unleashing the potential of novel bacterial fatty acid hydratases.

BACKGROUND: Hydroxy fatty acids represent an emerging class of compounds with promising applications in the chemical, medicinal and functional food sectors. The challenges associated with their chemical synthesis have spurred exploration of biological synthesis as an alternative route, particularly through the use of fatty acid hydratases. Fatty acid hydratases catalyse the regioselective addition of a hydrogen atom and a hydroxyl group from a water molecule to the carbon-carbon cis-double bond of unsaturated fatty acids to form hydroxy fatty acids. Despite having been discovered in the early 1960s, previous research has primarily focused on characterizing single fatty acid hydratase variants with a limited range of substrates. Comprehensive studies that systematically examine and compare the characteristics of multiple variants of fatty acid hydratases are still lacking. RESULTS: In this study, we employed an integrated bioinformatics workflow to identify 23 fatty acid hydratases and characterized their activities against nine unsaturated fatty acid substrates using whole-cell biotransformation assays. Additionally, we tested a dual-protein system involving two fatty acid hydratases of distinct regioselectivity and demonstrated its suitability in enhancing the biosynthesis of di-hydroxy fatty acids. CONCLUSIONS: Our study demonstrates that fatty acid hydratases can be classified into three subtypes based on their regioselectivity and provides insights into their preferred substrate structures. These understandings pave ways for the design of optimal fatty acid hydratase variants and bioprocesses for the cost-efficient biosynthesis of hydroxy fatty acids.

Tunable cell differentiation via reprogrammed mating-type switching.

This study introduces a synthetic biology approach that reprograms the yeast mating-type switching mechanism for tunable cell differentiation, facilitating synthetic microbial consortia formation and cooperativity. The underlying mechanism was engineered into a genetic logic gate capable of inducing asymmetric sexual differentiation within a haploid yeast population, resulting in a consortium characterized by mating-type heterogeneity and tunable population composition. The utility of this approach in microbial consortia cooperativity was demonstrated through the sequential conversion of xylan into xylose, employing haploids of opposite mating types each expressing a different enzyme of the xylanolytic pathway. This strategy provides a versatile framework for producing and fine-tuning functionally heterogeneous yet isogenic yeast consortia, furthering the advancement of microbial consortia cooperativity and offering additional avenues for biotechnological applications.

Proposal for reclassification of the species Hungatella xylanolytica as Lacrimispora xylanisolvens nom. nov. and transfer of the genus Hungatella to the family Lachnospiraceae.

Hungatella xylanolytica X5-1T is an anaerobic, xylan-fermenting bacterium first isolated from methane-producing cattle manure. Initially identified as Bacteroides xylanolyticus, this species was later reclassified as H. xylanolytica in 2019. Although this reclassification found support through Genome blast Distance Phylogeny analysis which placed H. xylanolytica X5-1T into the same clade as Hungatella effluvii DSM 24995T, it was contradicted by 16S rRNA gene phylogenetic analysis, which associated it with a set of misnamed Clostridium species later reassigned into the genus Lacrimispora. To ascertain its taxonomic position, comparative analyses were performed to re-examine the relationship between H. xylanolytica X5-1T and all species of the genera Hungatella and Lacrimispora. The ranges of 16S rRNA gene sequence similarity, average amino acid identity, and percentage of conserved protein prediction values were higher between H. xylanolytica X5-1T and species of the genus Lacrimispora than Hungatella. In addition, H. xylanolytica X5-1T was found to harbour genes and pathways conserved and exclusive to species within the genus Lacrimispora but not Hungatella. Essentially, in both the 16S rRNA gene phylogenetic tree and the core-genome phylogenomic tree, H. xylanolytica X5-1T clustered into the same clade as species of the genus Lacrimispora, distinct from species of the genus Hungatella. It is thus clear that H. xylanolytica X5-1T represents a species within the genus Lacrimispora, which we propose to reclassify as Lacrimispora xylanisolvens nom. nov. Finally, based on the results from the phylogenetic and comparative analyses, the genus Hungatella was transferred to the family Lachnospiraceae.

Lactiplantibacillus brownii sp. nov., a novel psychrotolerant species isolated from sauerkraut.

A Gram-stain-positive, rod-shaped, facultatively anaerobic and homofermentative strain, named WILCCON 0030T, was isolated from sauerkraut (fermented cabbage) collected from a local market in the Moscow region of Russia. Comparative analyses based on 16S rRNA gene sequence similarity and whole genome relatedness indicated that strain WILCCON 0030T was most closely related to the type strains Lactiplantibacillus nangangensis NCIMB 15186T, Lactiplantibacillus daoliensis LMG 31171T and Lactiplantibacillus pingfangensis LMG 31176T. However, the average nucleotide identity and digital DNA-DNA hybridization prediction values with these closest relatives only ranged from 84.6 to 84.9 % and from 24.1 to 24.7 %, respectively, and were below the 95.0 and 70.0% thresholds for species delineation. Substantiated by further physiological and biochemical analyses, strain WILCCON 0030T represents a novel species within the genus Lactiplantibacillus for which we propose the name Lactiplantibacillus brownii sp. nov. (type strain WILCCON 0030T=DSM 116485T=LMG 33211T).

Whole-Genome Sequence of Ligilactobacillus faecis WILCCON 0062, Isolated from Feces of a Wild Boar (Sus scrofa).

We report the whole genome of a strain of Ligilactobacillus faecis. The complete circular chromosome and plasmid of strain WILCCON 0062 were obtained through a combination of short- and long-read sequencing and may be used to derive unprecedented insights into the genome-level phylogeny and functional capacities of Ligilactobacillus faecis.

Ligilactobacillus ubinensis sp. nov., a novel species isolated from the wild ferment of a durian fruit (Durio zibethinus).

A Gram-stain-positive, rod-shaped, non-spore-forming, catalase-negative, urease-negative, homofermentative and facultatively anaerobic strain, named WILCCON 0076T, was isolated from a wild ferment of pieces of a 'Kampung' durian fruit collected on the island of Ubin (Pulau Ubin), Singapore. The durian had fallen to the ground from a durian tree (Durio zibethinus), on which a group of long-tailed macaques had been observed picking and eating the fruits. Comparative analyses of 16S rRNA gene sequences indicated that WILCCON 0076T potentially represented a novel species within the genus Ligilactobacillus, with the most closely related type strain being Ligilactobacillus agilis DSM 20509T (16S rRNA gene sequence similarity of 97.2 %). Average nucleotide identity and digital DNA-DNA hybridization prediction values were only 86.0% and 18.9 %, respectively. On the basis of the results of a polyphasic approach that included phylogenomic, chemotaxonomic and morphological analyses, we propose a novel species with the name Ligilactobacillus ubinensis sp. nov. (type strain WILCCON 0076T=DSM 114293T=LMG 32698T).

Development of destabilized mCherry fluorescent proteins for applications in the model yeast Saccharomyces cerevisiae.

Fluorescent proteins are widely used molecular reporters in studying gene expression and subcellular protein localization. To enable the monitoring of transient cellular events in the model yeast Saccharomyces cerevisiae, destabilized green and cyan fluorescent proteins have been constructed. However, their co-utilization is limited by an overlap in their excitation and emission spectra. Although red fluorescent protein is compatible with both green and cyan fluorescent proteins with respect to spectra resolution, a destabilized red fluorescent protein is yet to be constructed for applications in S. cerevisiae. To realize this, we adopted a degron-fusion strategy to prompt destabilization of red fluorescent protein. Specifically, we fused two degrons derived from Cln2, a G1-specific cyclin that mediates cell cycle transition, to the N- or C-terminus of mCherry to generate four destabilized fluorescent proteins that are soluble and functional in S. cerevisiae. Importantly, the four mCherry fluorescent proteins are highly differential with regards to fluorescence half-life and intensity, which provides a greater choice of tools available for the study of dynamic gene expression and transient cellular processes in the model yeast.

Synthetic biology toolkits and applications in Saccharomyces cerevisiae.

Synthetic biologists construct biological components and systems to look into biological phenomena and drive a myriad of practical applications that aim to tackle current global challenges in energy, healthcare and the environment. While most tools have been established in bacteria, particularly Escherichia coli, recent years have seen parallel developments in the model yeast strain Saccharomyces cerevisiae, one of the most well-understood eukaryotic biological system. Here, we outline the latest advances in yeast synthetic biology tools based on a framework of abstraction hierarchies of parts, circuits and genomes. In brief, the creation and characterization of biological parts are explored at the transcriptional, translational and post-translational levels. Using characterized parts as building block units, the designing of functional circuits is elaborated with examples. In addition, the status and potential applications of synthetic genomes as a genome level platform for biological system construction are also discussed. In addition to the development of a toolkit, we describe how those tools have been applied in the areas of drug production and screening, study of disease mechanisms, pollutant sensing and bioremediation. Finally, we provide a future outlook of yeast as a workhorse of eukaryotic genetics and a chosen chassis in this field.