Characterisation and substrate binding modes of exopolygalacturonase PGQ1 from Saccharobesus litoralis.

Among the enzymes required for the efficient utilisation of pectin is polygalacturonase. Saccharobesus litoralis harbours two polygalacturonases belonging to glycoside hydrolase family 28 (GH28). One of them, PGQ1, cleaved polygalacturonate exolytically at the non-reducing end into monomeric units. It was most active at 60 °C and pH 8, with Km and kcat values of 2.3 mg/ml and 6.4 s-1 respectively. Its homology model of a right-handed parallel ß-helix core consisted of Asp297 as the general acid and either Asp276 or Asp298 as the general base. By inferring the substrate binding modes at the -1 and +1 subsites from known crystal structures, a hexagalacturonate could be docked into the highly electropositive binding cleft. Interestingly, while no residues were present in the vicinity to make up the +2 and +4 subsites, Arg361 and Arg430 could readily bind to the carboxyl groups of the galacturonates at the +3 and +5 subsites respectively. Structural comparison suggested that this binding pattern with missing subsites might be unique to closely related exopolygalacturonases. As S. litoralis grew much more slowly on extracellular galacturonate due to the lack of a transporter for the monosaccharide, PGQ1 probably functioned in the periplasm to help degrade oligopectates completely.Communicated by Ramaswamy H. Sarma.

Genome Sequence of Vibrio sp. Strain CCB-PB317, Isolated from a Malaysian Mangrove, and Its Arsenic Resistance Mechanisms.

Vibrio sp. strain CCB-PB317 with potential arsenic detoxification was isolated from a mangrove in Pulau Betong, Malaysia. Here, we report a draft genome sequence of strain CCB-PB317, which comprised 5,157,574 bp with a G+C content of 44.9%. The genome contains genes related to an arsenic resistance system coupled with glycolytic metabolism.

Genome sequence data of Bacillus sp. CCB-MMP212 isolated from Malaysian mangrove: A potential strain in arsenic resistance with ArsI, C•As lyase.

Bacillus sp. CCB-MMP212 is a Gram-positive bacterium isolated from mangrove sediment in Matang Perak, Malaysia (4.85496°E, 100.73495°N). Genome sequencing was performed using the Oxford Nanopore and Illumina platforms. The assembled genome was annotated using the rapid annotation subsystem technology server (RAST) (rast.nmpdr.org). The genome size of the Bacillus sp. CCB-MMP212 was 6,151,644 base pairs (bp) with a G+C content of 34.75%. The genome includes 6,311 coding sequences and 58 RNAs. The sequence has been deposited at Genbank with the accession number of JALDQE000000000. Interestingly, an arsenic resistance (ars) operon consisted of arsenic resistance operon repressor (arsR), ACR3 family arsenite efflux transporter (arsB), and arsenate reductase (arsC) genes were found in the genome. In addition, the arsenic inducible gene (arsI), which encoded a dioxygenase with C•As lyase activity, was also found in the ars operon. The enzyme is crucial for the methylation of methylarsonous acid [MAs(III)] and trivalent roxarsone [Rox(III)]. This dataset reveals the genetic ability of this strain in arsenic resistance. To the best of our knowledge, the arsI encoding C•As lyase is rarely reported within the genus Bacillus. Therefore, the dataset presented in this manuscript provides further insight into the arsenic resistance mechanisms of the genus Bacillus.

Comparative Genomic Analyses of the Genus Photobacterium Illuminate Biosynthetic Gene Clusters Associated with Antagonism.

The genus Photobacterium is known for its ecophysiological versatility encompassing free-living, symbiotic, and pathogenic lifestyles. Photobacterium sp. CCB-ST2H9 was isolated from estuarine sediment collected at Matang Mangrove, Malaysia. In this study, the genome of CCB-ST2H9 was sequenced, and the pan-genome of 37 Photobacterium strains was analysed. Phylogeny based on core genes showed that CCB-ST2H9 clustered with P. galatheae, forming a distinct clade with P. halotolerans, P. salinisoli, and P. arenosum. The core genome of Photobacterium was conserved in housekeeping functions, while the flexible genome was well represented by environmental genes related to energy production and carbohydrate metabolism. Genomic metrics including 16S rRNA sequence similarity, average nucleotide identity, and digital DNA-DNA hybridization values were below the cut-off for species delineation, implying that CCB-ST2H9 potentially represents a new species. Genome mining revealed that biosynthetic gene clusters (BGCs) involved in producing antimicrobial compounds such as holomycin in CCB-ST2H9 could contribute to the antagonistic potential. Furthermore, the EtOAc extract from the culture broth of CCB-ST2H9 exhibited antagonistic activity against Vibrio spp. Intriguingly, clustering based on BGCs profiles grouped P. galatheae, P. halotolerans, P. salinisoli, P. arenosum, and CCB-ST2H9 together in the heatmap by the presence of a large number of BGCs. These BGCs-rich Photobacterium strains represent great potential for bioactive secondary metabolites production and sources for novel compounds.

Enhanced production of polyhydroxyalkanoate with manipulable and reproducible 3-hydroxyvalerate fraction by high alcohol tolerant Cupriavidus malaysiensis USMAA2-4 transformant.

The domination of high-cost organic acids over other 3-hydroxyvalerate (3HV) precursors due to the wide preference among polyhydroxyalkanoates (PHA)-producing bacteria has limited the development of diverse poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production processes. 1-pentanol is a low-cost 3HV precursor but is rarely employed due to the relatively low tolerance among PHA-producing bacteria. This study demonstrated P(3HB-co-3HV) production with manipulable and reproducible 3HV composition and 3HV yield from palm olein and 1-pentanol. Cupriavidus malaysiensis USMAA2-4ABH16 is the transformant strain with acquired lipase genes that retains the high tolerance towards 1-pentanol of its wild-type, with a preference for 1-pentanol over valeric acid indicated by the sixfold higher 3HV yield than that from valeric acid. C. malaysiensis USMAA2-4ABH16 was able to tolerate up to 0.15 wt% C 1-pentanol. Upon optimization using response surface methodology, 0.41‒0.52 g/g P(3HB-co-3HV) yield and 72‒89 wt% PHA content was achieved for 7, 9, 12 and 16 mol% 3HV, with 3HV yields of 0.30 g/g, 0.26 g/g, 0.23 g/g and 0.23 g/g, respectively. Up-scaling batch production by adopting the optimized concentrations of substrates for 12 mol% 3HV resulted in reproducible 3HV composition and 3HV yield on a 120-fold larger scale. The P(3HB-co-12 mol% 3HV) produced displayed higher flexibility than polypropylene and P(3HB-co-3HV) produced from different carbon sources. C. malaysiensis USMAA2-4ABH16 could be practically applicable for sustainable and economically feasible P(3HB-co-3HV) production on an industrial scale from used palm olein with relatively similar oleic acid content with palm olein and 1-pentanol, with higher 3HV compositions achievable through fed-batch strategies owing to its high 1-pentanol tolerance.

Genes for degradation and utilization of uronic acid-containing polysaccharides of a marine bacterium Catenovulum sp. CCB-QB4.

BACKGROUND: Oligosaccharides from polysaccharides containing uronic acids are known to have many useful bioactivities. Thus, polysaccharide lyases (PLs) and glycoside hydrolases (GHs) involved in producing the oligosaccharides have attracted interest in both medical and industrial settings. The numerous polysaccharide lyases and glycoside hydrolases involved in producing the oligosaccharides were isolated from soil and marine microorganisms. Our previous report demonstrated that an agar-degrading bacterium, Catenovulum sp. CCB-QB4, isolated from a coastal area of Penang, Malaysia, possessed 183 glycoside hydrolases and 43 polysaccharide lyases in the genome. We expected that the strain might degrade and use uronic acid-containing polysaccharides as a carbon source, indicating that the strain has a potential for a source of novel genes for degrading the polysaccharides. METHODS: To confirm the expectation, the QB4 cells were cultured in artificial seawater media with uronic acid-containing polysaccharides, namely alginate, pectin (and saturated galacturonate), ulvan, and gellan gum, and the growth was observed. The genes involved in degradation and utilization of uronic acid-containing polysaccharides were explored in the QB4 genome using CAZy analysis and BlastP analysis. RESULTS: The QB4 cells were capable of using these polysaccharides as a carbon source, and especially, the cells exhibited a robust growth in the presence of alginate. 28 PLs and 22 GHs related to the degradation of these polysaccharides were found in the QB4 genome based on the CAZy database. Eleven polysaccharide lyases and 16 glycoside hydrolases contained lipobox motif, indicating that these enzymes play an important role in degrading the polysaccharides. Fourteen of 28 polysaccharide lyases were classified into ulvan lyase, and the QB4 genome possessed the most abundant ulvan lyase genes in the CAZy database. Besides, genes involved in uronic acid metabolisms were also present in the genome. These results were consistent with the cell growth. In the pectin metabolic pathway, the strain had genes for three different pathways. However, the growth experiment using saturated galacturonate exhibited that the strain can only use the pathway related to unsaturated galacturonate.

Enhanced degradation of polyhydroxyalkanoates (PHAs) by newly isolated Burkholderia cepacia DP1 with high depolymerase activity.

The contribution of microbial depolymerase has received much attention because of its potential in biopolymer degradation. In this study, the P(3HB) depolymerase enzyme of a newly isolated Burkholderia cepacia DP1 from soil in Penang, Malaysia, was optimized using response surface methodology (RSM). The factors affecting P(3HB) depolymerase enzyme production were studied using one-variable-at-a-time approach prior to optimization. Preliminary experiments revealed that the concentration of nitrogen source, concentration of carbon source, initial pH and incubation time were among the main factors influencing the enzyme productivity. An increase of 9.4 folds in enzyme production with an activity of 5.66 U/mL was obtained using optimal medium containing 0.028% N of di-ammonium hydrogen phosphate and 0.31% P(3HB-co-21%4HB) as carbon source at the initial pH of 6.8 for 38 h of incubation. Moreover, the RSM model showed great similarity between predicted and actual enzyme production indicating a successful model validation. This study warrants the ability of P(3HB) degradation by B. cepacia DP1 in producing higher enzyme activity as compared to other P(3HB) degraders being reported. Interestingly, the production of P(3HB) depolymerase was rarely reported within genus Burkholderia. Therefore, this is considered to be a new discovery in the field of P(3HB) depolymerase production.