Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module.

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

Structure of the microbial carboxypeptidase T complexed with the transition state analog N-sulfamoyl-l-lysine.

Carboxypeptidase T (CPT) from Thermoactinomyces vulgaris (EC 3.4.17.18) has a broad substrate specificity, the mechanism of which remains unclear. It cleaves off arginine residues by 10, and lysine residues by 100 times worse than hydrophobic leucine residues despite the presence of negatively charged Asp260 at the bottom of the primary specificity pocket. To study the relationship between the structure and specificity the 3D structure of CPT in complex with the stable transition state analog N-sulfamoyl-l-lysine (SLys) was determined in which the S-atom imitates the sp3-hybridized carbon in the scissile-bond. Crystals grown in microgravity has the symmetry of space group P6322. The present complex structure was compared with the previously reported complex structure of CPT and N-sulfamoyl-L-arginine (SArg). The location/binding of SLys in the active site of CPT very closely resembled that of SArg, and the positively charged N-atom of SLys was at the same position as the corresponding positively charged N-atom of SArg. The SLys complex is stabilized by the hydrogen bond between the nitrogen atom and OH-group of Thr257. The contact areas of the residues Tyr255, Leu211, and Thr262 with SLys were reduced in comparison with the same of SArg. This difference in bonding of SArg and SLys side chains in the primary specificity pocket induces shifts differences within the catalytic center (especially Tyr255-O20 and S18-Arg129 N1 gap) that may influence the enzyme's catalytic reaction. Therefore, this information may be useful for the design of carboxypeptidases with improved selectivity towards Arg/Lys for biotechnological applications.

The complete genome sequence of the thermophilic bacterium Laceyella sacchari FBKL4.010 reveals the basis for tetramethylpyrazine biosynthesis in Moutai-flavor Daqu.

The genus Laceyella consists of a thermophilic filamentous bacteria. The pure isolate of Laceyella sacchari FBKL4.010 was isolated from Moutai-flavor Daqu, Guizhou Province, China. In this study, the whole genome was sequenced and analyzed. The complete genome consists of one 3,374,379-bp circular chromosome with 3,145 coding sequences (CDSs), seven clustered regularly interspaced short palindromic repeat (CRISPR) regions of 12 CRISPRs. Moreover, we identified that the genome contains genes encoding key enzymes such as proteases, peptidases, and acetolactate synthase (ALS) of the tetramethylpyrazine metabolic pathway. Metabolic pathways relevant to tetramethylpyrazine synthesis were also reconstructed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database. Annotation and syntenic analyses using antiSMASH 4.0 also revealed the presence of two gene clusters in this strain that differ from known tetramethylpyrazine synthesis clusters, with one encoding amino acid dehydrogenase (ADH) and the other encoding transaminase in tetramethylpyrazine metabolism. The results of this study provide flavor and genomic references for further research on the flavor-producing functions of strain FBKL4.010 in the Moutai liquor-making process.

Thermoactinomyces khenchelensis sp. nov., a filamentous bacterium isolated from soil sediment of a terrestrial hot spring.

A novel thermophilic filamentous bacterium, designated strain T36(T), was isolated from soil sediment sample from a hot spring source collected in Khenchela province, Algeria. Strain T36(T) was identified as a member of the genus Thermoactinomyces by a polyphasic approach. Strain T36(T) was observed to form white aerial mycelium and non-coloured to pale yellow substrate mycelium, both producing endospores, sessile or borne by short sporophores. The optimum growth temperature and pH were found to be 37-55 °C and 7.0-9.0, respectively and the optimum NaCl concentration for growth was found to be 0-7 % (w/v). The diagnostic diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid. The predominant menaquinone of strain T36(T) was identified as MK-7 (H0). The major fatty acids were found to be iso-C15:0 and iso-C17:0. The phospholipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphoglycolipid. The chemotaxonomic properties of strain T36(T) are consistent with those shared by members of the genus Thermoactinomyces. 16S rRNA gene sequence analysis indicated that the sequence similarities between strain T36(T) and Thermoactinomyces species with validly published names were less than 98 %. Based on the combined genotypic and phenotypic evidence, it is proposed that strain T36(T) should be classified as representative of a novel species, for which the name Thermoactinomyces khenchelensis sp. nov. is proposed. The type strain is T36(T) (=DSM 45951(T) = CECT 8579(T)).

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and ß-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

Inoculation of food waste with the thermo-tolerant lipolytic actinomycete Thermoactinomyces vulgaris A31 and maturity evaluation of the compost.

The ability of the thermo-tolerant lipolytic actinomycete, Thermoactinomyces vulgaris A31, to efficiently decompose food waste into mature compost was studied. Using a range of chemical parameters (pH, total organic carbon content (TOC), total nitrogen content, C/N ratio), CO(2) evolution, enzymatic activities (dehydrogenase, polyphenol oxidase, urease) and germination assays, the composition, stability and maturity of the compost produced were assessed. Inoculation reduced crude fat and decreased the maturation time of the compost when compared with the control. TOC, C/N ratio, CO(2) evolution, and enzymatic activities (dehydrogenase, polyphenol oxidase, urease) decreased, pH, total nitrogen content, germination rate, and germination index increased. The dehydrogenase, polyphenol oxidase, and urease activities were shown to be useful indicators for the stability of food waste composts. Based on germination assays, the food waste composts were phytotoxicity free and matured after composting for 2 months. Therefore, inoculation of food waste with the thermo-tolerant lipolytic actinomycete, T. vulgaris A31, presents as a feasible strategy to convert food wastes into mature compost efficiently.