Identification of the Cirratiomycin Biosynthesis Gene Cluster in Streptomyces Cirratus: Elucidation of the Biosynthetic Pathways for 2,3-Diaminobutyric Acid and Hydroxymethylserine.

Cirratiomycin, a heptapeptide with antibacterial activity, was isolated and characterized in 1981; however, its biosynthetic pathway has not been elucidated. It contains several interesting nonproteinogenic amino acids, such as (2S,3S)-2,3-diaminobutyric acid ((2S,3S)-DABA) and α-(hydroxymethyl)serine, as building blocks. Here, we report the identification of a cirratiomycin biosynthetic gene cluster in Streptomyces cirratus. Bioinformatic analysis revealed that several Streptomyces viridifaciens and Kitasatospora aureofaciens strains also have this cluster. One S. viridifaciens strain was confirmed to produce cirratiomycin. The biosynthetic gene cluster was shown to be responsible for cirratiomycin biosynthesis in S. cirratus in a gene inactivation experiment using CRISPR-cBEST. Interestingly, this cluster encodes a nonribosomal peptide synthetase (NRPS) composed of 12 proteins, including those with an unusual domain organization: a stand-alone adenylation domain, two stand-alone condensation domains, two type II thioesterases, and two NRPS modules that have no adenylation domain. Using heterologous expression and in vitro analysis of recombinant enzymes, we revealed the biosynthetic pathway of (2S,3S)-DABA: (2S,3S)-DABA is synthesized from l-threonine by four enzymes, CirR, CirS, CirQ, and CirB. In addition, CirH, a glycine/serine hydroxymethyltransferase homolog, was shown to synthesize α-(hydroxymethyl)serine from d-serine in vitro. These findings broaden our knowledge of nonproteinogenic amino acid biosynthesis.

Involvement of a putative acyltransferase gene in sporangium formation in Actinoplanes missouriensis.

The actinomycete Actinoplanes missouriensis forms branched substrate mycelia during vegetative growth and produces terminal sporangia, each of which contains a few hundred spherical flagellated spores, from the substrate mycelia through short sporangiophores. Based on the observation that remodeling of membrane lipid composition is involved in the morphological development of Streptomyces coelicolor A3(2), we hypothesized that remodeling of membrane lipid composition is also involved in sporangium formation in A. missouriensis. Because some acyltransferases are presumably involved in the remodeling of membrane lipid composition, we disrupted each of the 22 genes annotated as encoding putative acyltransferases in the A. missouriensis genome and evaluated their effects on sporangium formation. The atsA (AMIS_52390) null mutant (ΔatsA) strain formed irregular sporangia of various sizes. Transmission electron microscopy revealed that some ΔatsA sporangiospores did not mature properly. Phase-contrast microscopy revealed that sporangium dehiscence did not proceed properly in the abnormally small sporangia of the ΔatsA strain, whereas apparently normal sporangia opened to release the spores. Consistently, the number of spores released from ΔatsA sporangia was lower than that released from wild-type sporangia. These phenotypic changes were recovered by introducing atsA with its own promoter into the ΔatsA strain. These results demonstrate that AtsA is required for normal sporangium formation in A. missouriensis, although the involvement of AtsA in the remodeling of membrane lipid composition is unlikely because AtsA is an acyltransferase_3 (AT3) protein, which is an integral membrane protein that usually catalyzes the acetylation of cell surface structures.IMPORTANCEActinoplanes missouriensis goes through a life cycle involving complex morphological development, including mycelial growth, sporangium formation and dehiscence, swimming as zoospores, and germination to mycelial growth. In this study, we carried out a comprehensive gene disruption experiment of putative acyltransferase genes to search for acyltransferases involved in the morphological differentiation of A. missouriensis. We revealed that a stand-alone acyltransferase_3 domain-containing protein, named AtsA, is required for normal sporangium formation. Although the molecular mechanism of AtsA in sporangium formation, as well as the enzymatic activity of AtsA, remains to be elucidated, the identification of a putative acyltransferase involved in sporangium formation is significant in the study of morphological development of A. missouriensis. This finding will contribute to our understanding of a complex system for producing sporangia, a rare multicellular organism in bacteria.

Identification of a putative cell wall-hydrolyzing amidase involved in sporangiospore maturation in Actinoplanes missouriensis.

Actinoplanes missouriensis is a filamentous bacterium that differentiates into terminal sporangia, each containing a few hundred spores. Previously, we reported that a cell wall-hydrolyzing N-acetylglucosaminidase, GsmA, is required for the maturation process of sporangiospores in A. missouriensis; sporangia of the gsmA null mutant (ΔgsmA) strain released chains of 2-20 spores under sporangium dehiscence-inducing conditions. In this study, we identified and characterized a putative cell wall hydrolase (AsmA) that is also involved in sporangiospore maturation. AsmA was predicted to have a signal peptide for the general secretion pathway and an N-acetylmuramoyl-l-alanine amidase domain. The transcript level of asmA increased during the early stages of sporangium formation. The asmA null mutant (ΔasmA) strain showed phenotypes similar to those of the wild-type strain, but sporangia of the ΔgsmAΔasmA double mutant released longer spore chains than those from the ΔgsmA sporangia. Furthermore, a weak interaction between AsmA and GsmA was detected in a bacterial two-hybrid assay using Escherichia coli as the host. Based on these results, we propose that AsmA is an enzyme that hydrolyzes peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in cooperation with GsmA in A. missouriensis.IMPORTANCEActinoplanes missouriensis produces sporangiospores as dormant cells. The spores inside the sporangia are assumed to be formed from prespores generated by the compartmentalization of intrasporangium hyphae via septation. Previously, we identified GsmA as a cell wall hydrolase responsible for the separation of adjacent spores inside sporangia. However, we predicted that an additional cell wall hydrolase(s) is inevitably involved in the maturation process of sporangiospores because the sporangia of the gsmA null mutant strain released not only tandemly connected spore chains (2-20 spores) but also single spores. In this study, we successfully identified a putative cell wall hydrolase (AsmA) that is involved in sporangiospore maturation in A. missouriensis.

The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis.

In Streptomyces, multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis, which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis. The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (ΔAmssgB) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB. Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) (ssgA-G) in the ΔAmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis. In contrast to the ΔAmssgB strain, the ftsZ-disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae. IMPORTANCE: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

Identification of the p-coumaric acid biosynthetic gene cluster in Kutzneria albida: insights into the diazotization-dependent deamination pathway.

Recently, we identified the biosynthetic gene cluster of avenalumic acid (ava cluster) and revealed its entire biosynthetic pathway, resulting in the discovery of a diazotization-dependent deamination pathway. Genome database analysis revealed the presence of more than 100 ava cluster-related biosynthetic gene clusters (BGCs) in actinomycetes; however, their functions remained unclear. In this study, we focused on an ava cluster-related BGC in Kutzneria albida (cma cluster), and revealed that it is responsible for p-coumaric acid biosynthesis by heterologous expression of the cma cluster and in vitro enzyme assays using recombinant Cma proteins. The ATP-dependent diazotase CmaA6 catalyzed the diazotization of both 3-aminocoumaric acid and 3-aminoavenalumic acid using nitrous acid in vitro. In addition, the high efficiency of the CmaA6 reaction enabled us to perform a kinetic analysis of AvaA7, which confirmed that AvaA7 catalyzes the denitrification of 3-diazoavenalumic acid in avenalumic acid biosynthesis. This study deepened our understanding of the highly reducing type II polyketide synthase system as well as the diazotization-dependent deamination pathway for the production of avenalumic acid or p-coumaric acid.

Architecture of Actinoplanes missouriensis sporangia and zoospores visualized using quick-freeze deep-etch electron microscopy.

The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface.

A unique sigma/anti-sigma system in the actinomycete Actinoplanes missouriensis.

Bacteria of the genus Actinoplanes form sporangia that contain dormant sporangiospores which, upon contact with water, release motile spores (zoospores) through a process called sporangium dehiscence. Here, we set out to study the molecular mechanisms behind sporangium dehiscence in Actinoplanes missouriensis and discover a sigma/anti-sigma system with unique features. Protein σSsdA contains a functional sigma factor domain and an anti-sigma factor antagonist domain, while protein SipA contains an anti-sigma factor domain and an anti-sigma factor antagonist domain. Remarkably, the two proteins interact with each other via the anti-sigma factor antagonist domain of σSsdA and the anti-sigma factor domain of SipA. Although it remains unclear whether the SipA/σSsdA system plays direct roles in sporangium dehiscence, the system seems to modulate oxidative stress responses in zoospores. In addition, we identify a two-component regulatory system (RsdK-RsdR) that represses initiation of sporangium dehiscence.

In vitro characterization of nonribosomal peptide synthetase-dependent O-(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine.

Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative azaserine biosynthetic gene (azs) cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the Streptomyces fragilis genome. Azaserine was produced by the heterologous expression of the azs cluster in Streptomyces albus. In vitro enzyme assays using recombinant Azs proteins revealed the azaserine biosynthetic pathway as follows. AzsSPTF and carrier protein (CP) AzsQ are used to synthesize the 2-hydrazineylideneacetyl (HDA) moiety attached to AzsQ from HAA. AzsD transfers the HDA moiety to the C-terminal CP domain of AzsN. The heterocyclization (Cy) domain of the nonribosomal peptide synthetase AzsO synthesizes O-(2-hydrazineylideneacetyl)serine (HDA-Ser) attached to its CP domain from l-serine and HDA moiety-attached AzsN. The thioesterase AzsB hydrolyzes it to yield HDA-Ser, which appears to be converted to azaserine by oxidation. Bioinformatics analysis of the Cy domain of AzsO showed that it has a conserved DxxxxD motif; however, two conserved amino acid residues (Thr and Asp) important for heterocyclization are substituted for Asn. Site-directed mutagenesis of two Asp residues in the DxxxxD motif (D193 and D198) and two substituted Asn residues (N414 and N447) indicated that these four residues are important for ester bond synthesis. These results showed that the diazo ester of azasrine is synthesized by the stepwise oxidation of the HAA moiety and provided another strategy to biosynthesize the diazo group.

Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.

The chromosomal evolutionary lineage of the genus Zygosaccharomyces.

Genome ploidy of Zygosaccharomyces rouxii is an intriguing topic in the field of industrial yeast research. However, the evolutionary relationship between the genome of Z. rouxii and other Zygosaccharomyces species is complex and not completely understood. In this study, we determined the genome sequences of Z. rouxii NCYC 3042, also referred to as 'Z. pseudorouxii,' and Z. mellis CBS 736T. We also conducted comparative analysis of the yeast genomes of a total of 21 strains, including 17 strains of nine Zygosaccharomyces species. This comparative genomics revealed that 17 Zygosaccharomyces strains are classified into four groups consisting of nine genome types: (i) Z. rouxii, Z. mellis, Z. sapae, Z. siamensis, and 'Candida versatilis' t-1 belong to the group Rouxii sharing four related genome types (Rouxii-1 to Rouxii-4), (ii) Z. bailii, Z. parabailii, and Z. pseudobailii belong to the group Bailii sharing three related genome types (Bailii-1 to Bailii-3), (iii and iv) Z. bisporus and Z. kombuchaensis belong to the groups Bisporus and Kombuchaensis, respectively, which each have haploid genomes. The Zygosaccharomyces genome seems to have acquired complexity and diversity through evolutionary events such as interspecies hybridization, reciprocal translocation, and diploidization of these nine genome types.

Engineering the Substrate Specificity of Toluene Degrading Enzyme XylM Using Biosensor XylS and Machine Learning.

Enzyme engineering using machine learning has been developed in recent years. However, to obtain a large amount of data on enzyme activities for training data, it is necessary to develop a high-throughput and accurate method for evaluating enzyme activities. Here, we examined whether a biosensor-based enzyme engineering method can be applied to machine learning. As a model experiment, we aimed to modify the substrate specificity of XylM, a rate-determining enzyme in a multistep oxidation reaction catalyzed by XylMABC in Pseudomonas putida. XylMABC naturally converts toluene and xylene to benzoic acid and toluic acid, respectively. We aimed to engineer XylM to improve its conversion efficiency to a non-native substrate, 2,6-xylenol. Wild-type XylMABC slightly converted 2,6-xylenol to 3-methylsalicylic acid, which is the ligand of the transcriptional regulator XylS in P. putida. By locating a fluorescent protein gene under the control of the Pm promoter to which XylS binds, a XylS-producing Escherichia coli strain showed higher fluorescence intensity in a 3-methylsalicylic acid concentration-dependent manner. We evaluated the 3-methylsalicylic acid productivity of XylM variants using the fluorescence intensity of the sensor strain as an indicator. The obtained data provided the training data for machine learning for the directed evolution of XylM. Two cycles of machine learning-assisted directed evolution resulted in the acquisition of XylM-D140E-V144K-F243L-N244S with 15 times higher productivity than wild-type XylM. These results demonstrate that an indirect enzyme activity evaluation method using biosensors is sufficiently quantitative and high-throughput to be used as training data for machine learning. The findings expand the versatility of machine learning in enzyme engineering.

Bacterial Avenalumic Acid Biosynthesis Includes Substitution of an Aromatic Amino Group for Hydride by Nitrous Acid Dependent Diazotization.

The diazo group is an important functional group that can confer biological activity to natural products owing to its high reactivity. Recent studies have revealed that diazo groups are synthesized from amino groups using nitrous acid in secondary metabolites of actinomycetes. However, genome database analysis indicated that there are still many diazo group-biosynthesizing enzymes for unknown biosynthetic pathways. Here, we discovered an avenalumic acid biosynthesis gene cluster in Streptomyces sp. RI-77 by genome mining of enzymes involved in diazo group formation. Through heterologous expression, the gene cluster was revealed to direct avenalumic acid (AVA) biosynthesis via 3-aminoavenalumic acid (3-AAA). In vitro enzyme assays showed that AvaA6 and AvaA7 catalyzed the diazotization of 3-AAA using nitrous acid and substitution of the diazo group for hydride to synthesize AVA, respectively. This study revealed an unprecedented pathway for amino group removal via diazotization.

Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states.

Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 12 C atoms, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-linking methods to capture and elucidate conformational transitions accompanying KS-mediated catalysis at near-atomic resolution.

Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence. IMPORTANCE BldC is a global transcriptional regulator that acts as a "brake" in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.

3-Amino-4-hydroxybenzoic acid production from glucose and/or xylose via recombinant Streptomyces lividans.

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

Enzymatic synthesis of non-natural flavonoids by combining geranyl pyrophosphate C6-methyltransferase and aromatic prenyltransferase.

Terpenoids are the largest class of natural products and are derived from C5 isoprene units. Recent discoveries of modification enzymes in native isoprene units before cyclization or transfer reactions have revealed that C5 units with additional carbon atoms are also used to produce terpenoids. These reports indicate that the utilization of these modification enzymes is useful for the enzymatic production of non-natural terpenoids. In this study, we have attempted to produce methylgeranyl polyphenols, which are not observed in nature, by combining a geranyl pyrophosphate C6 methyltransferase, BezA, which was discovered from the benzastatin biosynthetic pathway, and the promiscuous prenyltransferase NphB, which catalyzes prenylation of various flavonoids. We successfully synthesized five methylgeranylated flavonoids from naringenin, apigenin, and genistein. This result demonstrates that BezA is a powerful tool for the synthesis of novel non-natural terpenoids.

The α/ß Hydrolase AzpM Catalyzes Dipeptide Synthesis in Alazopeptin Biosynthesis Using Two Molecules of Carrier Protein-Tethered Amino Acid.

During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/ß hydrolase AzpM synthesizes the DON-DON dipeptide using DON tethered to the carrier protein AzpF (DON-AzpF). However, whether AzpM catalyzes the condensation of DON-AzpF with DON or DON-AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS). We found that AzpM catalyzed the condensation between AZS-AzpF and DON-AzpF, but not between AZS-AzpF and DON. Possible reaction intermediates, DON-DON-AzpF and AZS-AZS-AzpF, were also detected during AzpM-catalyzed dipeptide formation from DON-AzpF and AZS-AzpF, respectively. From these results, we concluded that AzpM catalyzed the condensation of the two molecules of DON-AzpF and subsequent hydrolysis to produce DON-DON. Thus, AzpM is an unprecedented α/ß hydrolase that catalyzes dipeptide synthesis from two molecules of a carrier protein-tethered amino acid.

Surface structure and nanomechanical properties of Actinoplanes missouriensis sporangia analyzed via atomic force microscopy.

The surface structures of the sporangia produced by Actinoplanes missouriensis were analyzed at high resolution in air and liquid via atomic force microscopy. Results revealed a dynamic change in sporangium surface structure in response to the amount of moisture. Furthermore, the Young's modulus of the sporangium surface (1.95 ± 0.92 GPa) was calculated by analyzing the force-distance curves in air.

Engineering of the Ligand Specificity of Transcriptional Regulator XylS by Deep Mutational Scanning.

Deep mutational scanning is a method for protein engineering. Here, we applied it to alter the ligand specificity of the transcriptional regulator XylS from Pseudomonas putida to recognize p-toluic acid instead of the native ligand m-toluic acid. For this purpose, we used an antibiotic resistance gene-based dual screening system, which was constructed for the directed evolution of XylS toward the above-mentioned ligand specificity. We constructed a xylS mutant library in which each codon for the amino acid residue of the putative ligand-binding domain (residues 1-213, except 7th residue) was randomized to generate all possible single amino acid-substituted XylS variants and introduced it into Escherichia coli harboring the selection plasmid for the screening system. The cells were cultured in the presence of appropriate antibiotics and m-toluic acid or p-toluic acid, and the frequency of each mutation present in the library was examined using a next-generation sequencer before and after cultivation. Heatmaps showing the enrichment score of each XylS variant were obtained. By searching for a p-toluic-acid-specific heatmap pattern, we focused on G71 and H77. Analysis of the ligand specificities of G71- or H77-substituted XylS variants revealed that several G71-substituted XylS variants responded specifically to p-toluic acid. Thus, the 71st residue was found to be an unprecedented residue that is important for switching ligand specificity. Our study demonstrated the usefulness of deep mutational scanning in engineering the ligand specificity of a transcriptional regulator without structural information. We also discussed the advantages and disadvantages of deep mutational scanning compared with directed evolution.

Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus.

Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. In vitro analysis of the first module revealed that its acyltransferase domain selects malonyl-CoA, although predicted to select methylmalonyl-CoA. Inactivation of seven tailoring enzyme-encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.