4002bp, contained an AT-rich region and eneoded several open reading frfimes, as deduced by the comp]ete nucleotide sequence. One of the plltative open reading frames showed homology with replication proteins of other plasmids. A shuttle vector of Escherichia coti and this strain was constructed by connecting pAH4 to pUCI8. Electroporation of the shllttle vector into the strain yielded 1 .7 x 10i ampicillin resistant transformants per pg DNA. The shuttle plasmid was yery stably maintained in the strain.

excess (% e.e.), repeated crysta]lization gave a higher purity (99.8% e.e.) of the S configuration product. Production of S-PLA was significantly increased when 2.0% (wlv) of calcium ch]oride were added to the reaction mixture for precipitation of S-PLA. Chemical mutagenesis yietded a mutant strain, named BC348-9, with 16 times higher activity (40mUIOD,,,), compaTed with that of the parent strain (2.5mUIOD63o). VVhen the mutant strain BC348-9 was used, approximately 18g/OD63. was produced, which is 12 times higher than that of the parent strain. The final accumulation of PLA exceeded 6.0% , 1 .2 times higher than that of the parent strain.

Protein acetylation, the reversible addition of an acetyl group to lysine residues, is a protein post-translational modification ubiquitous in living cells. Although the involvement of protein acetylation in the regulation of primary metabolism has been revealed, the function of protein acetylation is largely unknown in secondary metabolism. Here, we characterized protein acetylation in Streptomyces griseus, a streptomycin producer. Protein acetylation was induced in the stationary and sporulation phases in liquid and solid cultures, respectively, in S. griseus. By comprehensive acetylome analysis, we identified 134 acetylated proteins with 162 specific acetylated sites. Acetylation was found in proteins related to primary metabolism and translation, as in other bacteria. However, StrM, a deoxysugar epimerase involved in streptomycin biosynthesis, was identified as a highly acetylated protein by 2-DE-based proteomic analysis. The Lys70 residue, which is critical for the enzymatic activity of StrM, was the major acetylation site. Thus, acetylation of Lys70 was presumed to abolish enzymatic activity of StrM. In accordance with this notion, an S. griseus mutant producing the acetylation-mimic K70QStrM hardly produced streptomycin, though the K70Q mutation apparently decreased the stability of StrM. A putative lysine acetyltransferase (KAT) SGR1683 in S. griseus, as well as the Escherichia coli KAT YfiQ acetylated Lys70 of StrM in vitro. Furthermore, absolute quantification analysis estimated that 13% of StrM molecules were acetylated in mycelium grown in solid culture for 3 days. These results indicate that StrM acetylation is of biological significance. We propose that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus. Biological significance: Protein acetylation has been extensively studied not only in eukaryotes, but also in prokaryotes. The acetylome has been analyzed in more than 14 bacterial species. Here, by comprehensive acetylome analysis, we showed that acetylation was found in proteins related to primary metabolism and translation in Streptomyces griseus, similarly to other bacteria. However, five proteins involved in secondary metabolism were also identified as acetylated proteins; these proteins are enzymes in the biosynthesis of streptomycin (StrB1 and StrS), grixazone (GriF), a nonribosomal peptide (NRPS1-2), and a siderophore (AIcC). Additionally, StrM in streptomycin biosynthesis was identified as a highly acetylated protein by 2-DE-based proteomic analysis; approximately 13% of StrM molecules were acetylated. The acetylation occurs at Lys70 to abolish the enzymatic activity of StrM, suggesting that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus. This is the first detailed analysis of protein acetylation of an enzyme involved in secondary metabolism. (C) 2016 Published by Elsevier B.V.

Background: Type III polyketide synthases (PKSs) show diverse cyclization specificity. Results: A single amino acid substitution in two Azotobacter type III PKSs reversed their cyclization specificity. Crystal structures were determined. Conclusion: The volume of the active site cavity is a crucial determinant of the cyclization specificity. Significance: An important insight into the cyclization specificity of type III PKSs was provided.Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 , respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl-CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C1014 fatty acyl-CoA unit, one malonyl-CoA unit and two methylmalonyl-CoA units. We identified these products as 4-hydroxy-3-methyl-6-(1-methyl-2-oxoalkyl)pyran-2-ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl-CoA as the extender substrate. In addition, in vitro reactions with 13C-labeled malonyl-CoA revealed that RpsA produced tetraketide 6-alkyl-4-hydroxy-1,5-dimethyl-2-oxocyclohexa-3,5-diene-1-carboxylic acids from C1420 fatty acyl-CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.

SummaryIn bacteria, the RNA polymerase holoenzyme comprises a five‐subunit core enzyme and a dissociable subunit, sigma factor, which is responsible for transcriptional initiation. The filamentous bacterium Streptomyces griseus has 52 sigma factors, including one essential ‘principal’ sigma factor (σHrdB) that is responsible for the transcription of housekeeping genes. Here we characterized an alternative sigma factor (σShbA), which is highly conserved within the genus Streptomyces. A σShbA‐deficient mutant showed a severe growth defect and transcriptome analysis indicated that many housekeeping genes were downregulated in response to insufficient σShbA production. Biochemical and genetic analyses proved that σShbA is a major determinant of transcription of the σHrdB gene. This observation of a principal sigma factor being governed by another sigma factor throughout growth is unprecedented. We found that increasing σShbA production with mycelial growth maintained a high σHrdB level late in growth. Furthermore, a hrdB‐autoregulatable σShbA‐deficient mutant, in which the principal sigma factor gene can be transcribed by RNA polymerase containing σHrdB itself, showed several defects: rapid mycelial lysis in stationary phase in liquid culture and delayed morphological development and impaired streptomycin production in solid culture. From these observations, we discuss the biological significance of control of σHrdB by σShbA in S. griseus.

Streptomyces griseus contains the srs operon, which is required for phenolic lipid biosynthesis. The operon consists of srsA, srsB, and srsC, which encode a type III polyketide synthase, an O-methyltransferase, and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13'-methyltetradecyl)-4-methylresorcinol, using iso-C(16) fatty acyl-coenzyme A (CoA) as a starter substrate and malonyl-CoA and methylmalonyl-CoA as extender substrates. An in vitro SrsA reaction using [(13)C(3)]malonyl-CoA confirmed that the order of extender substrate condensation was methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product rather than an alkylresorcinol. The functional SrsB protein was produced in the membrane fraction in Streptomyces lividans and used for the in vitro SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of S-adenosyl-l-methionine (SAM). SrsB was incapable of catalyzing the O-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely, by both the O-methylation of the hydroxyl group (C-6) and the decarboxylation of the neighboring carboxyl group (C-1). O-methylated alkylresorcylic acid was not detected in the in vitro SrsAB reaction, although it was presumably stable, indicating that O-methylation did not precede decarboxylation. We therefore postulated that O-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To the best of our knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.

Streptomyces griseus AdpA is the central transcription factor in the A-factor regulatory cascade and activates a number of genes that are required for both secondary metabolism and morphological differentiation, leading to the onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. The DNA-binding domain of AdpA consists of two helix-turn-helix DNA-binding motifs and shows low nucleotide-sequence specificity. To reveal the molecular basis of the low nucleotide-sequence specificity, an attempt was made to obtain cocrystals of the DNA-binding domain of AdpA and several kinds of duplex DNA. The best diffracting crystal was obtained using a 14-mer duplex DNA with two-nucleotide overhangs at the 5'-ends. The crystal diffracted X-rays to 2.8 Å resolution and belonged to space group C222(1), with unit-cell parameters a = 76.86, b = 100.96, c = 101.25 Å. The Matthews coefficient (V(M) = 3.71 Å(3) Da(-1)) indicated that the crystal was most likely to contain one DNA-binding domain of AdpA and one duplex DNA in the asymmetric unit, with a solvent content of 66.8%.

(JB06406-11R) 1 Full-length paper 2 Section: Enzymes and Proteins 3 4 The O-methyltransferase SrsB catalyzes the decarboxylative methylation of 5 alkylresorcylic acid during phenolic lipid biosynthesis by Streptomyces griseus 6 7 8 Chiaki Nakano, Nobutaka Funa, Yasuo Ohnishi*, and Sueharu Horinouchi. 9 10 Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The 11 University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. 12 13 Running title: Decarboxylative methylation of alkylresorcylic acid 14 15 16 *Corresponding author: 17 Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The 18 University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 19 Tel: +81 3 5841-5123; Fax: +81 3 5841-8021; E-mail: ayasuo@mail.ecc.u-tokyo.ac.jp 20 21 Deceased on 12th July 2009. 22 23 24 25 Copyright © 2012, American Society for Microbiology. All Rights Reserved. J. Bacteriol. doi:10.1128/JB.06406-11 JB Accepts, published online ahead of print on 13 January 2012

Actinoplanes missouriensis Couch 1963 is a well-characterized member of the genus Actinoplanes, which is of morphological interest because its members typically produce sporangia containing motile spores. The sporangiospores are motile by means of flagella and exhibit chemotactic properties. It is of further interest that members of Actinoplanes are prolific sources of novel antibiotics, enzymes, and other bioactive compounds. Here, we describe the features of A. missouriensis 431(T), together with the complete genome sequence and annotation. The 8,773,466 bp genome contains 8,125 protein-coding and 79 RNA genes.

AdpA is a global transcriptional activator triggering morphological differentiation and secondary metabolism in Streptomyces griseus. AdpA influences the expression of >1000 genes; however, the overall picture of the AdpA regulon remains obscure. Here, we took snapshots of the distribution of AdpA across the chromosome in living S. griseus cells using chromatin immunoprecipitation/chromatin affinity precipitation-seq analysis. In both liquid and solid cultures, AdpA bound to >1200 similar sites, which were located on not only in putative regulatory regions (65%), but also in regions (35%) that appeared not to affect transcription. Transcriptome analysis indicated that ∼40% of the AdpA-binding sites in putative regulatory regions were involved in gene regulation. AdpA was indicated to act as a transcriptional repressor as well as an activator. Expression profiles of AdpA-target genes were very different between liquid and solid cultures, despite their similar AdpA-binding profiles. We concluded that AdpA directly controls >500 genes in cooperation with other regulatory proteins. A comprehensive competitive gel mobility shift assay of AdpA with 304 selected AdpA-binding sites revealed several unique characteristics of the DNA-binding property of AdpA. This study provides the first experimental insight into the extent of the AdpA regulon, indicating that many genes are under the direct control of AdpA.

The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b = 69.35, c = 77.71 Å, β = 96.63°, and diffracted X-rays to 2.22 Å resolution.

Adenosine 5′-monophosphate (AMP) deaminase (AMPD; EC 3.5.4.6), an enzyme that catalyzes the deamination of AMP to produce inosine 5′-monophosphate (IMP), plays an important role in purine/urate metabolism in higher eukaryotes (Morisaki and Morisaki, 2008). In humans, metabolic myopathy is known to be associated with a lack of AMPD in skeletal muscle (Morisaki et al., 1992; Rubio et al., 2000). In plants, AMPD has been shown to be a target of herbicides (Dancer et al., 1997; Sabina et al., 2007). In addition to the important physiological and metabolic functions of AMPD in living cells, AMPD from Aspergillus melleus has been applied to the production of yeast extract with enhanced taste (Tabata et al., 1994). It converts tasteless AMP, which is produced by the digestion of yeast RNA by RNases, into IMP, which has an “umami” fl avor. Thus, AMPD is an industrially important enzyme. Amano Enzyme, Inc. carried out microbial screening for thermostable AMPDs that would be more suitable for industrial application. As a result, two thermostable AMPDs were found, one from Aspergillus fumigatus (Moriguchi, 2006) and one from Streptomyces murinus IFO 14082 (= NBRC 14082) (Mizuguchi et al., 2005). This study deals with the thermostable AMPD gene found from S. murinus. To the best of our knowledge, this is the fi rst bacterial AMPD to be identifi ed. S. murinus is known to produce a thermostable xylose isomerase, which has been used for isomerization in sugar manufacturing for many years (Ashby et al., 1987; Bandlish et al., 2002). As mentioned above, Mizuguchi et al. (2005) found a thermostable AMPD that is not denatured and that remains active at up to 65°C at its optimum pH of 5.6 in the culture medium of S. murinus. The AMPD appeared to function as a monomer and the molecular weight of the protein was estimated to be 48±2 kDa and 60±3 kDa by gel fi ltration and SDS-PAGE, respectively (Mizuguchi et al., 2005). The N-terminal and internal amino acid sequences of the purifi ed AMPD were determined by protein sequencing (Mizuguchi et al., 2005). The 3.8 kb NotI DNA fragment, which was assumed to contain the whole AMPD gene, was cloned by conventional methods, including PCR amplifi cation of part of the AMPD gene, Southern hybridization, and colony hybridization (Mizuguchi et al., 2005). When the resulting plasmid was introduced into a conventional Streptomyces host, Streptomyces lividans TK24, the AMPD protein was J. Gen. Appl. Microbiol., 58, 65‒70 (2012)

Recent bacterial genome sequencing projects have shown the presence of many putative sesquiterpene cyclase (SC) genes, especially in the Gram-positive, filamentous bacterial genus Streptomyces . We describe here the characterization of a SC gene (SGR6065, named gecA ) from Streptomyces griseus . Overexpression of gecA in Streptomyces lividans produced a sesquiterpene, which was isolated and determined to be (+)-epicubenol using spectroscopic analyses. The N-terminal histidine-tagged GecA protein was produced in Escherichia coli . Incubation of the recombinant GecA protein with farnesyl diphosphate (FPP) yielded (+)-epicubenol as the major product. The K m value for FPP and the k cat value for (+)-epicubenol formation were calculated to be 254±7.1 n M and 0.026±0.001 s −1 , respectively. The k cat / K m value (0.10 s −1 μ M −1 ) was broadly comparable to those reported for known bacterial SCs. (+)-Epicubenol was detected in the crude cell lysate of wild-type S. griseus , but not in a gecA -knockout mutant, indicating that GecA is a genuine (+)-epicubenol synthase. Although (+)-epicubenol synthases have been previously purified and characterized from the liverwort Heteroscyphus planus and Streptomyces sp. LL-B7, no (+)-epicubenol synthase gene has been cloned to date. The gecA gene is thus the first example of an (+)-epicubenol synthase-encoding gene. (+)-Epicubenol production was not controlled by the microbial hormone A-factor that induces morphological differentiation and production of several secondary metabolites in S. griseus .

A polyketide biosynthesis gene cluster ( agq ) was found on the genome of a rare actinomycete, Actinoplanes missouriensis. Streptomyces lividans expressing agqA encoding a type III polyketide synthase produced alkylresorcinols mainly from C 16–17 fatty acids. Heterologous expression of the agq genes in S. lividans indicated the function of cognate polyketide modification enzymes; a monooxygenase AgqB hydroxylates the alkylresorcinols to yield 6‐alkyl‐2‐hydroxyhydroquinones, a methyltransferase AgqC catalyzes O ‐methylation of the alkyl‐hydroxyhydroquinones to yield 6‐alkyl‐2‐methoxyhydroquinones, and a UbiA‐like prenyltransferase AgqD attaches a prenyl group to the C‐4 hydroxy group of the alkyl‐methoxyhydroquinones to yield 6‐alkyl‐4‐ O ‐geranyl‐2‐methoxyhydroquinones and 6‐alkyl‐4‐ O ‐dihydrofarnesyl‐2‐methoxyhydroquinones derived from C 16–17 fatty acids. In contrast, A. missouriensis was found to produce 6‐alkyl‐4‐ O ‐dihydrogeranyl‐2‐methoxyhydroquinones derived from C 16–18 fatty acids by the function of the agq gene cluster. All of these prenylated phenolic lipids were novel compounds.