Yasuo Ohnishi

A new pathway for polyketide synthesis in microorganisms

Chalcone synthases, which biosynthesize chalcones (the starting materials for many flavonoids,), have been believed to be specific to plants. However, the rppA gene from the Gram-positive, soil-living filamentous bacterium Streptomyces griseus encodes a 372-amino-acid protein that shows significant similarity to chalcone synthases. Several rppA-like genes are known, but their functions and catalytic properties have not been described. Here we show that a homodimer of RppA catalyses polyketide synthesis: it selects malonyl-coenzyme-A as the starter, carries out four successive extensions and releases the resulting pentaketide to cyclize to 1,3,6,8-tetrahydroxynaphthalene (THN). Site-directed mutagenesis revealed that, as in other chalcone synthases,, a cysteine residue is essential for enzyme activity. Disruption of the chromosomal rppA gene in S. griseus abolished melanin production in hyphae, resulting in ‘albino’ mycelium. THN was readily oxidized to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which then randomly polymerized to form various coloured compounds. THN formed by RppA appears to be an intermediate in the biosynthetic pathways for not only melanins but also various secondary metabolites containing a naphthoquinone ring. Therefore, RppA is a chalcone-synthase-related synthase that synthesizes polyketides and is found in the Streptomyces and other bacteria.

The A‐factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus : identification of a target gene of the A‐factor receptor

In Streptomyces griseus, A‐factor (2‐isocapryloyl‐3R‐hydroxymethyl‐γ‐butyrolactone) at an extremely low concentration triggers streptomycin biosynthesis and cell differentiation by binding a repressor‐type receptor protein (ArpA) and dissociating it from DNA. An A‐factor‐responsive transcriptional activator (AdpA) able to bind the promoter of strR, a pathway‐specific regulatory gene responsible for transcription of other streptomycin biosynthetic genes, was purified to homogeneity and adpA was cloned by PCR on the basis of amino acid sequences of purified AdpA. adpA encoding a 405‐amino‐acid protein containing a helix‐turn‐helix DNA‐binding motif at the central region showed sequence similarity to transcriptional regulators in the AraC/XylS family. The −35 and −10 regions of the adpA promoter were found to be a target of ArpA; ArpA bound the promoter region in the absence of A‐factor and exogenous addition of A‐factor to the DNA–ArpA complex immediately released ArpA from the DNA. Consistent with this, S1 nuclease mapping showed that adpA was transcribed only in the presence of A‐factor and strR was transcribed only in the presence of intact adpA. Furthermore, adpA disruptants produced no streptomycin and overexpression of adpA caused the wild‐type S. griseus strain to produce streptomycin at an earlier growth stage in a larger amount. On the basis of these findings, we propose here a model to demonstrate how A‐factor triggers streptomycin biosynthesis at a late exponential growth stage.

AdpA, a Central Transcriptional Regulator in the A-Factor Regulatory Cascade That Leads to Morphological Development and Secondary Metabolism in Streptomyces griseus

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a microbial hormone that triggers aerial mycelium formation and secondary metabolism in Streptomyces griseus. A-factor produced in a growth-dependent manner switches on the transcription of adpA encoding a transcriptional activator by binding to the A-factor receptor protein (ArpA), which has bound the adpA promoter, and dissociating the DNA-bound ArpA from the DNA. AdpA then activates a number of genes with various functions required for morphological development and secondary metabolism, forming an AdpA regulon. AdpA, which contains a ThiJ/PfpI/DJ-1-like dimerization domain at its N-terminal portion and an AraC/XylS-type DNA-binding domain at its C-terminal portion, is a representative of a large subfamily of the AraC/XylS family. AdpA binds various positions with respect to the transcriptional start points of the target genes and recruits RNA polymerase to the specific promoter region, and facilitates the isomerization of the RNA polymerase-DNA complex into an open complex competent for transcriptional initiation. The AdpA-binding consensus sequence is 5′-TGGCSNGWWY-3′ (S: G or C; W: A or T; Y: T or C; N: any nucleotide). The DNA-binding specificity of AdpA in conjunction with that of other AraC/XylS family members is also discussed.

Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster

ABSTRACT In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins—PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata—were constructed. The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (PT7) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single PT7 in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both PT7 and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.

Biosynthesis of γ-butyrolactone autoregulators that switch on secondary metabolism and morphological development in Streptomyces

A factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a representative of the γ-butyrolactone autoregulators that trigger secondary metabolism and morphogenesis in the Gram-positive, filamentous bacterial genus Streptomyces. Here, we report the A factor biosynthesis pathway in Streptomyces griseus. The monomeric AfsA, containing a tandem repeat domain of ≈80 aa, catalyzed β-ketoacyl transfer from 8-methyl-3-oxononanoyl-acyl carrier protein to the hydroxyl group of dihydroxyacetone phosphate (DHAP), thus producing an 8-methyl-3-oxononanoyl-DHAP ester. The fatty acid ester was nonenzymatically converted to a butenolide phosphate by intramolecular aldol condensation. The butenolide phosphate was then reduced by BprA that was encoded just downstream of afsA. The phosphate group on the resultant butanolide was finally removed by a phosphatase, resulting in formation of A factor. The 8-methyl-3-oxononanoyl-DHAP ester produced by the action of AfsA was also converted to A factor in an alternative way; the phosphate group on the ester was first removed by a phosphatase and the dephosphorylated ester was converted nonenzymatically to a butenolide, which was then reduced by a reductase different from BprA, resulting in A factor. Because introduction of afsA alone into Escherichia coli caused the host to produce a substance having A factor activity, the reductase(s) and phosphatase(s) were not specific to the A factor biosynthesis but commonly present in bacteria. AfsA is thus the key enzyme for the biosynthesis of γ-butyrolactones.

An A-factor-dependent extracytoplasmic function sigma factor (sigma(AdsA)) that is essential for morphological development in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function sigma factor belonging to a subgroup of the primary sigma(70) family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named sigma(AdsA). Transcription of adsA depended on A-factor and AdpA, since adsA was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that sigma(AdsA) is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomyces species examined by Southern hybridization suggests a common role in morphogenesis in this genus.

DNA‐binding specificity of AdpA, a transcriptional activator in the A‐factor regulatory cascade in Streptomyces griseus

AdpA, belonging to the AraC/XylS family, is the key transcriptional activator for a number of genes of various functions in the A‐factor regulatory cascade in Streptomyces griseus. It consists of a ThiJ/PfpI/DJ‐1‐like dimerization domain at its N‐terminal portion and a DNA‐binding domain with two helix–turn–helix motifs at its C‐terminal portion, representing a large subgroup of the AraC/XylS family. Uracil interference assay and missing T and GA interference assays on several AdpA binding sites, followed by gel mobility shift assays on systematically mutated binding sites, revealed a consensus AdpA‐binding sequence, 5′‐TGGCSNGWWY‐3′ (S: G or C; W: A or T; Y: T or C; N: any nucleotide). A dimer of AdpA bound a site including the two consensus sequences, with a space of 13–14 bp, as an inverted repeat (type I) at various positions, for example more than 200 bp upstream (−200) and 25 bp downstream (+25) from the transcriptional start point of the target gene. In addition, AdpA also bound a site including the consensus sequence in a single copy (type II) at positions, in most cases, from −40 to −50 and from −50 to −60. For transcriptional activation, some genes required simultaneous binding of a dimer of AdpA to type I and II sites, but others required only a single type I or type II site. AdpA bound mutated type I sites with various distances between the two consensus sequences with significant affinities, although the optimal distances for AdpA to bind were 13–14 bp and 2 bp. The DNA‐binding domain is therefore connected to the ThiJ/PfpI/DJ‐1‐like dimerization domain with a flexible linker. The DNA‐binding specificity of AdpA in conjunction with that of other AraC/XylS family members is discussed.

Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus

RppA, a chalcone synthase-related polyketide synthase (type III polyketide synthase) in the bacteriumStreptomyces griseus, catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA. The K m value for malonyl-CoA and thek cat value for THN synthesis were determined to be 0.93 ± 0.1 μm and 0.77 ± 0.04 min−1, respectively. RppA accepted aliphatic acyl-CoAs with the carbon lengths from C4 to C8as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield α-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2′,4′,6′-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five molecules of malonyl-CoA, suggesting that the size of the active site cavity of RppA is larger than any other chalcone synthase-related enzymes found so far in plants and bacteria. RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the starter and methylmalonyl-CoA as an extender. Thus, the broad substrate specificity of RppA yields a wide variety of products.

Transcriptional Switch On of ssgA by A-Factor, Which Is Essential for Spore Septum Formation in Streptomyces griseus

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers morphological development and secondary metabolism in Streptomyces griseus. A transcriptional activator (AdpA) in the A-factor regulatory cascade switches on a number of genes required for both processes. AdBS11 was identified in a library of the DNA fragments that are bound by AdpA and mapped upstream of ssgA, which is essential for septum formation in aerial hyphae. Gel mobility shift assays and DNase I footprinting revealed three AdpA-binding sites at nucleotide positions about -235 (site 1), -110 (site 2), and +60 (site 3) with respect to the transcriptional start point, p1, of ssgA. ssgA had two transcriptional start points, one starting at 124 nucleotides (p1) and the other starting at 79 nucleotides (p2) upstream of the start codon of ssgA. Of the three binding sites, only sites 1 and 2 were required for transcriptional activation of p1 and p2 by AdpA. The transcriptional switch on of ssgA required the extracytoplasmic function sigma factor, sigma(AdsA), in addition to AdpA. However, it was unlikely that sigma(AdsA) recognized the two ssgA promoters, since their -35 and -10 sequences were not similar to the promoter sequence motifs recognized by sigma(BldN), a sigma(AdsA) homologue of Streptomyces coelicolor A3(2). An ssgA disruptant formed aerial hyphae, but did not form spores, irrespective of the carbon source of the medium, which indicated that ssgA is a member of the whi genes. Transcriptional analysis of ssfR, located just upstream of ssgA and encoding an IclR-type transcriptional regulator, suggested that no read-through from ssfR into ssgA occurred, and ssgA was transcribed in the absence of ssfR. ssgA was thus found to be controlled by AdpA and not by SsfR to a detectable extent. SsfR appeared to regulate spore septum formation independently of SsgA or through interaction with SsgA in some unknown way, because an ssfR disruptant also showed a whi phenotype.

GriC and GriD Constitute a Carboxylic Acid Reductase Involved in Grixazone Biosynthesis in Streptomyces griseus

In grixazone biosynthesis by Streptomyces griseus, a key intermediate 3-amino-4-hydroxybenzoic acid (3,4-AHBA) is converted to another key intermediate 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL). Two genes griC and griD in the grixazone biosynthesis gene cluster were found to be responsible for this conversion, because disruption of each gene resulted in the extracellular accumulation of 3-acetylamino-4-hydroxybenzoic acid, a shunt product from 3,4-AHBA. Significant sequence similarity of GriC to AMP-binding proteins and of GriD to NAD(P)-dependent aldehyde dehydrogenases suggested that GriC and GriD constituted an ATP- and NAD(P)-dependent carboxylic acid reductase (CAR) catalyzing reduction of 3,4-AHBA to produce 3,4-AHBAL through acyl-AMP formation, as is found for the reactions catalyzed by some CARs. griG encoding a benzoate transporter homologue in the grixazone biosynthesis gene cluster was nonessential for grixazone biosynthesis but probably enhanced the membrane permeability for 3,4-AHBA. Simultaneous overexpression of griC, griD, and griG in S. griseus mutant cells deficient in an acetyltransferase responsible for N-acetylation of 3,4-AHBA led to efficient bioconversion of exogenously added 3,4-AHBA to 3,4-AHBAL. This system also turned out to be useful for reduction of some aryl carboxylates to the corresponding aryl aldehydes.