Takuya Nihira

A complex role for the γ-butyrolactone SCB1 in regulating antibiotic production in Streptomyces coelicolor A3(2)

Many streptomycetes produce extracellular γ‐butyrolactones. In several cases, these have been shown to act as signals for the onset of antibiotic production. Synthesis of these molecules appears to require a member of the AfsA family of proteins (AfsA is required for A‐factor synthesis of the γ‐butyrolactone A‐factor and consequently for streptomycin production in Streptomyces griseus). An afsA homologue, scbA, was identified in Streptomyces coelicolor A3(2) and was found to lie adjacent to a divergently transcribed gene, scbR, which encodes a γ‐butyrolactone binding protein. Gel retardation assays and DNase I footprinting studies revealed DNA binding sites for ScbR at − 4 to − 33 nt with respect to the scbA transcriptional start site, and at − 42 to − 68 nt with respect to the scbR transcriptional start site. Addition of the γ‐butyrolactone SCB1 of S. coelicolor resulted in loss of the DNA‐binding ability of ScbR. A scbA mutant produced no γ‐butyrolactones, yet overproduced two antibiotics, actinorhodin (Act) and undecylprodigiosin (Red), whereas a deletion mutant of scbR also failed to make γ‐butyrolactones and showed delayed Red production. These phenotypes differ markedly from those expected by analogy with the S. griseus A‐factor system. Furthermore, transcription of scbR increased, and that of scbA was abolished, in an scbR mutant, indicating that ScbR represses its own expression while activating that of scbA. In the scbA mutant, expression of both genes was greatly reduced. Addition of SCB1 to the scbA mutant induced transcription of scbR, but did not restore scbA expression, indicating that the deficiency in scbA transcription in the scbA mutant is not solely due to the inability to produce SCB1, and that ScbA is a positive autoregulator in addition to being required for γ‐butyrolactone production. Overall, these results indicate a complex mechanism for γ‐butyrolactone‐mediated regulation of antibiotic biosynthesis in S. coelicolor.

Application of immobilized lipase to hydrolysis of triacylglyceride

SummaryLipase from Candida cylindracea was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers, and by covalent binding or by adsorption to different types of porous inorganic or organic supports. All of the immobilized lipase preparations thus obtained showed some activity for hydrolysis of olive oil. Lipase entrapped with a hydrophobic photo-cross-linkable resin prepolymer exhibited the highest activity, which was about 30% of that of the free counterpart. Entrapment method enabled lipase to gain operational stability. Semicontinuous hydrolysis of olive oil using immobilized lipase was also accomplished in a packed-bed reactor with a recycling system. In this reactor, immobilized lipase was observed to have the sufficient activity and stability.

Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus

ABSTRACT Citrinin produced by Aspergillus, Penicillium, and Monascus species is a polyketide compound that has nephrotoxic activity in mammals and is bactericidal toward gram-positive bacteria. To avoid the risk of citrinin contamination in other fermentation products produced by Monascus purpureus, knowledge of the citrinin biosynthetic genes is needed so that citrinin-nonproducing strains can be generated. We cloned a polyketide synthase (PKS) gene from M. purpureus with degenerate primers designed to amplify the conserved region of a ketosynthase domain of a fungal PKS. A 13-kb genomic DNA fragment was identified that contained a full-length PKS gene (pksCT) of 7,838 bp with a single 56-bp intron. pksCT encodes a 2,593-amino-acid protein that contains putative domains for ketosynthase, acyltransferase, acyl carrier protein (ACP), and a rare methyltransferase. There was no obvious thioesterase domain, which usually is downstream of the ACP domain in multi-aromatic-ring PKSs. pksCT transcription was correlated with citrinin production, suggesting that the pksCT gene product was involved in citrinin biosynthesis. Homologous recombination between the wild-type allele and a truncated disruption construct resulted in a pksCT-disrupted strain of M. purpureus. The disruptant did not produce citrinin, but a pksCT revertant generated by successive endogenous recombination events in the pksCT disruptant restored citrinin production, indicating that pksCT encoded the PKS responsible for citrinin biosynthesis in M. purpureus.

A bacterial hormone (the SCB1) directly controls the expression of a pathway‐specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor

Gamma‐butyrolactone signalling molecules are produced by many Streptomyces species, and several have been shown to regulate antibiotic production. In Streptomyces coelicolor A3(2) at least one γ‐butyrolactone (SCB1) has been shown to stimulate antibiotic production, and genes encoding proteins that are involved in its synthesis (scbA) and binding (scbR) have been characterized. Expression of these genes is autoregulated by a complex mechanism involving the γ‐butyrolactone. In this study, additional genes influenced by ScbR were identified by DNA microarray analysis, and included a cryptic cluster of genes for a hypothetical type I polyketide. Further analysis of this gene cluster revealed that the pathway‐specific regulatory gene, kasO, is a direct target for regulation by ScbR. Gel retardation and DNase I footprinting analyses identified two potential binding sites for ScbR, one at −3 to −35 nt and the other at −222 to −244 nt upstream of the kasO transcriptional start site. Addition of SCB1 eliminated the DNA binding activity of ScbR at both sites. The expression of kasO was growth phase regulated in the parent (maximal during transition phase), undetectable in a scbA null mutant, and constitutively expressed in a scbR null mutant. Addition of SCB1 to the scbA mutant restored the expression of kasO, indicating that ScbR represses kasO until transition phase, when presumably SCB1 accumulates in sufficient quantity to relieve kasO repression. Expression of the cryptic antibiotic gene cluster was undetectable in a kasO deletion mutant. This is the first report with comprehensive in vivo and in vitro data to show that a γ‐butyrolactone‐binding protein directly regulates a secondary metabolite pathway‐specific regulatory gene in Streptomyces.

Lipase catalyzed synthesis of macrocyclic lactones in organic solvents

Abstract A new method for the preparation of macrocyclic lactones from ω-hydroxyacid methyl esters is described. The approach utilizes intramolecular transesterification catalyzed by lipase in organic solvents. This procedure is also applicable to the synthesis of asymmetric lactones.

Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis

Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing >70% of commercially important antibiotics. The production of these compounds is often regulated by low-molecular-weight bacterial hormones called autoregulators. Although 60% of Streptomyces strains may use γ-butyrolactone–type molecules as autoregulators and some use furan-type molecules, little is known about the signaling molecules used to regulate antibiotic production in many other members of this genus. Here, we purified a signaling molecule (avenolide) from Streptomyces avermitilis—the producer of the important anthelmintic agent avermectin with annual world sales of

Construction of a citrinin gene cluster expression system in heterologous Aspergillus oryzae.

850 million—and determined its structure, including stereochemistry, by spectroscopic analysis and chemical synthesis as (4S,10R)-10-hydroxy-10-methyl-9-oxo-dodec-2-en-1,4-olide, a class of Streptomyces autoregulator. Avenolide is essential for eliciting avermectin production and is effective at nanomolar concentrations with a minimum effective concentration of 4 nM. The aco gene of S. avermitilis, which encodes an acyl-CoA oxidase, is required for avenolide biosynthesis, and homologs are also present in Streptomyces fradiae, Streptomyces ghanaensis, and Streptomyces griseoauranticus, suggesting that butenolide-type autoregulators may represent a widespread and another class of Streptomyces autoregulator involved in regulating antibiotic production.

Isolation and structure of a new butyrolactone autoregulator from Streptomyces sp. FRI-5

Filamentous fungi are considered an attractive resource for the discovery and production of bioactive compounds. To facilitate molecular breeding, biosynthetic genes must be rapidly identified. But, even after the chemical structure of a compound is identified, finding the corresponding biosynthetic genes in the fungal genome still remains a challenge. In an attempt to overcome this difficulty and to easily characterize each gene in a cluster, we constructed a heterologous expression system using Aspergillus oryzae. The approximate region covering the citrinin biosynthetic gene cluster from Monascus purpureus was introduced into A. oryzae via an Aspergillus-Escherichia coli shuttle cosmid vector without manipulating the gene structure. The transformants showed slight but reproducible citrinin production and definite transcription of the biosynthetic genes. Introducing additional copies of an activator gene (ctnA), controlled by the Aspergillus nidulans trpC promoter, into the citrinin-cluster-containing transformants enhanced the transcription of all the genes in the cluster and resulted in an almost 400-fold higher citrinin production compared to that of the parental transformant. This result suggested that CtnA controlled citrinin production in this system in the same way as in the native strain. This is the first report documenting the heterologous production of functional fungal secondary metabolites in A. oryzae following the introduction of an entire gene cluster.

Characterization of a regulatory gene, aveR, for the biosynthesis of avermectin in Streptomyces avermitilis.

A hormone-like factor, which triggers blue pigment production in Streptomyces sp. FRI-5, was purified 170,000-fold from a culture broth of the strain with 14% activity recovery. The compound, named IM-2, was effective at a concentration of 0.6 ng/ml in initiating blue pigment-production. The structure was determined to be 2, 3-trans-2-(1′-hydroxybutyl)-3-(hydroxymethyl)butanolide (10) on the basis of spectral data and chemical synthesis.

Identification and in vivo functional analysis by gene disruption of ctnA, an activator gene involved in citrinin biosynthesis in Monascus purpureus.

Avermectin is an important macrocyclic polyketide produced by Streptomyces avermitilis and widely used as an anthelmintic agent in the medical, veterinary, and agricultural fields. The avermectin biosynthetic gene cluster contains aveR, which belongs to the LAL-family of regulatory genes. In this study, aveR was inactivated by gene replacement in the chromosome of S. avermitilis, resulting in the complete loss of avermectin production. The aveR mutant was unable to convert an avermectin intermediate to any avermectin derivatives, and complementation by intact aveR and its proper upstream region restored avermectin production in the mutant, suggesting that AveR is a positive regulator controlling the expression of both polyketide biosynthetic genes and postpolyketide modification genes in avermectin biosynthesis. Despite the general concept that an increased amount of a positive pathway-specific regulator leads to higher production, a higher amount of aveR resulted in complete loss of avermectin, indicating that there is a maximum threshold concentration of aveR for the production of avermectin.