In Vitro Production of Coenzyme A Using Thermophilic Enzymes.

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level, since it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for the high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using CoA-insensitive type III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using design of experiments (DOE) was employed to rationally determine the enzyme loading ratio to maximize the CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK toward ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type I and type II pantothenate kinase (PanK). Type III PanK is found only in bacteria and is generally insensitive to CoA. Previously, type III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic and instead shows inhibition toward CoA. In the present study, phylogenetic analysis combined with functional analysis of type III PanK from thermophiles revealed that the CoA-sensitive behavior of type III PanK from T. maritima is uncommon. We cloned type III PanKs from Thermus thermophilus and Geobacillus sp. strain 30 and showed that neither enzyme's activities were inhibited by CoA. Furthermore, we utilized type III PanK for a one-pot cascade reaction to produce CoA.

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum.

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5 times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20 times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

Identification of biosynthetic genes for the ß-carboline alkaloid kitasetaline and production of the fluorinated derivatives by heterologous expression.

ß-Carboline alkaloids exhibit a broad spectrum of pharmacological and biological activities and are widely distributed in nature. Genetic information on the biosynthetic mechanism of ß-carboline alkaloids has not been accumulated in bacteria, because there are only a few reports on the microbial ß-carboline compounds. We previously isolated kitasetaline, a mercapturic acid derivative of a ß-carboline compound, from the genetically modified Kitasatospora setae strain and found a plausible biosynthetic gene cluster for kitasetaline. Here, we identified and characterized three kitasetaline (ksl) biosynthetic genes for the formation of the ß-carboline core structure and a gene encoding mycothiol-S-conjugate amidase for the modification of the N-acetylcysteine moiety by using heterologous expression. The proposed model of kitasetaline biosynthesis shows unique enzymatic systems for ß-carboline alkaloids. In addition, feeding fluorotryptophan to the heterologous Streptomyces hosts expressing the ksl genes led to the generation of unnatural ß-carboline alkaloids exerting novel/potentiated bioactivities.

Butenolides from Streptomyces albus J1074 Act as External Signals To Stimulate Avermectin Production in Streptomyces avermitilis.

In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as Streptomyces hormones based on their extremely low effective concentrations (nM) and the involvement of specific receptor proteins. Our previous distribution study revealed that butenolide-type Streptomyces hormones, including avenolide, are a general class of signaling molecules in streptomycetes and that Streptomyces albus strain J1074 may produce butenolide-type Streptomyces hormones. Here, we describe metabolite profiling of a disruptant of the S. albusaco gene, which encodes a key biosynthetic enzyme for butenolide-type Streptomyces hormones, and identify four butenolide compounds from S. albus J1074 that show avenolide activity. The compounds structurally resemble avenolide and show different levels of avenolide activity. A dual-culture assay with imaging mass spectrometry (IMS) analysis for in vivo metabolic profiling demonstrated that the butenolide compounds of S. albus J1074 stimulate avermectin production in another Streptomyces species, Streptomyces avermitilis, illustrating the complex chemical interactions through interspecies signals in streptomycetes.IMPORTANCE Microorganisms produce external and internal signaling molecules to control their complex physiological traits. In actinomycetes, Streptomyces hormones are low-molecular-weight signals that are key to our understanding of the regulatory mechanisms of Streptomyces secondary metabolism. This study reveals that acyl coenzyme A (acyl-CoA) oxidase is a common and essential biosynthetic enzyme for butenolide-type Streptomyces hormones. Moreover, the diffusible butenolide compounds from a donor Streptomyces strain were recognized by the recipient Streptomyces strain of a different species, resulting in the initiation of secondary metabolism in the recipient. This is an interesting report on the chemical interaction between two different streptomycetes via Streptomyces hormones. Information on the metabolite network may provide useful hints not only to clarification of the regulatory mechanism of secondary metabolism, but also to understanding of the chemical communication among streptomycetes to control their physiological traits.

Discovery of a new diol-containing polyketide by heterologous expression of a silent biosynthetic gene cluster from Streptomyces lavendulae FRI-5.

The genome of streptomycetes has the ability to produce many novel and potentially useful bioactive compounds, but most of which are not produced under standard laboratory cultivation conditions and are referred to as silent/cryptic secondary metabolites. Streptomyces lavendulae FRI-5 produces several types of bioactive compounds. However, this strain may also have the potential to biosynthesize more useful secondary metabolites. Here, we activated a silent biosynthetic gene cluster of an uncharacterized compound from S. lavendulae FRI-5 using heterologous expression. The engineered strain carrying the silent gene cluster produced compound 5, which was undetectable in the culture broth of S. lavendulae FRI-5. Using various spectroscopic analyses, we elucidated the chemical structure of compound 5 (named lavendiol) as a new diol-containing polyketide. The proposed assembly line of lavendiol shows a unique biosynthetic mechanism for polyketide compounds. The results of this study suggest the possibility of discovering more silent useful compounds from streptomycetes by genome mining and heterologous expression.

Characterization of AvaR1, a butenolide-autoregulator receptor for biosynthesis of a Streptomyces hormone in Streptomyces avermitilis.

Streptomyces hormones, sometimes called as autoregulators, are important signaling molecules to trigger secondary metabolism across many Streptomyces species. We recently identified a butenolide-type autoregulator (termed avenolide) as a new class of Streptomyces hormone from Streptomyces avermitilis that produces important anthelmintic agent avermectin. Avenolide triggers the production of avermectin with minimum effective concentration of nanomolar. Here, we describe the characterization of avaR1 encoding an avenolide receptor in the regulation of avermectin production and avenolide biosynthesis. The disruption of avaR1 resulted in transcriptional derepression of avenolide biosynthetic gene with an increase in avenolide production, with no change in the avermectin production profile. Moreover, the avaR1 mutant showed increased transcription of avaR1. Together with clear DNA-binding capacity of AvaR1 toward avaR1 upstream region, it suggests that AvaR1 negatively controls the expression of avaR1 through the direct binding to the promoter region of avaR1. These findings revealed that the avenolide receptor AvaR1 functions as a transcriptional repressor for avenolide biosynthesis and its own synthesis.

Differential contributions of two SARP family regulatory genes to indigoidine biosynthesis in Streptomyces lavendulae FRI-5.

The Streptomyces antibiotic regulatory protein (SARP) family regulators have been shown to control the production of secondary metabolites in many Streptomyces species as the most downstream regulators in the regulatory cascade. Streptomyces lavendulae FRI-5 produces a blue pigment (indigoidine) together with two types of antibiotics: D-cycloserine and the nucleoside antibiotics. The production of these secondary metabolites is governed by a signaling system consisting of a γ-butyrolactone, IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl-γ-butanolide], and its cognate receptor, FarA. Here, we characterized two regulatory genes of the SARP family, farR3 and farR4, which are tandemly located in the proximal region of farA. farR3 is transcribed both as a monocistronic RNA and as a bicistronic farR4-farR3 mRNA, and the expression profile is tightly controlled by the IM-2/FarA system. Loss of farR3 delayed and decreased the production of indigoidine without any changes in the transcriptional profile of other far regulatory genes, indicating that FarR3 positively controls the biosynthesis of indigoidine and is positioned in the downstream region of the IM-2/FarA signaling system. Meanwhile, loss of farR4 induced the early production of IM-2 by increasing transcription of an IM-2 biosynthetic gene, farX, indicating that FarR4 negatively controls the biosynthesis of IM-2. Thus, our results suggested differential contributions of the SARP family regulators to the regulation of secondary metabolism in S. lavendulae FRI-5. This is the first report to show that an SARP family regulator is involved in the biosynthesis of a signaling molecule functioning at the most upstream region of the regulatory cascade for Streptomyces secondary metabolism.

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 $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.

SdrA, a new DeoR family regulator involved in Streptomyces avermitilis morphological development and antibiotic production.

The SAV3339 (SdrA) protein of Streptomyces avermitilis, a member of the DeoR family of regulators, was assessed to determine its in vivo function by gene knockdown through the use of cis-encoded noncoding RNA and knockout of the sdrA gene. These analyses revealed that SdrA represents another class of Streptomyces regulator that controls morphological development and antibiotic production.

Pleiotropic control of secondary metabolism and morphological development by KsbC, a butyrolactone autoregulator receptor homologue in Kitasatospora setae.

The γ-butyrolactone autoregulator signaling cascades have been shown to control secondary metabolism and/or morphological development among many Streptomyces species. However, the conservation and variation of the regulatory systems among actinomycetes remain to be clarified. The genome sequence of Kitasatospora setae, which also belongs to the family Streptomycetaceae containing the genus Streptomyces, has revealed the presence of three homologues of the autoregulator receptor: KsbA, which has previously been confirmed to be involved only in secondary metabolism; KsbB; and KsbC. We describe here the characterization of ksbC, whose regulatory cluster closely resembles the Streptomyces virginiae barA locus responsible for the autoregulator signaling cascade. Deletion of the gene ksbC resulted in lowered production of bafilomycin and a defect of aerial mycelium formation, together with the early and enhanced production of a novel ß-carboline alkaloid named kitasetaline. A putative kitasetaline biosynthetic gene cluster was identified, and its expression in a heterologous host led to the production of kitasetaline together with JBIR-133, the production of which is also detected in the ksbC disruptant, and JBIR-134 as novel ß-carboline alkaloids, indicating that these genes were biosynthetic genes for ß-carboline alkaloid and thus are the first such genes to be discovered in bacteria.

Jomthonic acid , a modified amino acid from a soil-derived Streptomyces.

Jomthonic acid A (1), a new modified amino acid, was isolated from the culture broth of a soil-derived actinomycete of the genus Streptomyces. The structure and absolute configuration of 1 were determined by spectroscopic analyses and chemical conversion. Jomthonic acid A (1) induced differentiation of preadipocytes into mature adipocytes at 2-50 µM.

VisG is essential for biosynthesis of virginiamycin S, a streptogramin type B antibiotic, as a provider of the nonproteinogenic amino acid phenylglycine.

A streptogramin type B antibiotic, virginiamycin S (VS), is produced by Streptomyces virginiae, together with a streptogramin type A antibiotic, virginiamycin M1 (VM), as its synergistic counterpart. VS is a cyclic hexadepsipeptide containing a nonproteinogenic amino acid, Lphenylglycine (L-pheGly), in its core structure. We have identified, in the left-hand extremity of the virginiamycin supercluster, two genes that direct VS biosynthesis with L-pheGly incorporation. Transcriptional analysis revealed that visF, encoding a nonribosomal peptide synthetase, and visG, encoding a protein with homology to a hydroxyphenylacetyl-CoA dioxygenase, are under the transcriptional regulation of virginiae butanolide (VB), a small diffusing signalling molecule that governs virginiamycin production. Gene deletion of visG resulted in complete loss of VS production without any changes in VM production, suggesting that visG is required for VS biosynthesis. The abolished VS production in the visG disruptant was fully recovered either by the external addition of pheGly or by gene complementation, which indicates that VisG is involved in VS biosynthesis as the provider of an L-pheGly molecule. A feeding experiment with L-pheGly analogues suggested that VisF, which is responsible for the last condensation step, has high substrate specificity toward L-pheGly.

The autoregulator receptor homologue AvaR3 plays a regulatory role in antibiotic production, mycelial aggregation and colony development of Streptomyces avermitilis.

The γ-butyrolactone autoregulator receptor has been shown to control secondary metabolism and/or morphological differentiation across many Streptomyces species. Streptomyces avermitilis produces an important anthelmintic agent (avermectin) and two further polyketide antibiotics, filipin and oligomycin. Genomic analysis of S. avermitilis revealed that this micro-organism has the clustered putative autoregulator receptor genes distant from the antibiotic biosynthetic gene clusters. Here, we describe the characterization of avaR3, one of the clustered receptor genes, which encodes a protein containing an extra stretch of amino acid residues that has not been found in the family of autoregulator receptors. Disruption of avaR3 resulted in markedly decreased production of avermectins, with delayed expression of avermectin biosynthetic genes, suggesting that AvaR3 positively controls the avermectin biosynthetic genes. Moreover, the disruption caused increased production of filipin without any changes in the transcriptional profile of the filipin biosynthetic genes, suggesting that filipin production is indirectly controlled by AvaR3. The avaR3 disruptant displayed fragmented growth in liquid culture and conditional morphological defects on solid medium. These findings demonstrated that AvaR3 acts as a global regulator that controls antibiotic production and cell morphology.

Germination stimulatory activity of bacterial butenolide hormones from Streptomyces albus J1074 on seeds of the root parasitic weed Orobanche minor.

Damage caused by Orobanchaceae root parasitic weeds is a substantial agricultural problem for global food security. Many studies have been conducted to establish practical methods of control, but efforts are still required for successful management. Seed germination of root parasitic weeds requires host-derived germination stimulants including strigolactones (SLs). Studies on SLs have revealed that a butenolide ring is the essential moiety for SL activity as a germination stimulant. Interestingly, recent studies have revealed that butenolide hormones regulate the biosynthesis of secondary metabolites and mediate communication in actinomycete bacteria. Because of the structural similarity between SLs and the bacterial butenolides, we evaluated the germination stimulatory activity of butenolides isolated from Streptomyces albus J1074 on root parasitic weeds. These butenolides were found to specifically induce seed germination of Orobanche minor. Our findings contribute to understanding the molecular mechanisms of germination stimulant perception and to the development of a method for their biological control.

Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica.

Methyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

Lavencidin, a polyene macrolide antibiotic from Streptomyces lavendulae FRI-5.

In our screening program for new biologically active compounds, a new polyene macrolide, lavencidin (1), along with known compound RKGS-A2215A (2), was isolated from the fermentation broth of Streptomyces lavendulae FRI-5 by changing the composition of liquid medium normally used for the strain. Their structures were elucidated by spectral methods (high-resolution fast-atom bombardment mass spectrometry (HRFABMS) and nuclear magnetic resonance (NMR)). Compound 1 includes a conjugated pentaene moiety together with six hydroxy groups and a carboxylic acid as a side chain. Lavencidin (1) showed moderate growth-inhibitory activity against yeast and was cytotoxic against human cancer cell lines with low-micromolar IC50 values.

Activation of cryptic milbemycin A4 production in Streptomyces sp. BB47 by the introduction of a functional bldA gene.

Streptomycetes are characterized by their ability to produce structurally diverse compounds as secondary metabolites and by their complex developmental life cycle, which includes aerial mycelium formation and sporulation. The production of secondary metabolites is growth-stage dependent, and generally coincides with morphological development on a solid culture. Streptomyces sp. BB47 produces several types of bioactive compounds and displays a bald phenotype that is devoid of an aerial mycelium and spores. Here, we demonstrated by genome analysis and gene complementation experiments that the bald phenotype arises from the bldA gene, which is predicted to encode the Leu-tRNAUUA molecule. Unlike the wild-type strain producing jomthonic acid A (1) and antarlide A (2), the strain complemented with a functional bldA gene newly produced milbemycin (3). The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as milbemycin A4, which is an insecticidal/acaricidal antibiotic. These results indicate that genetic manipulation of genes involved in morphological development in streptomycetes is a valuable way to activate cryptic biosynthetic pathways.

Null mutation analysis of an afsA-family gene, barX, that is involved in biosynthesis of the {gamma}-butyrolactone autoregulator in Streptomyces virginiae.

Virginiae butanolide (VB) is a gamma-butyrolactone autoregulator that triggers production of the streptogramin antibiotic virginiamycin in Streptomyces virginiae. Our previous studies suggested that the barX gene, an afsA-family gene, is likely to participate in the regulatory pathway for the production of VB, rather than in the biosynthetic pathway of VB itself, in contrast to the function of other afsA-family genes. Mutation analysis now shows that BarX at least plays an enzymic role in the VB biosynthetic pathway. Heterologous expression of the afsA gene from Streptomyces griseus into the barX mutant partially restored the deficiency of virginiamycin production, suggesting that afsA-family genes have a common ability to synthesize the gamma-butyrolactone autoregulators. Taken together with previous works relating to the function of an afsA-family gene, these results support the idea that streptomycetes have two biosynthetic pathways for the gamma-butyrolactone autoregulators.

Control of secondary metabolism by farX, which is involved in the gamma-butyrolactone biosynthesis of Streptomyces lavendulae FRI-5.

The gamma-butyrolactone signaling system is distributed widely among streptomycetes as an important regulatory mechanism of antibiotic production and/or morphological differentiation. IM-2 [(2R,3R,1'R)-2-(1'-hydroxybutyl)-3-hydroxymethyl-gamma-butanolide] is a gamma-butyrolactone that switches off the production of D: -cycloserine but switches on the production of several nucleoside antibiotics as well as blue pigment in Streptomyces lavendulae FRI-5. farX is a member of the afsA-family genes, which are proposed to encode enzymes involved in gamma-butyrolactone biosynthesis. Disruption of farX caused overproduction of D: -cycloserine, and abolished production of nucleoside antibiotic and blue pigment with the loss of IM-2 production. The finding that all phenotypic changes observed in the farX disruptant were restored by the addition of exogenous IM-2 suggested that FarX plays a biosynthetic role in IM-2 production. Transcriptional comparison between the wild-type strain and the farX disruptant revealed that, in addition to already known genes farR1 and farR2, several other genes (farR4, farD, and farE) are under the transcriptional regulation of IM-2. Furthermore, the fact that farX transcription is under the control of IM-2 suggested that S. lavendulae FRI-5 has a fine-tuning system to control gamma-butyrolactone production.

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

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.