Overexpression of ribosome recycling factor causes increased production of avermectin in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> strains

Ribosome recycling factor (RRF), encoded by frr gene, is involved in the release of ribosomes from the translational post-termination complex for a new round of initiation. In this study, the frr gene with either its own promoter or with ermE*p was cloned into a multi-copy vector, pKC1139, and a single-site integrative vector, pSET152, respectively. The resulting plasmids were transformed into Streptomyces avermitilis wild-type strain ATCC31267, avermectin high-producing mutant strain 76-02-e, and the engineered strain GB-165 that produces only avermectin B. The results showed that overexpression of frr increased avermectin yield (by 3- to 3.7-fold in the wild-type strain) and revealed an frr gene “copy number effect”; i.e., multiple copies of frr had a greater promoting effect on avermectin production than a single copy in each of the three transformed S. avermitilis strains. Comparison of the growth and expression of the ave genes in an frr-overexpressing strain and wild-type ATCC31267 indicated that frr overexpression promoted cell growth as well as the expression of ave genes (including pathway-specific positive regulatory gene aveR for avermectin biosynthesis and ave structural genes), leading in turn to avermectin overproduction. These findings provide an effective approach for the improvement of antibiotic production in Streptomyces.     

Inactivation of the extracytoplasmic function sigma factor Sig6 stimulates avermectin production in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

The role of the extracytoplasmic function (ECF) σ factor Sig6 (SAV663) in avermectin production by Streptomyces avermitilis was investigated by gene-deletion, complementation and over-expression experiments. Inactivation of Sig6 had no major effect on growth, stress responses, or morphology. Avermectin yield was increased 2- to 2.7-fold (~680 μg/ml) relative to the wild-type strain by deletion of the sig6 gene, and was restored to the wild-type level by introduction of a single copy of sig6. Introduction of extra multi-copy or integrative sig6 vectors into the wild-type decreased avermectin yield by 56–63%. Taken together, these findings indicate that Sig6 plays a negative regulatory role in avermectin production in S. avermitilis. RT-PCR analysis demonstrated that this role of Sig6 is mediated by the pathway-specific activator gene aveR.     

Characterization of a regulatory gene, <Emphasis Type="Italic">aveR</Emphasis>, for the biosynthesis of avermectin in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

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.     

Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains

To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.     

Enhanced rapamycin production in <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> by integrative expression of <Emphasis Type="Italic">aveR</Emphasis>, a LAL family transcriptional regulator

Rapamycin produced by Streptomyces hygroscopicus displays immunosuppressive, antifungal, anti-tumor, anti-inflammatory and neuro-regenerative properties. To enhance production of rapamycin, aveR, a stimulator in Streptomyces avermitilis, was integrated into the chromosome of S. hygroscopicus TYQ0915. This resulted in a 274.9% increase of rapamycin production in an exconjugant S. hygroscopicus AVH1124. Introduction of aveR acts pleiotropically by affecting growth and sporulation of S. hygroscopicus AVH1124, although aveR is verified to be a pathway-specific regulator in S. avermitilis. This study proved that introduction of a homologous regulatory gene from the same protein family enhanced rapamycin production.     

Targeted gene disruption of the avermectin B O-methyltransferase gene in Streptomyces avermitilis

The biological activity of avermectin B components is superior to that of avermectin A components, which are derived from avermectin B by avermectin B 5-O-methyltransferase. Gene disruption, targeting avermectin B 5-O-methyltransferase gene in Streptomyces avermitilis, was carried out to obtain a strain of avermectin B producer. Phenotype analysis of the mutant with the disrupted O-methyltransferase gene showed that only avermectin B components were produced with a significant increase in production     

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression,antibiotic biosynthesis,and morphological development in Streptomyces avermitilis

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5′-TTKAMKHSTTSAV-3′, was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.     

Screening of spontaneous and induced mutants in Streptomyces avermitilis enhances avermectin production

Because of the loss of productivity in industrial strains (as a consequence of genetic instability), the selection of spontaneous and induced mutants in Streptomyces might generate enhanced producers of bioactive compounds. In this work, a spontaneously high producing mutant of Streptomyces avermitilis, strain 267/2H, was isolated. This mutant produced 8.2 times more avermectin B₁ than the wild type and it was treated with methyl methanesulphonate (MMS) in order to obtain better avermectin producers. One mutant, strain IPT-85, produced about 16 times more avermectin than the wild-type strain ATCC 31267 and twice as much as the parental strain 267/2H. Reversion studies showed that avermectin production by the IPT-85 mutant was unstable and required constant selection to maintain high levels of avermectin B₁ production. Upon a second MMS treatment of IPT-85, a new avermectin-aglycone-producing mutant, strain IPT 85-62, was isolated. Received: 2 March 1999 / Received revision: 16 June 1999 / Accepted: 27 June 1999     

Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus

Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus. It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics.     

Genome mining of the Streptomyces avermitilis genome and development of genome-minimized hosts for heterologous expression of biosynthetic gene clusters

To date, several actinomycete genomes have been completed and annotated. Among them, Streptomyces microorganisms are of major pharmaceutical interest because they are a rich source of numerous secondary metabolites. S. avermitilis is an industrial microorganism used for the production of an anthelmintic agent, avermectin, which is a commercially important antiparasitic agent in human and veterinary medicine, and agricultural pesticides. Genome analysis of S. avermitilis provides significant information for not only industrial applications but also understanding the features of this genus. On genome mining of S. avermitilis, the microorganism has been found to harbor at least 38 secondary metabolic gene clusters and 46 insertion sequence (IS)-like sequences on the genome, which have not been searched so far. A significant use of the genome data of Streptomyces microorganisms is the construction of a versatile host for heterologous expression of exogenous biosynthetic gene clusters by genetic engineering. Since S. avermitilis is used as an industrial microorganism, the microorganism is already optimized for the efficient supply of primary metabolic precursors and biochemical energy to support multistep biosynthesis. The feasibility of large-deletion mutants of S. avermitilis has been confirmed by heterologous expression of more than 20 exogenous biosynthetic gene clusters.     

Enhancing macrolide production in Streptomyces by coexpressing three heterologous genes

Antibiotic production in Streptomyces can often be increased by introducing heterologous genes into strains that contain an antibiotic biosynthesis gene cluster. A number of genes are known to be useful for this purpose. We chose three such genes and cloned them singly or in combination under the control of the strong constitutive ermE* promoter into a ?C31-derived integrating vector that can be transferred efficiently by conjugation from Escherichia coli to Streptomyces. The three genes are adpA, a global regulator from Streptomyces coelicolor, metK, encoding S-adenosylmethionine synthetase from S. coelicolor, and, VHbS, hemoglobin from Vitreoscilla. The substitutions with GC in VHbS was intended to convert codons from lower usage to higher, yet causing no change to the encoded amino acid. Plasmids containing either one of these genes or genes in various combinations were introduced into Streptomyces sp. FR-008, which produces the macrolide antibiotic FR-008-III (also known as candicidin D). The largest increase in FR-008-III production was achieved by the plasmid containing all three genes. This plasmid also increased avermectin production in Streptomyces avermitilis, and is likely to be generally useful for improving antibiotic production in Streptomyces.     

Identification of avermectin-high-producing strains by high-throughput screening methods

Avermectins produced by Streptomyces avermitilis are potent against a broad spectrum of nematode and arthropod parasites with low-level side effects on the host organisms. This study was designed to investigate a high-throughput screening strategy for the efficient identification of avermectin high-yield strains. The production protocol was miniaturized in 96 deep-well microplates. UV absorbance at 245 nm was used to monitor avermectin production. A good correlation between fermentation results in both 96 deep-well microplates and conventional Erlenmeyer flasks was observed. With this protocol, the production of avermectins was determined in less than 10 min for a full plate without compromising accuracy. The high-yield strain selected through this protocol was also tested in 360 m³ batch fermentation with 1.6-fold improved outcome. Thus, the development of this protocol is expected to accelerate the selection of superior avermectin-producing strains.     

Avermectin: biochemical and molecular basis of its biosynthesis and regulation

Avermectin and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents in the field of animal health, agriculture, and human infections. They are 16-membered pentacyclic lactone compounds derived from polyketide and linked to a disaccharide of the methylated deoxysugar l-oleandrose. Labeling studies, analyses of the biosynthetically blocked mutants, and the identification of the avermectin gene cluster allows characterization of most of the biosynthetic pathway. Recent completion of S. avermitilis genome sequencing is also expected to help in revealing the precise biosynthetic sequence and the complicated regulatory mechanism for avermectin biosynthesis, which has been long-awaited to be elucidated. The well characterized avermectin biosynthetic pathway and availability of S. avermitilis genome information in combination with the recent development of combinatorial biosynthesis should allow us to redesign more potent avermectin analogues and to engineer S. avermitilis as a more efficient host for the production of important commercial analogues.