Avermectin Production Research Articles

Yeast screening from avermectins wastewater and investigation on the ability of its fermentation

Wastewater from avermectin production is refractory with high output, high chemical oxygen demand (COD) concentration and high cost further. The current wastewater treatment technology, with little reuse of the organic material, needs high dilution ratio during treatment. Yeast single cell protein was produced from the wastewater of avermectins fermentation in this research. First, the yeast strain (H-1) which show enough tolerance to avermectins residue was screened from the wastewater, and it was identified as being most closely related to Candida tropicalis (100%, EF120592.1) using 18S rDNA gene sequence analysis. Second, its growth characteristics in the avermectins wastewater were studied. The dry biomass reached the maximal point of 10g/L, the COD removal was up to 66.67%, and avermectins removal in the wastewater was 99.48% at the optimal condition that it was liquid volume 50ml (250ml flask), pH 4.0, temperature 30°C, inoculum volume 10% (V/V) and fermentation period 20h. Third, the nutritional contents of dry yeast powder were determined, in which the water content was 8.12%, ash content was 5.18% and the crude protein was 40.02%. The dry yeast powder from avermectin waste liquor was promising to be used as a raw material or nitrogen source for commercial production of avermectins. The project was economically feasible by primary cost accounting.

Inactivation of the extracytoplasmic function sigma factor Sig6 stimulates avermectin production in Streptomyces avermitilis

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.

Enhancement of avermectin and ivermectin production by overexpression of the maltose ATP-binding cassette transporter in Streptomyces avermitilis

We investigated the function of maltose ABC transporter system encoded by malEFG-a and the effect of its overexpression on antibiotic production in Streptomyces avermitilis. A malEFG-a deletion mutant was unable to grow in a minimal medium with maltose as sole carbon source and produce avermectin. Maltose utilization and avermectin production were restored by introduction of a single copy of malEFG-a. RT-PCR analysis showed that the expression of malE-a was induced by maltose, and was strongly repressed by glucose. When multi-copy, integrative malEFG-a gene expression vectors were introduced into wild-type strain ATCC31267 and ivermectin-producer OI-31, antibiotic production increased by 2.6- to 3.3-fold and the time required for fermentation decreased by about 10%. The overexpression of malEFG-a improved the utilization rate of starch, and thereby enhanced avermectin production. Such an approach would be useful for the improvement of commercial antibiotic production using starch as the main carbon source in the fermentation process.

Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are widely used in the field of animal health, agriculture, and human health. Here we have adopted a practical approach to successfully improve avermectin production in an industrial overproducer. Transcriptional levels of the wild-type strain and industrial overproducer in production cultures were monitored using microarray analysis. The avermectin biosynthetic genes, especially the pathway-specific regulatory gene, aveR, were up-regulated in the high-producing strain. The upstream promoter region of aveR was predicted and proved to be directly recognized by sigma(hrdB) in vitro. A mutant library of hrdB gene was constructed by error-prone PCR and selected by high-throughput screening. As a result of evolved hrdB expressed in the modified avermectin high-producing strain, 6.38 g/L of avermectin B1a was produced with over 50% yield improvement, in which the transcription level of aveR was significantly increased. The relevant residues were identified to center in the conserved regions. Engineering of the hrdB gene can not only elicit the overexpression of aveR but also allows for simultaneous transcription of many other genes. The results indicate that manipulating the key genes revealed by reverse engineering can effectively improve the yield of the target metabolites, providing a route to optimize production in these complex regulatory systems.

Overexpression of ribosome recycling factor causes increased production of avermectin in Streptomyces avermitilis 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.

The pathway-specific regulator AveR from Streptomyces avermitilis positively regulates avermectin production while it negatively affects oligomycin biosynthesis

The function of the regulatory protein AveR in Streptomyces avermitilis was examined. An aveR deletion mutant abolished avermectin production and produced more oligomycin, and its phenotype was complemented by a single copy of the aveR gene. Removal of the C-terminal HTH domain of AveR abolished avermectin biosynthesis, indicating the importance of HTH domain for AveR function. Promoter titration and promoter probe assays suggested that the transcription of aveA1, encoding polypeptide AVES1 of avermectin PKS, was activated by AveR. Chromatin immunoprecipitation (ChIP) assay showed that the predicted promoter regions of both the ave cluster and the olm cluster were target sites of AveR, and the DNA-binding activity of AveR was dependent on its HTH domain. RT-PCR analysis revealed that the transcriptions of ave structural genes were dependent on AveR, but that of olm structural genes and putative pathway-specific regulatory genes increased in the aveR mutants. Consistent with these observations, overexpression of aveR successfully increased avermectin production. These results indicated that aveR encodes a pathway-specific activator essential for avermectin biosynthesis and it also negatively affects oligomycin biosynthesis.

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 m(3) 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.

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.

Functional Expression of SAV3818, a Putative TetR-Family Transcriptional Regulatory Gene from Streptomyces avermitilis, Stimulates Antibiotic Production in Streptomyces Species

Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. Previously, comparative transcriptome analysis between the low-producer S. avermitilis ATCC31267 and the high-producer S. avermitilis ATCC31780 using a S. avermitilis whole genome chip revealed that 50 genes were overexpressed at least two-fold higher in S. avermitilis ATCC31780. To verify the biological significance of some of the transcriptomics-guided targets, five putative regulatory genes were individually cloned under the strong-andconstitutive promoter of the Streptomyces expression vector pSE34, followed by the transformation into the lowproducer S. avermitilis ATCC31267. Among the putative genes tested, three regulatory genes including SAV213, SAV3818, and SAV4023 exhibited stimulatory effects on avermectin production in S. avermitilis ATCC31267. Moreover, overexpression of SAV3818 also stimulated actinorhodin production in both S. coelicolor M145 and S. lividans TK21, implying that the SAV3818, a putative TetR-family transcriptional regulator, could be a global upregulator acting in antibiotic production in Streptomyces species.

Effect of mycelial morphology on bioreactor performance and avermectin production of Streptomyces avermitilis in submerged cultivations

Factors affecting the morphology of Streptomyces avermitilis and avermectin production in submerged cultivation, including nitrogen sources, inoculum level and DO (dissolved oxygen) tension in the broth were investigated in a 50-L bioreactor. It was found that a combination of soybean meal and yeast meal as nitrogen sources and 4.3% inoculum led to pellet formation, and the pellet morphology facilitated to maintain DO > 20% in the early stage of fermentation. With the aid of image analysis tools, area and density of pellets in different batches were calculated. Results show that higher dissolved oxygen tension was favorable for pellet formation and avermectin production.

Isolation of soluble proteins from an industrial strain Streptomyces avermitilis in complex culture medium for two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) is a core proteomic technique to study protein expression and function in living organisms. Although 2-DE has been extensively used for the investigation of bacteria, yeast, animal and plant tissue cells, the isolation of proteins from the organisms and elimination of salt, nucleotide, polysaccharide, lipids and other contaminations from the samples often limit its application. In this study, the protocol for protein isolation from cells of Streptomyces avermitilis cultivated in partially insoluble complex medium was investigated. The goal was to make the obtained extraction samples suitable for the two-dimensional electrophoresis, thus make the further proteome analysis possible. Compared to non-denatured procedure, the denatured one, precipitating with 10% TCA in acetone, efficiently eliminated the interference substances from the cell lysate. Thiourea in the rehydration solution enhanced the resolubilization of protein pellets but led to heavy horizontal streaking in the 2-DE gels. High protein loading amount improved the resolution of some low abundance proteins but did not adapt to the high abundance proteins. And it was also important to collect cells at appropriate culture time according to the analysis target. With the optimized protein extraction protocol, the protein expression patterns of S. avermitilis during the onset of avermectin production in complex medium were analyzed.

Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion

Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. Intraspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveD− and olmA− (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, respectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the production of other avermectin components and oligomycin will make F23 and F29 useful strains for avermectin production. Strain F29’s tolerance of fermentation conditions will also make it suitable for industrial applications.

An adpA homologue in Streptomyces avermitilis is involved in regulation of morphogenesis and melanogenesis

In Streptomyces griseus, AdpA, the key transcriptional activator in the A-factor regulatory cascade, switches on the transcription of multiple genes required for secondary metabolism and morphological differentiation. Streptomyces avermitilis also contains an ortholog of adpA, which is named adpA-a. To clarify the in vivo function of adpA-a, an adpA-a-disrupted strain was constructed by double crossover recombination. No difference in avermectin production was found between the adpA-a-disruptant and the wild-type strain. However, this disruptant neither formed spores nor produced melanin and its phenotype was restored to the original wild-type by a single copy of the adpA-a gene integrated into the chromosome. This report shows that adpA-a is involved in regulation of morphological differentiation and melanin production in S. avermitilis.

Catalytic Domain of AfsKav Modulates Both Secondary Metabolism and Morphologic Differentiation in Streptomyces avermitilis ATCC 31272

Genetic characterization of afsK-av (SAV3816) in Streptomyces avermitilis ATCC 31272 was performed to evaluate the role(s) of this eukaryotic-type serine-threonine protein kinase (STPK) in the regulation of morphologic differentiation and secondary metabolism. The afsK-av::neo mutant (SJW4001) was defective in sporulation, melanogenesis, and avermectin production. These phenotypic defects were complemented by introduction of either the intact afsK-av or the 900-nt catalytic domain region. The catalytic domain restored sporulation and melanogenesis to SJW4001 whereas it partially recovered avermectin production. This study reveals that AfsKav is a pleiotropic regulator and demonstrates in vivo that the C-region of AfsKav is not essential for its regulatory role in S. avermitilis differentiations.

Modulation of Serine/Threonine Protein Kinase Activity in Chloramphenicol-Resistant Mutants of Streptomyces avermitilis

A mutation to chloramphenicol resistance (Cmlr) stimulates production of macrolide avermectin in Streptomyces avermitilis; production starts in early stationary growth. By labeling in vivo, the Cmlr mutation was shown to stimulate phosphorylation of Ser and Thr in several proteins in the same growth phase. Autophosphorylation of active protein kinases (PK) was analyzed in gel after one- or two-dimensional PAGE for the original S. avermitilis strain ATCC 31272, its Cmlr mutant, and a Cmls revertant. An increase in in vivo phosphorylation was associated with an increase in autophosphorylation of Ser/Thr-PK 41K, 45K, 52K, 62K, and 85K and complete suppression of autophosphorylation of PK 66K. Comparison of the PK molecular weights and pI with the parameters deduced for putative PK encoded by S. avermitilis genes identified the 41K, 45K, 52K, 62K, and 85K PK as pkn 24, pkn 32, pkn 13, pkn 12, and pkn 5, respectively. Prenylamine lactate, a modulator of calmodulin-dependent processes, substantially reduced the avermectin production, impaired the Cml resistance, and selectively inhibited Ca2+-dependent PK 85K in the Cmlr mutant. It was assumed that PK 85K is involved in regulating the avermectin production.

Ethanol evolution rate: a new parameter to determine the feeding rate for the production of avermectins by Streptomyces avermitilis.

A new parameter ethanol evolution rate (EER) was developed to aid in the determination of glucose feeding rate in avermectin production. The EER characterized the level of primary metabolism and its value was affected mainly by the supply of O2 and glucose. In an abnormal batch, over-feeding of glucose led to 2.5-fold increase of the maximum EER value compared to the normal one, and the production was thus decreased by nearly 80%. Together with other criteria, the EER helped to control utilization of substrate, so it has been successfully used to control glucose feeding in an industrial process.

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

Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various <i>Streptomyces avermitilis</i> Strains

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.

Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis

The global regulatory gene, afsR2, from Streptomyces lividans was previously reported to highly stimulate two structurally unrelated antibiotics, actinorhodin and undecylprodigiosin, in both S. lividans and its close relative S. coelicolor. Production of eight avermectin components was also improved in S. avermitilis: the use of wild-type S. avermitilis and its high-producing mutant, transformed by introduction of multiple copies of afsR2, increased the total avermectin productions by 2.3-fold and 1.5-fold, respectively.

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.