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.     

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.     

Protoplast fusion inStreptomyces avermitilis

Summary The power of protoplast fusion as a generally applicable method for obtaining genetic recombination is demonstrated by the recombination of genes involved in avermectin biosynthesis. A backcross ofStreptomyces avermitilis strain MA6202, an improved mutant that had lost the ability to carry out the methylation of the C-5 hydroxyl of the avermectin molecule, with the original soil isolate MA4680 resulted in the recovery of at least one unambiguous recombinant class despite the instability of rifampicin resistance, one of two markers initially used for recombinant selection. Such intrinsic instability is frequently encountered in streptomycete genetics, and this result delineates the utility of protoplast fusion as a genetic tool. Other difficulties addressed include recovery of complementary recombinant classes, differences in recombination frequency due to colony density on regeneration medium, and alteration in plating efficiency on diagnostic media following protoplasting and regeneration. The results of a cross between a nicotinamide auxotroph MRG1003 and a lysine auxotroph MRG 1004 are included to aid in the elucidation of these problems as well as to support the finding of homologous recombination inS. avermitilis.     

Engineering the aveC gene to enhance the ratio of doramectin to its CHC-B2 analogue produced in Streptomyces avermitilis

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC-B1), which is sold commercially as Dectomax is co-produced during fermentation with the undesired analogue CHC-B2 at a CHC-B2:CHC-B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The aveC gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site-specific mutagenesis and error-prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC-B1, produced relative to the undesired CHC-B2 fermentation component. High-throughput (HTP) screening of cultures grown on solid-phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An aveC gene with mutations that result in a 4-fold improvement in the ratio of doramectin to CHC-B2 was identified. Subsequent integration of the enhanced aveC gene into the chromosome of the S. avermitilis production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

The avermectin-producing Streptomyces avermitilis possesses an inducible valine dehydrogenase

Summary Valine dehydrogenase (VDH) is believed to be absent in Streptomyces avermitilis. In the present study, a VDH (M _r, 72 000) was detected by activity measurement and activity staining on a native-PAGE gel. The enzyme activity was induced by L-valine and repressed by ammonia. VDH activity was found to be significantly lower than L-valine transaminase activity. The results suggest that one active VDH does exist in S. avermitilis, and plays a role in valine catabolism and avermectin biosynthesis.     

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.     

Production of 4′-epidaunorubicin by metabolic engineering of Streptomyces coeruleorubidus strain SIPI-1482

Streptomyces coeruleorubidus strain SIPI-1482 is an important industrial microbial strain which produces daunorubicin, the precursor for semi-synthesis of first-line anti-tumor antibiotics doxorubicin and epirubicin. dnmV, the C4 ketoreductase gene in the biosynthetic pathway of TDP-l-daunosamine was successfully disrupted by homologue recombination. The SIPI-1482 dnmV-blocked mutant lost the ability to produce daunorubicin and aggregate the intermediate ε-rhodomycinone. By introducing dnmV, the daunorubicin biosynthetic pathway in S. coeruleorubidus was reconstituted. Further more, aveBIV from S. avermitilis, as well as oleU from S. antibiotics, and novS from S. niveus were introduced into the dnmV-blocked mutant. The SIPI-1482 dnmV::aveBIV mutant could produce 4′-epidaunorubicin instead of daunorubicin, but dnmV::oleU and dnmV::novS mutant could not. Our study showed that the genetically engineered strain had a different fermentation condition and extraction protocol compared with the wild type daunorubicin producer. These results suggest that metabolic engineering is a powerful tool to produce novel hybrid antibiotics and a good alternative to chemical synthesis.     

Production of avermectin A2a and monoglycosides A2a and B2a by a strain ofStreptomyces avermitilis

A strain ofStreptomyces avermitilis producing almost predominantly avermectin A2a and monoglycosides A2a and B2a was described. Methods of analytical and preparative high performance liquid chromatography were used for comparison of chromatographic profiles and product isolation, respectively. Identification of isolated compounds was based on¹³C-NMR spectrometric results.     

Oxygen uptake rate regulation during cell growth phase for improving avermectin B<Subscript>1a</Subscript> batch fermentation on a pilot scale (2 m<Superscript>3</Superscript>)

Avermectin B_1a batch fermentation of Streptomyces avermitilis in a 2 m³ fermentor was investigated by oxygen uptake rate (OUR) regulation during cell growth phase. OUR was controlled by adjusting of aeration and agitation. Result showed that OUR strongly affected cell growth and antibiotics production. Avermectin B_1a biosynthesis could be effectively enhanced when OUR was stably regulated at an appropriate level in batch fermentation of S. avermitilis. Avermectin B_1a yield reached 5568 ± 111 mg/l by controlling maximal OUR between 15 and 20 mmol/l/h during cell growth phase, which was increased by 21.8% compared with the control (maximal OUR above 20 mmol/l/h). The stimulation effect on avermectin B_1a production could be attributed to the improved supply of propionic acid and acetic acid, the precursors of avermectin B_1a, in the cells. Hence, this OUR control method during cell growth phase may be a simple and applicable way to improve industrial production of avermectin.     

Characterization of glucose transport inSchizosaccharomyces pombe

Conclusions GHT1 was isolated as suppressor ofd-glucose uptake deficiency ofS. pombe mutant YGS-5. The correspondingS. pombe DNA encodes a putative protein with significant amino acid sequence identity to theS. cerevisiae HXT transporters. Heterologous expression ofGHT1 inS. cerevisiae hxt mutant RE700A (strain HLY709) enabled the mutant to grow ond-glucose as the sole carbon source. HLY709 cells take up hexoses with similar specificity toS. pombe wild strain and accumulate the non-metabolizable analogues of glucose (2DG and 6DG) intracellularly, thus matchingS. pombe wild strain. Southern blot analysis revealed the existence of other putative glucose transporters inS. pombe and the search for related transporter genes inS. pombe genome is in progress.     

Identification of a gene cluster encoding meilingmycin biosynthesis among multiple polyketide synthase contigs isolated from<Emphasis Type="Italic"> Streptomyces nanchangensis</Emphasis> NS3226

A cluster encoding genes for the biosynthesis of meilingmycin, a macrolide antibiotic structurally similar to avermectin and milbemycin 11, was identified among seven uncharacterized polyketide synthase gene clusters isolated from Streptomyces nanchangensis NS3226 by hybridization with PCR products using primers derived from the sequences of aveE, aveF and a thioesterase domain of the avermectin biosynthetic gene cluster. Introduction of a 24.1-kb deletion by targeted gene replacement resulted in a loss of meilingmycin production, confirming that the gene cluster encodes biosynthesis of this important anthelminthic antibiotic compound. A sequenced 8.6-kb fragment had aveC and aveE homologues (meiC and meiE) linked together, as in the avermectin gene cluster, but the arrangement of aveF (meiF) and the thioesterase homologues differed. The results should pave the way to producing novel insecticidal compounds by generating hybrids between the two pathways.     

Draft genome sequence of Streptomyces coelicoflavus ZG0656 reveals the putative biosynthetic gene cluster of acarviostatin family α-amylase inhibitors

Aims: The aims of this study are to obtain the draft genome sequence of Streptomyces coelicoflavus ZG0656, which produces novel acarviostatin family α‐amylase inhibitors, and then to reveal the putative acarviostatin‐related gene cluster and the biosynthetic pathway. Methods and Results: The draft genome sequence of S. coelicoflavus ZG0656 was generated using a shotgun approach employing a combination of 454 and Solexa sequencing technologies. Genome analysis revealed a putative gene cluster for acarviostatin biosynthesis, termed sct‐cluster. The cluster contains 13 acarviostatin synthetic genes, six transporter genes, four starch degrading or transglycosylation enzyme genes and two regulator genes. On the basis of bioinformatic analysis, we proposed a putative biosynthetic pathway of acarviostatins. The intracellular steps produce a structural core, acarviostatin I00‐7‐P, and the extracellular assemblies lead to diverse acarviostatin end products. Conclusions: The draft genome sequence of S. coelicoflavus ZG0656 revealed the putative biosynthetic gene cluster of acarviostatins and a putative pathway of acarviostatin production. Significance and Impact of the Study: To our knowledge, S. coelicoflavus ZG0656 is the first strain in this species for which a genome sequence has been reported. The analysis of sct‐cluster provided important insights into the biosynthesis of acarviostatins. This work will be a platform for producing novel variants and yield improvement.     

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.     

Studies on the biosynthesis of avermectins

To elucidate the pathway of avermectin biosynthesis, the biosynthetic relationships of avermectins A1a, A2a, B1a, B2a, and their respective monosaccharides and aglycones were studied. 14C-labeled avermectin compounds prepared from [1-14C]acetate were fed to Streptomyces avermitilis strain MA5502 and their metabolites were determined. Two furan ring-free aglycones, 6,8a-seco-6,8a-deoxy-5-keto avermectin B1a and B2a, have been isolated from the fermentation broth of a blocked mutant of S. avermitilis. Addition of the compounds and a semisynthetic compound, 5-keto avermectin B2a aglycone, to the fermentation medium of a second blocked mutant established that the two compounds are intermediates in the avermectin biosynthetic pathway immediately preceding avermectin aglycones.     

The growth and EPA synthesis ofShewanella oneidensis MR-1 and expectation of EPA biosynthetic pathway

Shewanella oneidensis MR-1 has the ability to inhale certain metals and chemical compounds and exhale these materials in an altered state; as a result, this microorganism has been widely applied in bioremediation protocols. However, the relevant characteristics of cell growth and biosynthesis of PuFAs have yet to be thoroughly investigated. Therefore, in this study, we have attempted to characterize the growth and fatty acid profiles ofS. oneidensis MR-1 under a variety of temperature conditions. The fastest growth ofS. oneidensis MR-1 was observed at 30°C, with a specific growth rate and doubling time of 0.6885 h⁻¹ and 1.007 h. The maximum cell mass of this microorganism was elicited at a temperature of 4°C. The eicosapentaenoic acid (EPA) synthesis ofS. oneidensis MR-1 was evaluated under these different culture temperatures.S. oneidensis MR-1 was found not to synthesize EPA at temperatures in excess of 30°C, but was shown to synthesize EPA at temperatures below 30°C. The EPA content was found to increase with decreases in temperature. We then evaluated the EPA biosynthetic pathway, using a phylogenetic tree predicted on 16s rRNA sequences, and the homology of ORFs betweenS. oneidensis MR-1 andShewanella putrefaciens SCRC-2738, which is known to harbor a polyketide synthase (PKS)-like module. The phylogenetic tree revealed that MR-1 was very closely related to bothMoritella sp., which is known to synthesize DHA via a PKS-like pathway, andS. putrefaciens, which has been reported to synthesize EPA via an identical pathway. The homology between the PKS-like module ofS. putrefaciens SCRC-2738 and the entire genome ofS. oneidensis MR-1 was also analyzed, in order to mine the genes associated with the PKS-like pathway inS. oneidensis MR-1. A putative PKS-like module for EPA biosynthesis was verified by this analysis, and was also corroborated by the experimental finding thatS. oneidensis MR-1 was able to synthesize EPA without the expression of dihomo-γ-linoleic acid (DGLA) and arachidonic acid (AA) formed during EPA synthesis via the FAS pathway.     

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