Rationally Improving Doramectin Production in Industrial Streptomyces avermitilis Strains.

Avermectins (AVMs), a family of 16-membered macrocyclic macrolides produced by Streptomyces avermitilis, have been the most successful microbial natural antiparasitic agents in recent decades. Doramectin, an AVM derivative produced by S. avermitilis bkd- mutants through cyclohexanecarboxylic acid (CHC) feeding, was commercialized as a veterinary antiparasitic drug by Pfizer Inc. Our previous results show that the production of avermectin and actinorhodin was affected by several other polyketide biosynthetic gene clusters in S. avermitilis and Streptomyces coelicolor, respectively. Thus, here, we propose a rational strategy to improve doramectin production via the termination of competing polyketide biosynthetic pathways combined with the overexpression of CoA ligase, providing precursors for polyketide biosynthesis. fadD17, an annotated putative cyclohex-1-ene-1-carboxylate:CoA ligase-encoding gene, was proven to be involved in the biosynthesis of doramectin. By sequentially removing three PKS (polyketide synthase) gene clusters and overexpressing FadD17 in the strain DM203, the resulting strain DM223 produced approximately 723 mg/L of doramectin in flasks, which was approximately 260% that of the original strain DM203 (approximately 280 mg/L). To summarize, our work demonstrates a novel viable approach to engineer doramectin overproducers, which might contribute to the reduction in the cost of this valuable compound in the future.

Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa.

Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA (Ser) gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa.

Recombinational cloning of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of Streptomyces coelicolor A3(2).

The model organism Streptomyces coelicolor A3(2) harbors a 356-kb linear plasmid, SCP1. We report here development of a recombinational cloning method for deleting large segment from one telomere of SCP1 followed by replacing with the telomere of pSLA2 and sequentially inserting with the overlapping cosmids in vivo. The procedure depends on homologous recombination coupled with cleavage at telomere termini by telomere terminal protein. Using this procedure, we cloned the 81-kb avermectin and the 76-kb spinosad biosynthetic gene clusters into SCP1. Heterologous expression of avermectin production in S. coelicolor was detected. These results demonstrate the utility of SCP1 for cloning large DNA segments such as antibiotic biosynthetic gene clusters.

Differential regulation of antibiotic biosynthesis by DraR-K, a novel two-component system in Streptomyces coelicolor.

A novel two-component system (TCS) designated as DraR-K (sco3063/sco3062) was identified to be involved in differential regulation of antibiotic biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow-pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR-K activates ACT but represses yCPK production directly through the pathway-specific activator genes actII-ORF4 and kasO, respectively, while its role on RED biosynthesis was independent of pathway-specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at -124 to -98 nt and -24 to -1 nt relative to the respective transcription start point of actII-ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR-binding sequence, 5'-AMAAWYMAKCA-3' (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel-retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR-K in primary metabolism. DraR-K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR-Ksav (sav_3481/sav_3480, homologue of draR-K) in the industrial model strain S. avermitilis NRRL-8165 led to similar abnormal antibiotic biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR-K-mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of antibiotic biosynthesis in Streptomyces.

An orphan histidine kinase, OhkA, regulates both secondary metabolism and morphological differentiation in Streptomyces coelicolor.

We report here the physiological and genetic characterization of an orphan histidine kinase (HK) (OhkA, SCO1596) in Streptomyces coelicolor and its homolog (OhkAsav, SAV_6741) in Streptomyces avermitilis. The physiological analysis showed that the ohkA mutant of S. coelicolor exhibits impaired aerial mycelium formation and sporulation and overproduction of multiple antibiotics on mannitol-soy flour (MS) medium, especially actinorhodin (ACT) and calcium-dependent antibiotic (CDA), and disruption of ohkAsav in S. avermitilis also led to the similar phenotypes of impaired morphological differentiation and significantly increased oligomycin A production. DNA microarray analysis combined with real-time reverse transcription-PCR (RT-PCR) and RNA dot blot assay in the S. coelicolor ohkA deletion mutant confirmed the physiological results by showing the upregulation of genes involved in the biosynthesis of ACT, CDA, undecylprodigiosin (RED), a yellow type I polyketide (CPK, SCO6273-6289), and a sesquiterpene antibiotic, albaflavenone (SCO5222-5223). The results also suggested that the increased production of ACT and RED in the mutant could be partly ascribed to the enhanced precursor malonyl coenzyme A (malonyl-CoA) supply through increased transcription of genes encoding acetyl-CoA carboxylase (ACCase). Interestingly, DNA microarray analysis also showed that deletion of ohkA greatly downregulated the transcription of chpABCDEFGH genes essential for aerial mycelium formation by S. coelicolor on MS medium but significantly increased transcription of ramS/C/R, which is responsible for SapB formation and regulation and is normally absent on MS medium. Moreover, many other genes involved in development, such as bldM/N, whiG/H/I, ssgA/B/E/G/R, and whiE, were also significantly downregulated upon ohkA deletion. The results clearly demonstrated that OhkA is an important global regulator for both morphological differentiation and secondary metabolism in S. coelicolor and S. avermitilis.

Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China.

A large number of isolates from the Fusarium graminearum clade representing all regions in China with a known history of Fusarium head blight (FHB) epidemics in wheat were assayed using PCR to ascertain their trichothecene mycotoxin chemotypes and associated phylogenetic species and geographical distribution. Of the 299 isolates assayed, 231 are from F. asiaticum species lineage 6, which produce deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON); deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON); and nivalenol and 4-acetylnivalenol (NIV) mycotoxins, with 3-AcDON being the predominant chemotype. Ninety-five percent of this species originated from the warmer regions where the annual average temperatures were above 15 degrees C, based on the climate data of 30 y during 1970-1999. However, 68 isolates within F. graminearum species lineage 7 consisted only of 15-AcDON producers, 59% of which were from the cooler regions where the annual average temperatures were 15 degrees C or lower. Identification of a new subpopulation of 15-AcDON producers revealed a molecular distinction between F. graminearum and F. asiaticum that produce 15-AcDON. An 11-bp repeat is present in F. graminearum within their Tri7 gene sequences but is absent in F. asiaticum, which could be directly used for differentiating the two phylogenetic species of the F. graminearum clade.