NsdB, a TPR-like-domain-containing protein negatively affecting production of antibiotics in Streptomyces coelicolor A3 (2).

Tetratricopeptide repeat (TPR) domains usually mediate protein-protein interactions. NsdA, one of the 70 proteins containing TPR-like domains in Streptomyces coelicolor A3 (2), was previously found to negatively control sporulation and antibiotic production. Here we show that elimination of SCO7252, which encodes another of these proteins, also caused overproduction of two antibiotics, actinorhodin and CDA, but did not affect morphological differentiation. Disruption of SCO1593, encoding another of the family, had no obvious phenotypic effects. In surface-grown cultures, expression of SCO7252, which was named nsdB, was initiated at about 30 h, like that of nsdA. Analysis in silico of the 70 predicted TPR-like-containing proteins of S. coelicolor showed that 32 of them contained only TPR-like domains, and 25 of the remainder contained additional DNA-binding domains, implying that they might control gene expression directly.

[Cloning of bldAa and the effect on morphological differentiation and avermectins production in Streptomyces avermitilis NRRL8165].

bldA encodes the only tRNA that efficiently translates the rare UUA leucine codon in Streptomyces coelicolor. bldA inactivation leaded to defection in morphological development and production of two of four known antibiotics in S. coelicolor. A bldA homologue, termed bldA. , has been identified in the sequenced genome of Streptomyces avermitilis MA4680. To investigate the function of bldA., genomic DNA of S. avermitilis NRRL8165 was digested with BamH I and the 5 - 6kb was fractioned and ligated with the BamH I digested E. coli plasmid vector pIJ4642 to yield a sub-library. A clone containing bldAa and its flanking sequence was obtained by screening from this genome sub-library. pHL358, a bldA, replacement plasmid, was constructed using the lambdaRED mediated PCR-targeting technique, and conjugated into S. avermitilis NRRL8165.Three bldA-disruption mutant strains (named TW10) were obtained, which showed a bald phenotype, indicating that bldA, controlled the morphological differentiation of S. avermitilis . HPLC analysis of the TW10 fermentation culture showed that TW10 did not synthesize avermectins anymore, suggesting that the synthesis of avermectins were dominated by bldAa . There are TTA codons within aveA3 and aveR of the avermectin biosynthesis gene cluster, suggesting that the translation of the two genes may depend on bldAa, which were consistent with the experimental results.

[Activation of silent antibiotic synthesis in Streptomyces lividans by disruption of a negative regulator nsdA, a gene conserved in Streptomyces].

The global regulatory gene, nsdA, negatively regulates antibiotics production in Streptomyces coelicolor. Southern blot experiment, using an nsdA fragment of S. coelicolor as probe, indicated that nsdA gene existed in many Streptomyces. Primers were designed based on the published sequences of S. coelicolor and S. avermitilis. PCR amplification and sequencing showed that nsdA in Streptomyces was conservative and that of S. lividans ZX64 has a 100% identity in the nucleotide sequence comparing with that of S. coelicolor A3 (2). The nsdA disrupted mutant of S. lividans was constructed named as WQ2. WQ2 was able to produce actinorhodin but the wild-type strain ZX64 did not, which has a silent gene cluster contributing to the biosynthesis of actinorhodin. However, the ability was lost when another copy of the wild nsdA gene was introduced into WQ2. All the results above indicate that nsdA homologous gene is wildly existent and conserved in Streptomyces. And it plays a role in negatively regulating the actinorhodin synthesis in S. lividans and disruption of it can activate the silent gene cluster.

[Effect of lat disruption on clavulanic acid production].

A 1.8kb fragment of lat was obtained from Streptomyces clavuligerus 27064, and replacement plasmid of pXAL1 and pXAL2 were constructed. PXAL1 and pXAL2 were used to disrupt the lat gene by bi-parental conjugation from E. coli to Streptomyces clavuligerus. A Am(r)Thio(S) transformant, named as XAL863, was obtained. The genome of Streptomyces clavuligerus 27064 and XAL863 was analyzed by southern blot technique, and the activity of lysine epsilon-aminotransferase in the two strains was also tested. Both results proved that the lat was disrupted in the XAL863. Streptomyces clavuligerus and XAL863 were cultured in the shaken flask respectively, and the production of clavulanic acid was analyzed by HPLC with the different incubation time interval, and the yield was approximately 1.8 times higher in the XAL863 at their highest production point.

[Construction of a temperature inducible shuttle expression vector and its application in Streptomyces].

pHZ1080, an E. coli-Streptomyces shuttle expression vector was constructed in order to explore the utilization of lambda phage regulated expression elements in Streptomyces. A 2.7 kb polyketide synthase (PKS) gene from Streptomyces sp. FR-008 was inserted into downstream of lambda phage promoter (PR) to give the shuttle plasmid, pHZ1067. The PKS protein was expressed in Streptomyces lividans carrying pHZ1067 in a heat-dependent manner, as it did in E. coli. The PKS protein expressed in both hosts with same molecular weight was detected by SDS-PAGE and Western-blot. The successful heat-induced expression of PKS suggested that pHZ1080 was useful and convenient for heat-induced expression of heterologous genes in both E. coli and Streptomyces.