Overexpression of ribosome recycling factor is responsible for improvement of nucleotide antibiotic-toyocamycin in Streptomyces diastatochromogenes 1628

Abstract

Ribosome recycling factor (RRF), a product of the frr gene, is responsible for the dissociation of ribosomes from messenger RNA after the termination of translation. In order to overexpress frr gene in the toyocamycin (TM) producer Streptomyces diastatochromogenes 1628, we cloned and placed the gene under the control of the constitutive promoter PermE(*). The resulting plasmid pIB139-frr was integrated into the chromosome of S. diastatochromogenes 1628 by conducting intergeneric conjugation. The strain S. diastatochromogenes 1628 containing pIB139-frr (1628-FRR) showed a 33.3 % increase in cell growth and a 46 % increase in TM production compared to wild-type strain 1628 when cultivated in a 7 l fermentor. In addition, it was possible to shorten the fermentation time from 84 to 72 h. Furthermore, by conducting reverse transcription polymerase chain reaction (RT-PCR) analysis, we discovered that the transcriptional levels of regulatory gene adpA-sd, toyF, and toyG involved in TM biosynthesis were enhanced in S. diastatochromogenes 1628-FRR compared to S. diastatochromogenes 1628. In addition, by using a fluorescent intensity reporter system, which is based on the green fluorescent protein (GFP), and by using Western blot analysis, we revealed that overexpression of frr also strongly promoted protein biosynthesis in late growth phase. These findings confirmed that by increasing copy number of frr gene, it is a useful approach to improve antibiotic production.

Engineering Streptomyces diastatochromogenes 1628 to increase the production of toyocamycin

“…The integration of plasmid pIB139‐ toy G into the chromosome of S. diastatochromogenes 1628 was verified by PCR. As determined by qPCR, copy number of toy G in S. diastatochromogenes 1628‐TOYG was two times as much as the copy number in the wild‐type strain (data no shown), indicating that the plasmid containing toyG had been integrated into the unique chromosomal attachment site att B, this was consistent with that of prior work . The specific activity of TOYG of S. diastatochromogenes 1628‐TOYG and wild‐type strain was 3.4 U/mg protein and 2.3 U/mg protein, respectively (Table ).…”

“…E. coli/Streptomyces shuttle vector pIB139 was gifted by Prof. Deng Z. X. Plasmids pIB139‐ vgb and pIB139‐ frr were previously constructed by our laboratory . Plasmid pIB139‐ toy G harboring toy G was constructed as the following: The toy G gene was amplified from genome DNA of S. diastatochromogenes 1628 using primers P1 and P2 (Table ), digested with Nde I and Not I, and ligated into Nde I/ Not I site of plasmid pIB139 (Fig.…”

“…The extraction of RNA and analysis of toy G gene transcriptional level were performed as described previously .…”

“…An increased expression of frr in S. diastatochromogenes 1628 was recently found to cause enhancement of protein synthesis during the late growth phase and subsequent over‐production of TM. It was observed that the transcriptional level of toy G was significantly improved in the recombinant strain S. diastatochromogenes 1628‐FRR compared to the wild strain and it was presumed that toy G was involved in TM biosynthesis in S. diastatochromogenes 1628.…”

Identification of a gene from Streptomyces rimosus M527 negatively affecting antibiotic biosynthesis and morphological differentiation

“…This result confirmed that NsdA sr is not only a homologue of NsdA sc but also has similar effects. Subsequently, by heterologous expression of nsdA sr , similar negative effects on morphological differentiation and antibiotic production were observed in S. diastatochromogenes 1628, which is considered a significant industrial producer of the nucleoside antibiotic, TM [35,36].…”