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.

Streptomyces diastatochromogenes 1628, capable of producing toyocamycin (TM), has exhibited a potential biocontrol effect in inhibiting the development of phytopathogens in the agriculture field. In this study, an efficient transformation system was developed using the intergeneric conjugation. This was achieved by optimization of experimental parameters. Under optimal conditions, a maximal conjugation frequency of 4.1 × 10(-4) per recipient was obtained. In order to heterologously express the gene vgb encoding Vitreoscilla hemoglobin in S. diastatochromogenes 1628, we placed vgb under the control of the constitutive promoter PermE(*) and constructed plasmid pIB139-vgb. This plasmid was integrated into the chromosome of S. diastatochromogenes 1628 using intergeneric conjugation established above. Finally, strain 1628-VHB-23 with the highest TM production was screened. Results indicated that expression of vgb gene had always significantly promoted the cell growth and TM production in S. diastatochromogenes 1628 under different dissolved oxygen conditions. In particular, under the limited aerobic condition, strain 1628-VHB-23 obtained 33.3 % more DCW and produced 210 % more TM in 7-l fermentor as compared with the wild-type strain.

Toyocamycin exhibits effective biological activities for use against plant pathogenic fungi thanks to its structural similarity to nucleoside. It has been recognized as a promising agricultural antibiotic utilized in controlling the occurrence of plant diseases. In our previous study, a strain that was isolated was identified and designated as Streptomyces diastatochromogenes whose major secondary metabolite was toyocamycin, but the production was largely limited. Protoplast transformation is a useful technique in the improvement of streptomycete. In this study, we optimized some key factors necessary for protoplast formation, regeneration, and transformation of S. diastatochromogenes. When mycelium was cultivated in CP medium with 1 % glycine, harvested at 48 h old, and then treated with 3 mg lysozyme/mL in P buffer for 1 h, the greatest regeneration frequency (42.5 %) of protoplasts was obtained. By using 1 × 10(9)/mL protoplasts with polyethylene glycol 1000 at a concentration of 30 % (w/v), the best performance of protoplast transformation efficiency was 4.8 × 10(3)/μg DNA transformants.

It is generally known that genes dhaB and dhaT are responsible for 1,3‐propanediol (1,3‐PD) production in the presence of glycerol in Klebsiella pneumoniae and these genes are organized in one operon. In the present study, a genetic means of increasing the enzyme activities of 1,3‐PD formation pathway through the over‐expression of 1,3‐PD opeorn was performed in K. pneumoniae S6. The recombinant strain S6‐PD showed 27‐ and 15‐fold increase in enzymatic activities of DhaB and DhaT, respectively with respect to wild‐type strain while failed to improve the 1,3‐PD yield due to the inadequacy of cofactor NADH. Therefore, in order to increase NADH availability, a NADH regeneration system was constructed by heterologous expression of NAD+‐dependent formate dehydrogenase gene (fdh1) from Candida boidinii and introduced into S6‐PD to investigate its effects on the glycerol utilization and 1,3‐PD formation. The results demonstrated that the increase of NADH availability could efficiently improve glycerol metabolism and promote 1,3‐PD yield.

Toyocamycin (TM) shows antifungal activity and can be used to treat plant diseases. TM is produced by Streptomyces diastatochromogenes 1628, but the production rate is always very low. Recently, the gene toyG was cloned; however, nothing was known about the influence of toyG on TM production. In this study, the rational combined co‐expression of toyG with frr encoding a ribosome recycling factor and vgb encoding Vitreoscilla hemoglobin was performed. Introduction of these genes in double‐ or triple‐combination into strain S. diastatochromogenes 1628 led to greatly increased TM production. Transcription analysis revealed that higher TM production in the constructed recombinant strains resulted from an increase in transcriptional levels of toyG gene. At the end, among all these different recombinant strains, the strain S. diastatochromogenes 1628‐VGF, harboring vgb, frr, and toyG placed under the control of PermE*, exhibited the largest increase in TM production. Notably, TM produced by S. diastatochromogenes 1628‐VGF reached the highest level of 319.2 mg/L at 72 h, while TM yield of the original strain 1628 was 147.1 mg/L at 84 h. The high‐yield, energy‐saving strain 1628‐VGF constructed in this study shall be beneficial for the industrial production of TM.