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

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.

Optimization of electroporation conditions for toyocamycin producer Streptomyces diastatochromogenes 1628.

Because of its structural similarity to nucleoside, toyocamycin exhibits potential of wide application and various biological activities. Streptomyces diastatochromogenes 1628, capable of producing toyocamycin, has exhibited a potential biocontrol effect in inhibiting the development of phytopathogens in the agriculture field. An efficient transformation system is a prerequisite for genetic and molecular study of S. diastatochromogenes 1628. In this study, we optimized experimental factors involved in the electroporation transformation process. Key features of this procedure, including collection of cells at the mid-log phase stage and the treatment of cells with lysozyme and penicillin G prior to the electroporation and recovery medium and time, produced the greatest increase in the efficiency and consistency of results. The transformation efficiency also depends on field strength, cell concentration, and plasmid DNA quantity. Under the optimal conditions, a maximal efficiency of (3 ± 0.4) × 10(4) µg(-1) DNA was obtained. The development of transformation method for S. diastatochromogenes 1628 will foster genetic manipulation of this important strain.

Formation, regeneration, and transformation of protoplasts of Streptomyces diastatochromogenes 1628.

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.

Development of intergeneric conjugal gene transfer system in Streptomyces diastatochromogenes 1628 and its application for improvement of toyocamycin 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.

Effects of NADH availability on the Klebsiella pneumoniae strain with 1,3-propanediol operon over-expression.

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.

Development of a novel recombinant strain Zygosacharomyces rouxii JL2011 for 1,3-propanediol production from glucose.

1,3-propanediol (1,3-PDO) is one of the most important industrial chemicals due to its highly desired properties and its wide applications as a key component of the emerging polymer industry. Biotechnology route has been one of the most interesting methods for 1,3-PDO production, whereas, the dha genes were essential to 1,3-PDO biosynthesis. In this study, we cloned and placed the dha cassettes under the control of a glyceraldehyde 3-phosphate dehydrogenase gene promoter pGAP and homologous ZrFPS1 gene promoter pZrfps1; these two promoters were further integrated into the chromosome of Z. rouxii JL2011 to generate recombinant strain JL2011-GZ and JL2011-ZZ, respectively. The results showed that the two strains could produce 1,3-PDO from glucose with a final yield of 6.9 and 10.3 g/l, respectively. The engineered strain JL2011-ZZ showed a 2.3- and 1.5-fold increase in the specific activities and final concentration of 1,3-PDO, respectively, with respect to JL2011-GZ. Batch fermentation with aerobic/micro-aerobic combined strategy of JL2011-ZZ resulted a titer of 17.1 g/l and a yield from glucose of 8.6 %. These results demonstrated that JL2011-ZZ would be a potential strain for 1,3-PDO production from glucose.

Antifeedant and insecticidal effects of mandelic acid on the brown planthopper Nilaparvata lugens Stål.

To study the effects of mandelic acid (MA) on the brown planthopper (BPH), Nilaparvata lugens, the survival rate and behaviour of BPH fed on an artificial diet with different dosages of MA was observed. The survival rate of BPH decreased with the increase of the MA concentration and feeding time. In contrast to the control, the survival rate of BPH 72 h after feeding decreased significantly. Electrical penetration graph (EPG) data indicated that MA absorbed by the rice plant from Kimura B solution significantly affected the feeding behaviour of BPH. At the concentrations of 0.1, 0.5, and 1.0 mg/ml, duration of the phloem ingestion of BPH decreased from 115.34 min (control) to 30.41, 7.63, and 0.36 min, respectively. Periods of xylem ingestion of MA-treated BPH were significantly shorter than those of the control (50.44 min). Moreover, BPH spent more time walking around or being at rest on MA-treated rice plants, as well as in stylet activities. The GST (glutathione S-transferase) activity of BPH increased with the increasing MA concentration, while the GPX (glutathione peroxidases) activity did not change significantly. The results indicate that MA has an antifeedant and insecticidal effect on BPH.