Engineered biosynthesis of milbemycins in the avermectin high-producing strain Streptomyces avermitilis.

BACKGROUND: Milbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis. Milbemycins possess strong acaricidal, insecticidal, and anthelmintic activities but low toxicity. Due to the high commercial value of the milbemycins and increasing resistance to the avermectins and their derivatives, it is imperative to develop an efficient combinatorial biosynthesis system exploiting an overproduction host strain to produce the milbemycins and novel analogs in large quantities. RESULTS: The respective replacement of AveA1 and AveA3 (or module 7 in AveA3) of the avermectin polyketide synthase (PKS) in the avermectin high-producing strain S. avermitilis SA-01 with MilA1 and MilA3 (or module 7 in MilA3) of the milbemycin PKS resulted in the production of milbemycins A3, A4, and D in small amounts and their respective C5-O-methylated congener milbemycins B2, B3, and G as major products with total titers of approximately 292 mg/l. Subsequent inactivation of the C5-O-methyltransferase AveD led to a production of milbemycins A3/A4 (the main components of the commercial product milbemectin) in approximately 225 and 377 mg/l in the flask and 5 l fermenter culture, respectively, along with trace amounts of milbemycin D. CONCLUSIONS: We demonstrated that milbemycin biosynthesis can be engineered in the avermectin-producing S. avermitilis by combinatorial biosynthesis with only a slight decrease in its production level. Application of a similar strategy utilizing higher producing industrial strains will provide a more efficient combinatorial biosynthesis system based on S. avermitilis for further enhanced production of the milbemycins and their novel analogs with improved insecticidal potential.

Medium optimization and application of an affinity column chromatography for streptomyces griseus trypsin production from the recombinant Streptomyces griseus.

The expression vector pWHM3-TR1R2, which contains sprT encoding Streptomyces griseus trypsin (SGT) and two positive regulatory genes (sgtR1 and sgtR2), was introduced into S. griseus IFO13350 and the productivity of SGT by the transformant was investigated in various media. Among the tested media, Ferm-0 gave 1.4 times more trypsin activity than C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 medium with 2% dextrin and 1% tryptone (named as Ferm-II medium) yielded significantly enhanced trypsin activity, by 4.1-fold, than that of Ferm-0. For simplifying the purification process, the cultural supernatant of S. griseus transformant in Ferm-II medium was fractionated with ammonium sulfate (25%-55%), and then applied to Hitrap benzamidine FF affinity column chromatography. The specific activity of the purified SGT by one-step column chromatography was 69,550 unit/mg protein, and the overall purification yield was above 8%, which was more effective than the methods of previous reports. The trypsin activity of the purified SGT was most active at pH 8.0 and 50 degrees C, and maintained their activities between pH 7.0 and pH 9.0, and up to 70 degrees C. These enzymatic properties were very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

Development of anangiogenesis-focused cDNA chip and validation of its functionality.

DNA chip has been used as a powerful tool to study the genetic reprogramming of cells and its link to cellular phenotype such as angiogenesis. To evaluate the angiogenesis related genetic reprogramming more efficiently, we here developed an angiogenesis-focused cDNA chip containing 153 angiogenesis related genes arrayed in duplicate on a slide glass. In order to validate the functionality of the angiogenesis-focused cDNA chip, we examined gene expression profiles in HT1080 cells treated with either fetal bovine serum, a well known pro-angiogenic factor, or trichostatin A, a known angiogenesis inhibitor, using the cDNA chip. All duplicate data from the analysis are well matched with each other and gene expression profiles are well consistent with previously reported data. These results demonstrate that the angiogenesis-focused cDNA chip developed here can be a useful tool towards angiogenesis- related researches.

Metabolic engineering of rational screened Saccharopolyspora spinosa for the enhancement of spinosyns A and D production.

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-O-methylated rhamnose which are derived from S-adenosylmethionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was 244 mg L(-1) and 129 mg L(-1), which was 4.88-fold and 4.77-fold higher than that in the wild-type (50 mg L(-1) and 27 mg L(-1)), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong ermE* promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Herewith, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to 372/217 mg L(-1) that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

Molecular cloning of low-temperature-inducible ribosomal proteins from soybean.

Three ribosomal protein genes induced by low-temperature treatment were isolated from soybean. GmRPS13 (742 bp) encodes a 17.1 kDa protein which has 95% identity with the 40S ribosomal protein S13 of Panax ginseng (AB043974). GmRPS6 (925 bp) encodes a 28.1 kDa protein which has 94% identity with the 40S ribosomal protein S6 of Asparagus officinalis (AJ277533). GmRPL37 (494 bp) encodes a 10.7 kDa protein which has 85% identity with the 60S ribosomal protein L37 of Arabidopsis thaliana (AF370216). The expression of these ribosomal protein genes started to increase 3 d after low-temperature treatment, whereas the cold-stress protein src1 was highly induced from the first day. Such late response of these ribosomal protein genes may be due to secondary signals during cold adaptation. The induction of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under low-temperature conditions.

Variability of chromosomal DNA contents in maize (Zea mays L.) inbred and hybrid lines.

The flow karyotypes of different maize (Zea mays L.) inbred and hybrid lines were analyzed. The accumulation and isolation of large quantities of high-quality metaphase chromosomes from root tips was achieved from many kinds of maize lines. The chromosome suspensions were prepared by a simple slicing method from synchronized maize root tips and analyzed by flow cytometry. Variations of experimental flow karyotypes were detected among inbred and hybrid lines in terms of the positions and/or the numbers of chromosome peaks. The 2C DNA amount among eight inbred lines ranged from 5.09 to 5.52 pg. The selection of appropriate maize lines is critical for sorting specific single chromosome types. At least five different chromosome types can be discriminated and sorted from five maize lines. The variability of DNA content in maize chromosome 1 was 9.1%, ranging from 0.685 to 0.747 pg. Differences were detected in the DNA content of homologous chromosome 1 of hybrid lines.

Flow karyotypes and chromosomal DNA contents of genus Triticum species and rye (Secale cereale).

The flow cytometry and chromosome imaging method were jointly used for analyzing genome content and chromosomal DNA content of hexaploid wheat (AABBDD), hexaploid triticale (AABBRR), tetraploid wheat (AABB), and AA, BB, DD genome donors and RR genome rye. Their genome sizes were 34.4 pg, 40.9 pg, 26.2 pg, 12.1 pg, 13.7 pg, 10.5 pg, and 16.9 pg, respectively. The 2C nuclear DNA content of BB genome donor with 13.7 pg was the highest value among the other genome donors, AA or DD. The genome content of tetraploid wheat, unlike hexaploid wheat or hexaploid triticale, was larger than the sum of the genomes of AA and BB genome donors. The DNA content of each chromosome ranged from 1.22 pg in DD genome donor to 2.61 pg in rye. Each chromosome peak was divided into three to four groups. Only one chromosome was included in the highest chromosomal DNA peak in hexaploid wheat, tetraploid wheat, DD genome donor and rye but two chromosomes in AA, BB genome donors, and hexaploid triticale. Correlation between 2C nuclear DNA content and chromosome density volume was the highest value compared with the other chromosomal parameters of chromosome area, or chromosome length.