A pair of two－component regulatory genes ecrA1／A2 in S. coelicolor

Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor. DNA microarray analysis revealed that ecrA1/A2, which mapped at distant sites from red locus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway, ecrA1 and ecrA2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover. Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain. However, the change of the actinorhodin (Act) production level was not significant compared with wild type. Thus, these experiment results confirmed that the two-component system ecrA 1/A2 was positive regulatory element for red gene cluster.

Improved production of spiramycin by mutant Streptomyces ambofaciens

Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S.ambofaciens XC 2-37 were studied. The potency orS. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m^3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

Scale-up of rifamycin B fermentation with Amycolatoposis mediterranei

Study of the effect of dissolved oxygen and shear stress on rifamycin B fermentation with A. mediterranei XC 9-25 showed that rifamycin B fermentation with Amycolatoposis mediterranei XC 9-25 needs high dissolved oxygen and is not very sensitive to shearing stress. The scale-up ofrifamycin B fermentation withA, mediterranei XC 9-25 from a shaking flask to a 15 L fermentor was realized by controlling the dissolved oxygen to above 25% of saturation in the fermentation process, and the potency of rifamycin B fermentation in the 15 L fermentor reached 10 g/L after 6-day batch fermentation. By continuously feeding glucose and ammonia in the fermentation process, the potency of rifamycin B fermentaion in the 15 L fermentor reached 18.67 g/L, which was 86.65% higher than that of batch fermentation. Based on the scale-up principle of constantly aerated agitation power per unit volume, the scale-up of rifamycin B fed-batch fermentation with continuous feed from a 15 L fermentor to a 7 m^3 fermentor and further to a 60 m^3 fermentor was realized successfully. The potency of rifamycin B fermentation in the 7 m^3 fermentor and in the 60 m^3 fermentor reached 17.25 g/L and 19.11 g/L, respectively.

Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia co/i and its “oxidizing” mutant

AIM: To study the influence of redox environment of Escherichia coli ( E. coli) cytoplasm on disulfide bond formation of recombinant proteins.METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF,and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3)and M15[pREP4]respectively. At the same time, both plasmids were transformedinto a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.RESULTS: All recombinant E. colistrains could efficiently produce target proteins. The level of BbFGF in BL21(DE3)was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm,and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3)was higher than its counterpart from BL21(DE3). The ED50of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami[pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv]was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from indusion body in M15[pQE-H Fv] was30-35 mg/L and the affinity constant was 1.98×107 mol/L.There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.

Genetic Analysis of Wild-type Hepatitis A Virus Strains

To clarify the distribution of hepatitis A virus (HAV)genotype in geographical regions of China.Methods Seventeen representative HAV strains were isolated from the stool or serum of hepatitis A patients in different geographical regions.Viral RNA was recovered from stool or serum by proteinase K digestion and phenol-chloroform extraction,followed by ethanol precipitation prior to reverse transcription and polymerase chain reaction(RT-PCR)amplification.The nucleotide sequences of VP1/2A junction region were tested by using a direct sequencing technique.Results A pairwise comparison of sequences within 168 bases at the VP1/2A junction revealed that all the sequences clustered within genotype I.About 53% of strains clustered in genotype IB,with less than 6% variability;while the others clustered in genotype IA,with less than 5.3% variability Sequence hore ology between genotype IA and IB varied from 88.7% to 92.3%. Conclusion Epidemic or sporadic HAV strains in China may belong to HAV genotype IA or IB, Epidemiologically related strains may be identical or closely related in sequence.

An O-glycoside of Sialic Acid Derivative that inhibits Both Hemagglutinin and Sialidase Activities of Influenza Viruses

The compound Neu5Ac3αF-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined.With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus,the inhibitory effect of Neu5Ac3αFDSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 μM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses.The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.