Department of Pharmaceutical Engineering, SunMoon University, Asansi 336-708, Korea.
J Microbiol Biotechnol. 2010 Jan;20(1):146-52.
Streptomyces clavuligerus NRRL3585 produces a clinically important ss-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into deleted mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared to the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared to the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.
棒链丝菌 NRRL3585 产生一种临床上重要的 ss-内酰胺酶抑制剂，克拉维酸（CA）。为了提高 CA 的产量，敲除棒链丝菌 NRRL3585 中的甘油醛-3-磷酸脱氢酶（GAPDH）基因以克服有限的甘油醛-3-磷酸池；共转化 ccaR（CA 生物合成操纵子的特异性调节因子）和 claR（Lys 型转录激活因子）基因的复制和整合表达到缺失突变体中以提高克拉维酸的产量。我们构建了两个重组质粒来增强棒链丝菌 NRRL3585 gap1 缺失突变体中 CA 的产量：pHN11 用于 ccaR-claR 的过表达，而 pHN12 则用于它们的染色体整合。与 gap1 缺失突变体相比，pHN11 和 pHN12 转化子分别将 CA 的产量提高了 2.59 倍和 5.85 倍。为了进一步提高 CA 的产量，我们向 pHN11 和 pHN12 转化子中添加了鸟氨酸和甘油。与 gap1 缺失突变体相比，鸟氨酸分别使 pHN11 和 pHN12 转化子中的 CA 产量增加了 3.24 倍和 6.51 倍，甘油分别增加了 2.96 倍和 6.21 倍，鸟氨酸和甘油一起分别使 CA 产量增加了 3.72 倍和 7.02 倍。
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