途径优化强化枯草芽胞杆菌合成肝素前体

Optimization of heparosan synthetic pathway in Bacillus subtilis 168

摘要:

肝素前体是化学酶法合成肝素的起点，肝素前体的微生物高效合成具有重要意义。在已构建的产肝素前体的枯草芽胞杆菌 ((1.71±0.08) g/L) 中，分析了UDP-葡萄糖醛酸 (UDP-GlcUA) 途径中关键酶基因 (pgcA、gtaB、tuaD) 以及UDP-乙酰氨基葡糖 (UDP-GlcNAc) 途径中关键酶基因 (glmS、glmM、glmU) 的过量表达对肝素前体产量及其分子量的影响。在此基础上，通过共表达tuaD、gtaB、glmU、glmM和glmS基因，摇瓶中肝素前体产量提高至(2.89±0.11) g/L，分子量为(75.90±1.18) kDa。通过在3 L发酵罐中进行补料分批发酵，肝素前体的产量最终积累到(7.25±0.36) g/L，分子量为(46.66±2.71) kDa，为工业化生产肝素奠定了基础。

Abstract:

Heparosan is the start point for chemoenzymatic synthesis of heparin and it is of great significance to efficiently synthesize heparosan in microorganisms. The effects of overexpressing key enzyme genes of the UDP-glucuronic acid (UDP-GlcUA) pathway (pgcA, gtaB and tuaD) or the UDP-N-acetyl-glucosamine (UDP-GlcNAc) pathway (glmS, glmM and glmU) on the heparosan production and molecular mass were analyzed in the constructed heparosan-producing Bacillus subtilis ((1.71±0.08) g/L). On this basis, heparosan production was increased to (2.89±0.11) g/L with the molecular mass of (75.90±1.18) kDa through co-overexpressing the tuaD, gtaB, glmU, glmM and glmS genes in shake flask cultivation. In the 3 L fed-batch fermentation, heparosan production was improved to (7.25±0.36) g/L with the molecular mass of (46.66±2.71) kDa, providing the potential for heparosan industrial production.