Key Laboratory of Biomass Chemical Engineering (Ministry of Education), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
Institute of Biological Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, People's Republic of China.
Appl Microbiol Biotechnol. 2017 Jun;101(12):4915-4922. doi: 10.1007/s00253-017-8259-8. Epub 2017 Apr 3.
The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (μ ) and specific PQQ formation rate (μ ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.
本研究系统考察了 pH 控制策略和发酵操作方式对甲基杆菌（Methylobacillus sp.）CCTCC M2016079 合成吡咯喹啉醌（PQQ）的影响。首先，在 5.3 至 7.8 等不同 pH 值条件下进行摇瓶培养和台式发酵。在对比细胞比生长速率（μ）和 PQQ 比生成速率（μ）的动力学分析后，观察到细胞生长和 PQQ 生物合成的最佳 pH 值存在差异，这促使我们开发了一种新型两段式 pH 控制策略。在此 pH 转换过程中，将发酵液的 pH 值控制在 6.8 以促进前 48 h 的细胞生长，然后切换至 5.8 以增强 PQQ 合成，直至发酵结束。通过应用该 pH 转换控制策略，在台式发酵罐中 PQQ 的最大产量提高到 158.61 mg/L，比最适恒 pH 发酵提高了约 44.9%。进一步的补料分批研究表明，通过在两段式 pH 过程中以 11.5 mL/h 的速度补加甲醇，可将 PQQ 的产量从 183.38 mg/L 提高到 272.21 mg/L。同时，产物得率也从 2.02 mg/L/h 提高到 2.84 mg/L/h。为了在 pH 转换阶段支持细胞生长，进行了甲醇和酵母提取物的联合补料，得到了最高的 PQQ 浓度（353.28 mg/L）和产物得率（3.27 mg/L/h）。本工作揭示了我们开发的简单、经济策略在大规模生产 PQQ 方面的潜力。
