大肠杆菌离子束诱变结合呼吸商在线实时调控高产聚唾液酸

Breeding of Escherichia coli by Ion Beam Mutagenesis Combined with RQ On-line Regulation for High-Efficient Production of Polysialic Acid

Escherichia coli ATCC13027能产生聚唾液酸(polysialic acid,PSA),为实现其产业化应用,采用菌种诱变结合发酵工艺优化以提高PSA产量。首先采用低能氮离子束注入诱变获取不同产量的变异菌株,然后比较3株不同产量菌株在发酵过程呼吸商(respiration quotient,RQ)的变化规律,最后根据玉米浆干粉脉冲式补入对RQ的影响制定相应的补料策略。结果表明:离子束注入量选择10×1014 ion/cm2时,得到1株PSA产量高达3.94 g/L的突变株,比出发菌株产量提高了36.81%。通过比较3株菌的RQ变化规律发现PSA生产速率最高时对应RQ值最低,而脉冲式补入玉米浆干粉对RQ会产生显著影响(P<0.01),因此在50 L罐上发酵16 h后开始流加玉米浆干粉将RQ控制在0.8,最终PSA产量达到12.83 g/L,比对照提高了45.30%,比出发菌株产量提高了88.95%。本研究为PSA产业化提供了优良的菌株及在线实时调控PSA生产的思路,为PSA产业化提供一定理论支持。

Escherichia coli ATCC13027 is a strain capable of producing polysialic acid(PSA). In order to put it into industrial application, strain mutagenesis combined with optimization of the fermentation process was used to increase the PSA yield. Firstly, low energy nitrogen ion beam injection was performed to obtain different strains with varied PSA yields and three different strains were chosen out. Then the respiration quotient(RQ) curves of the three strains were compared during the fermentation process. Finally, a strategy for feeding corn steep powder(CSP) was formulated based on the effect of pulsed feeding of CSP on the RQ. Results showed that the highest PSA titer attained was 3.94 g/L, which was increased by 36.81% as compared with that of the wild strain when the ion beam dose was set as 10 × 1014 ion/cm2. The RQ value of the strain with the highest yield was the lowest during the rapid synthesis phase of PSA. Considering that pulsed feeding of CSP had a significant effect on RQ(P < 0.01), CSP was fed starting from 16 h to control RQ around 0.8 in a 50 L fermentor and as a result, PSA titer reached 12.83 g/L, which was 45.30% and 88.95% higher than that of the control and the original strain, respectively. The high PSA yield strain and the strategy of real-time online regulation for PSA production presented in this study provide theoretical support for PSA industrialization.