Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, London, UK.
Biotechnol Bioeng. 2012 Sep;109(9):2179-89. doi: 10.1002/bit.24512. Epub 2012 Apr 17.
This article describes the first reported microwell whole-cell bioconversion using a water immiscible substrate that matches the specific activity and yield achieved in a 1.2 L stirred tank bioreactor. Maximum yields of 0.6 g/L(total) 1-dodecanol achieved in 24 h compare favorably to 0.28 g/L(total) 1-dodecanol after 48 h obtained in a stirred tank reactor. Using the microwell platform we present a rapid and systematic approach to identify the key bottlenecks in the bio-oxidation of long-chain alkanes using Escherichia coli expressing the alkane hydroxylase (alkB) complex. The results indicate that mass transfer rates limit productivity in the n-dodecane bio-oxidation system, rather than inherent enzyme activity. Furthermore, substrate solubility, oxygen availability and glucose concentration act cooperatively to affect the amount of by-product, dodecanoic acid. Optimizing these factors using response surface methodology enabled specific yields of 1-dodecanol to increase eightfold and overoxidation to dodecanoic acid to be reduced from 95% to 55%. This resulted in specific activities of 10.4 µmol/min/g(dcw) on n-dodecane; approximately 50% of the 21 µmol/min/g(dcw) obtained with n-octane. For the first time, this in vivo rate difference is within the range reported for the purified enzyme. Finally, the results obtained also provide strong evidence that the mechanism of E. coli interaction with alkanes is mainly via uptake of alkanes dissolved in the aqueous phase rather than by direct cell-droplet contact.
本文描述了首次使用不溶于水的基质进行微孔全细胞生物转化的报道，该基质的比活性和产率与在 1.2 L 搅拌罐生物反应器中获得的结果相匹配。在 24 h 内达到的 1-十二醇最大产率为 0.6 g/L（总量），与在搅拌罐反应器中 48 h 后获得的 0.28 g/L（总量）1-十二醇相比，这是有利的。使用微孔平台，我们提出了一种快速而系统的方法，用于鉴定大肠杆菌表达烷烃羟化酶（alkB）复合物的长链烷烃生物氧化中的关键瓶颈。结果表明，在正十二烷生物氧化体系中，传质速率限制了生产力，而不是固有酶活性。此外，底物溶解度、氧气可用性和葡萄糖浓度协同作用，影响副产物十二酸的量。通过响应面法优化这些因素，使 1-十二醇的比产率提高了 8 倍，过氧化物化为十二酸的比例从 95%降低到 55%。这使得 n-十二烷的比活性达到 10.4 µmol/min/g（dcw），接近 n-辛烷的 21 µmol/min/g（dcw）的 50%。这是首次将这种体内速率差异报告在纯化酶的范围内。最后，结果还提供了强有力的证据，证明大肠杆菌与烷烃相互作用的机制主要是通过吸收溶解在水相中的烷烃，而不是通过直接的细胞液滴接触。
