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Abstract：
The rising demand of bio-vanillin and the possibility to use microbial biotransformation to produce this compound from agroindustrial byproducts are economically attractive. However, there are still several bottlenecks, including substrate and product toxicity, formation of undesired products and genetic stability of the recombinant strains, that impede an efficient use of recombinant Escherichia coli strains to make the whole process cost effective. To overcome these problems, we developed a new E. coli strain, named FR13, carrying the Pseudomonas genes encoding feruloyl-CoA synthetase and feruloyl-CoA hydratase/aldolase integrated into the chromosome and, using resting cells, we demonstrated that the vanillin yield and selectivity were strongly affected by the physiological state of the cells, the temperature used for the growth and the recovery of the biomass and the composition and pH of the bioconversion buffer. The substrate consumption rate and the vanillin yield increased using a sodium/potassium phosphate buffer at pH 9.0 as bioconversion medium. Optimization of the bioprocess variables, using response surface methodology, together with the use of a two-phase (solid-liquid) system for the controlled release of ferulic acid allowed us to increase the vanillin yield up to 28.10 ± 0.05 mM. These findings showed that recombinant plasmid-free E. coli strains are promising candidates for the production of vanillin at industrial scale and that a reduction of the cost of the bioconversion process requires approaches that minimize the toxicity of both ferulic acid and vanillin.
摘要：
对生物香兰素的需求不断增长，以及使用微生物生物转化从农业工业副产品生产该化合物的可能性在经济上具有吸引力。然而,还有几个瓶颈，包括底物和产品毒性，不需要的产物的形成和重组菌株的遗传稳定性，这阻碍了重组大肠杆菌菌株的有效利用，从而使整个过程具有成本效益。为了克服这些问题,我们开发了一种新的大肠杆菌菌株，名为FR13，携带整合到染色体中的编码阿魏酸辅酶A合成酶和阿魏酸辅酶A水合酶/醛缩酶的假单胞菌基因，使用静息细胞，我们证明了香草醛的产量和选择性受到细胞生理状态的强烈影响，用于生长和回收生物质的温度以及生物转化缓冲液的组成和pH。使用pH9.0的磷酸钠/钾缓冲液作为生物转化培养基，底物消耗速率和香草醛产量增加。生物过程变量的优化，使用响应面方法，与使用两相（固-液）系统控制阿魏酸的释放一起，使我们能够将香草醛的产量提高到28.10±0.05mM。这些发现表明,无重组质粒的大肠杆菌菌株是工业规模生产香草醛的有希望的候选物,并且生物转化过程的成本的降低需要使阿魏酸和香草醛的毒性最小化的方法。
