Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France.
Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Universite Evry, Université Paris-Saclay, Evry, France.
Biotechnol Bioeng. 2019 Nov;116(11):2852-2863. doi: 10.1002/bit.27133. Epub 2019 Sep 3.
The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L ·h ) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L ·h ).
通过独立控制每种酶的生产水平，提高了一种多功能体内级联反应的效率，该级联反应涉及一种从生物多样性搜索中获得的、具有广泛底物特异性的醇脱氢酶和 Baeyer-Villiger 单加氧酶，以生产 ε-己内酯和 3,4-二氢香豆素。通过调整大肠杆菌质粒的每个细胞的拷贝数来实现这一目标。我们从观察到的一般与产生的酶的量相关的现象开始，并证明通过明智地选择质粒，可以改进体内多酶系统，其中限速步骤的酶的较低活性被更高的浓度所平衡。通过对质粒类型的无预设选择方法，我们观察到了积极和消极的协同效应，有时是出乎意料的，这取决于酶和质粒的组合。培养条件的实验优化使我们能够在 0.5-L 规模上实现环己醇（16mM）和 1-茚醇（7.5mM）的完全转化。环己醇的转化率为 80%（0.7g ε-己内酯，生产力为 244mg·L ·h ），而 1-茚醇的转化率为 60%（0.3g 3,4-二氢香豆素，生产力为 140mg·L ·h ）。
