BACKGROUND Caffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far. RESULTS The herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive. CONCLUSIONS The caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

中文翻译：

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大肠杆菌中异源咖啡酸的生物合成受细菌细胞色素P450的酪氨酸氨裂解酶和氧化还原伴侣的选择的影响。

背景技术咖啡酸因其抗氧化活性而在工业上得到认可，因此其可用作抗炎，抗癌，抗病毒，抗糖尿病和抗抑郁药的潜力。传统上，它是在能源密集和苛刻的化学提取条件下与木质化植物材料隔离的。但是，在过去的十年中，微生物细胞工厂已经建立了自下而上的生物合成方法，它们有潜力实现更量身定制和可持续的生产。这些方法中的一种已经在大肠杆菌中实施，并且仅需要通过酪氨酸氨裂合酶和细菌细胞色素P450单加氧酶的作用，将补充的L-酪氨酸进行两步转化。尽管与其他相比，中间体的进料显示出这种异源酶组合的巨大潜力，但迄今为止，利用细菌细胞色素P450仍无法从葡萄糖从头合成咖啡酸。结果本文所述的工作旨在提高该两步转化的效率，以便从葡萄糖确定从头形成咖啡酸。我们实施了酪氨酸氨裂解酶的替代品，据报道，酪氨酸氨裂解酶显示出优异的底物结合亲和力和选择性，并通过改变供电子的氧化还原系统提高了细胞色素P450的效率。通过这种策略，我们能够在96小时内在其他以葡萄糖为唯一碳源的野生型大肠杆菌MG1655（DE3）菌株中实现超过300 µM或47 mg / L咖啡酸的最终滴度。我们观察到氧化还原系统的选择和基因剂量强烈影响细胞色素P450的催化作用。此外，我们成功地应用了网络共享策略，该策略甚至使实际上无效的细胞色素P450 /氧化还原系统组合也具有生产力。结论在这项研究中获得的咖啡酸滴度比报道的未添加L-酪氨酸的野生型大肠杆菌中其他异源咖啡酸途径的滴度高约10％。应用于细胞色素P450的拴系策略似乎对于非天然细胞色素P450 /氧化还原伴侣组合特别有用，并且可能对利用细菌细胞色素P450的其他重组途径有用。我们成功地应用了网络共享策略，即使实际上几乎没有生产能力的Cytochrome P450 /氧化还原系统组合也可以生产。结论在这项研究中获得的咖啡酸滴度比报道的未添加L-酪氨酸的野生型大肠杆菌中其他异源咖啡酸途径的滴度高约10％。应用于细胞色素P450的束缚策略似乎对于非天然细胞色素P450 /氧化还原伴侣组合特别有用，并且可能对利用细菌细胞色素P450的其他重组途径有用。我们成功地应用了网络共享策略，即使实际上几乎没有生产能力的Cytochrome P450 /氧化还原系统组合也可以生产。结论在这项研究中获得的咖啡酸滴度比报道的未添加L-酪氨酸的野生型大肠杆菌中其他异源咖啡酸途径的滴度高约10％。应用于细胞色素P450的束缚策略似乎对于非天然细胞色素P450 /氧化还原伴侣组合特别有用，并且可能对利用细菌细胞色素P450的其他重组途径有用。结论在这项研究中获得的咖啡酸滴度比报道的未添加L-酪氨酸的野生型大肠杆菌中其他异源咖啡酸途径的滴度高约10％。应用于细胞色素P450的束缚策略似乎对于非天然细胞色素P450 /氧化还原伴侣组合特别有用，并且可能对利用细菌细胞色素P450的其他重组途径有用。结论在这项研究中获得的咖啡酸滴度比报道的未添加L-酪氨酸的野生型大肠杆菌中其他异源咖啡酸途径的滴度高约10％。应用于细胞色素P450的束缚策略似乎对于非天然细胞色素P450 /氧化还原伴侣组合特别有用，并且可能对利用细菌细胞色素P450的其他重组途径有用。