Department of Solar Materials, Helmholtz-Centre for Environmental Research - UFZ, Permoserstraße 15, Leipzig, Saxony, 04318, Germany.
Biotechnol J. 2020 Nov;15(11):e2000091. doi: 10.1002/biot.202000091. Epub 2020 Sep 6.
The current industrial production of polymer building blocks such as ε-caprolactone (ε-CL) and 6-hydroxyhexanoic acid (6HA) is a multi-step process associated with critical environmental issues such as the generation of toxic waste and high energy consumption. Consequently, there is a demand for more eco-efficient and sustainable production routes. This study deals with the generation of a platform organism that converts cyclohexane to such polymer building blocks without the formation of byproducts and under environmentally benign conditions. Based on kinetic and thermodynamic analyses of the individual enzymatic steps, a 4-step enzymatic cascade in Pseudomonas taiwanensis VLB120 is rationally engineered via stepwise biocatalyst improvement on the genetic level. It is found that the intermediate product cyclohexanol severely inhibits the cascade which could be optimized by enhancing the expression level of downstream enzymes. The integration of a lactonase enables exclusive 6HA formation without side products. The resulting biocatalyst shows a high activity of 44.8 ± 0.2 U g and fully converts 5 mm cyclohexane to 6HA within 3 h. This platform organism can now serve as a basis for the development of greener production processes for polycaprolactone and related polymers.
目前，ε-己内酯（ε-CL）和 6-羟基己酸（6HA）等聚合物单体的工业生产是一个多步骤的过程，与产生有毒废物和高能耗等关键环境问题有关。因此，人们需要更环保、更可持续的生产途径。本研究涉及生成一种平台生物，该生物可在无副产物和环境友好条件下将环己烷转化为这些聚合物单体。基于对单个酶步骤的动力学和热力学分析，通过逐步在遗传水平上改进生物催化剂，对 Pseudomonas taiwanensis VLB120 中的 4 步酶级联反应进行了合理设计。研究发现，中间产物环己醇会严重抑制级联反应，通过提高下游酶的表达水平可以对其进行优化。整合内酯酶可以实现无副产物的 6HA 选择性形成。所得生物催化剂的活性为 44.8±0.2 U g，可在 3 小时内完全将 5mm 环己烷转化为 6HA。该平台生物可作为开发更环保的聚己内酯和相关聚合物生产工艺的基础。
