Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14, 8010, Graz, Austria.
Molecular Plant Biology, Department of Life Technologies, University of Turku, 20014, Turku, Finland.
Microb Cell Fact. 2024 Aug 12;23(1):227. doi: 10.1186/s12934-024-02474-2.
The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.
Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U g was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U g. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.
Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.
氧化还原酶的大规模生物催化应用需要一种经济高效的方法来再生氧化还原辅因子。这些辅因子是工业生物生产的主要瓶颈，也是一个重要的成本因素。在这项工作中，通过共表达来自恶臭假单胞菌的转化酶和 Baeyer-Villiger 单加氧酶的基因，使大肠杆菌 WΔcscR 和大肠杆菌 BL21（DE3）能够有效地将环己酮转化为聚合物前体ε-己内酯，使用蔗糖作为电子供体来再生氧化还原辅因子，其速率可与葡萄糖相媲美。大肠杆菌 WΔcscR 具有天然的 csc 调控子，能够利用蔗糖，并通过删除抑制基因（cscR）而被去调控，因此即使在低于 6mM（2g/L）的浓度下也能够吸收蔗糖。另一方面，广泛用作表达宿主的大肠杆菌 BL21（DE3）不含 csc 调控子。
本文展示了一个概念验证，其中共表达两种大肠杆菌宿主的转化酶足以有效地利用蔗糖，以维持 Baeyer-Villiger 氧化环己酮过程中的辅因子再生。使用大肠杆菌 WΔcscR，获得了 37 U/g 的比酶活，证明了该菌株适合于重组基因共表达和随后的全细胞生物转化。此外，还将相同的共表达盒转移并与大肠杆菌 BL21（DE3）进行了研究，该酶在大肠杆菌 BL21（DE3）中表达的比酶活为 17 U/g。最后，使用大肠杆菌 WΔcscR 表达 BVMO 对来源于聚球藻 S02 的光合衍生蔗糖进行生物转化，3 小时后环己酮完全转化，特别是在周质表达转化酶基因的菌株中。
结果表明，蔗糖可以作为替代电子供体，驱动重组大肠杆菌菌株的全细胞生物转化，为可持续的化学生产开辟了新的策略。
