背景:氧化还原酶的大规模生物催化应用需要用于氧化还原辅因子的成本有效且有效的再生的系统。这些是工业生物生产的主要瓶颈和重要的成本因素。在这项工作中,来自伯克霍尔德氏菌的转化酶和Baeyer-Villiger单加氧酶基因共表达到大肠杆菌WΔcscR和大肠杆菌BL21(DE3),使环己酮有效地生物转化为聚合物前体,使用蔗糖作为氧化还原辅因子再生的电子源的ε-己内酯,速率与葡萄糖相当。大肠杆菌WΔcscR具有能够利用蔗糖的天然csc调节子,并且通过抑制基因(cscR)的缺失而失调,因此,即使在浓度低于6mM(2gL-1)时也能吸收蔗糖。另一方面,大肠杆菌BL21(DE3),它被广泛用作表达宿主,不包含csc调节子。
结果:这里,我们展示了一个概念的证明,其中两个大肠杆菌宿主的转化酶共表达足以有效利用蔗糖以维持环己酮的Baeyer-Villiger氧化中的辅因子再生。使用大肠杆菌WΔcscR,获得了37UgDCW-1的比活性,证明该菌株适用于重组基因共表达和随后的全细胞生物转化。此外,将相同的共表达盒转移并用大肠杆菌BL21(DE3)进行研究,显示17UgDCW-1的比活性。最后,使用来自集胞藻S02的光合作用来源的蔗糖与表达BVMO的大肠杆菌WΔcscR的生物转化在3小时后显示环己酮的完全转化,特别是在周质中表达转化酶基因的菌株。
结论:结果表明,蔗糖可以作为驱动重组大肠杆菌菌株全细胞生物转化的替代电子源,为可持续化学生产开辟了新的策略。
BACKGROUND: 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-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.
RESULTS: 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 gDCW-1 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 gDCW- 1. 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.
CONCLUSIONS: 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.