Ma Weichao, Cao Weijia, Zhang Bowen, Chen Kequan, Liu Quanzhen, Li Yan, Ouyang Pingkai
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, P.R. China.
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, P.R. China.
Sci Rep. 2015 Oct 22;5:15630. doi: 10.1038/srep15630.
Although the routes of de novo pyridoxal 5'-phosphate (PLP) biosynthesis have been well described, studies of the engineering of an intracellular PLP supply are limited, and the effects of cellular PLP levels on PLP-dependent enzyme-based whole-cell biocatalyst activity have not been described. To investigate the effects of PLP cofactor availability on whole-cell biocatalysis, the ribose 5-phosphate (R5P)-dependent pathway genes pdxS and pdxT of Bacillus subtilis were introduced into the lysine decarboxylase (CadA)-overexpressing Escherichia coli strain BL-CadA. This strain was then used as a whole-cell biocatalyst for cadaverine production from L-lysine. Co-expression strategies were evaluated, and the culture medium was optimised to improve the biocatalyst performance. As a result, the intracellular PLP concentration reached 1144 nmol/gDCW, and a specific cadaverine productivity of 25 g/gDCW/h was achieved; these values were 2.4-fold and 2.9-fold higher than those of unmodified BL-CadA, respectively. Additionally, the resulting strain AST3 showed a cadaverine titre (p = 0.143, α = 0.05) similar to that of the BL-CadA strain with the addition of 0.1 mM PLP. These approaches for improving intracellular PLP levels to enhance whole-cell lysine bioconversion activity show great promise for the engineering of a PLP cofactor to optimise whole-cell biocatalysis.
尽管从头合成5'-磷酸吡哆醛（PLP）的途径已得到充分描述，但关于细胞内PLP供应工程的研究却很有限，而且细胞内PLP水平对基于PLP依赖性酶的全细胞生物催化剂活性的影响也尚未见报道。为了研究PLP辅因子可用性对全细胞生物催化的影响，将枯草芽孢杆菌中依赖于5-磷酸核糖（R5P）的途径基因pdxS和pdxT导入过表达赖氨酸脱羧酶（CadA）的大肠杆菌菌株BL-CadA中。然后将该菌株用作从L-赖氨酸生产尸胺的全细胞生物催化剂。评估了共表达策略，并对培养基进行了优化以提高生物催化剂性能。结果，细胞内PLP浓度达到1144 nmol/gDCW，尸胺的比生产率达到25 g/gDCW/h；这些值分别比未修饰的BL-CadA高2.4倍和2.9倍。此外，所得菌株AST3的尸胺产量（p = 0.143，α = 0.05）与添加0.1 mM PLP的BL-CadA菌株相似。这些提高细胞内PLP水平以增强全细胞赖氨酸生物转化活性的方法对于工程化PLP辅因子以优化全细胞生物催化具有很大的前景。
