1.Hangzhou Hizyme Biotech Co., Ltd.;2.Tianjin university of science and technology, College of biotechnology
以枯草芽孢杆菌（Bacillus subtilis）为宿主，以脱氧胸苷和腺嘌呤为底物，异源组合表达来源于大肠杆菌（Escherichia coli）的嘧啶核苷磷酸化酶与嘌呤核苷磷酸化酶作为催化酶源，全细胞催化合成2’-脱氧腺苷。首先，通过对比不同宿主来源的核苷磷酸化酶确定最适的酶组合表达，并对核糖体结合位点（Ribosome binding site, RBS）序列进行优化，获得全细胞催化合成2’-脱氧腺苷的重组B. subtilis , 2’-脱氧腺苷的产量为133.4 g/L，脱氧胸苷的摩尔转化率为64.3%；其次，利用互作短肽构建自组装多酶复合物，显著提升了底物转化效率，2’-脱氧腺苷的产量达到179.6 g/L；最后，对全细胞催化条件细胞添加量及温度进行优化，进一步提高了2’-脱氧腺苷的产量，最终优化后的2’-脱氧腺苷的产量达到200.3 g/L，底物脱氧胸苷的摩尔转化率为96.6%。
In this study, Bacillus subtilis was used as the host to express the heterologous enzyme for biotechnological production of 2’-deoxyadenosine. The pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP) from Escherichia coli as biocatalyst to convert thymidine and adenine into 2’-deoxyadenosine with high efficiency. Firstly, the enzyme combination expression and ribosome binding site (RBS) optimization of nucleoside phosphorylases were performed, and the recombinant B. subtilis that synthesized 2’-deoxyadenosine by whole-cell catalysis was constructed. The yield of 2’-deoxyadenosine was 133.4 g/L, and the molar conversion rate of thymidine was 64.3%. Then, to further improve the yield and molar conversion rate, a short peptide pair was used to form self-assembling multienzyme complexes in vivo. The yield of 2’-deoxyadenosine was significantly improved, reaching to 179.6 g/L. Finally, the conditions of whole-cell catalysis were optimized to further improve the production of 2’-deoxyadenosine. The optimized production of 2’-deoxyadenosine reached to 200.3 g/L and the molar conversion rate of substrate thymidine was 96.6%.