S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)是一种重要的代谢中间体,对机体代谢活动的正常运行起重要作用,它是由底物L-甲硫氨酸和ATP在SAM合酶催化下生成的,目前主要应用于医药行业。该研究首先以蜡状芽孢杆菌Bacillus cereus和谷氨酸棒杆菌Corynebacterium glutamicum基因组为模板扩增出SAM合酶编码基因BcmetK和CgmetK,以pETDueT1质粒为载体,以大肠杆菌E.coli BL21(DE3)为底盘细胞,构建了SAM合成菌株E.coli BL21/pETDuet1-BcmetK和E.coli BL21/pETDuet1-CgmetK。其次,针对SAM合成菌株全细胞催化合成体系条件进行了优化,重组大肠杆菌E.coli BL21/pETDuet1-BcmetK在最优条件[L-甲硫氨酸浓度50 mmol/L、ATP浓度50 mmol/L、1.2% (体积分数) 的OD₆₀₀值约9.0的细胞悬浮液、600 mmol/L pTSoNa、50 mmol/L MgSO₄、100 mmol/L K₂SO₄、45 ℃、pH 8.0]下,连续转化5 h,获得了464 mg/L的SAM。重组大肠杆菌E.coli BL21/pETDuet1-CgmetK在最优条件[L-甲硫氨酸浓度50 mmol/L、ATP浓度40 mmol/L、0.8%(体积分数)的OD₆₀₀值约9.0的细胞悬浮液、800 mmol/L pTSoNa、50 mmol/L MgSO₄、100 mmol/L K₂SO₄、45 ℃、pH 8.5]下,连续转化5 h,获得了528 mg/L的SAM,可见,谷氨酸棒杆菌来源的SAM合酶催化能力优于蜡状芽孢杆菌来源的,更有利于SAM的合成。该研究成功构建了SAM合成菌株,虽然产量水平不高,但为SAM的可持续生物合成提供了重要借鉴。

Reactions that require the transfer of chemical functional groups are crucial to the survival and viability of living organisms. One such functional group is the methyl group. Methylation of genomic DNA is critical for the control of gene expression. The natural donor of the methyl group for such reaction is S-adenosylmethionine (SAM). Besides the methyl group, SAM is able to donate all groups surrounding its sulphur atom in metabolism. It is produced in almost all living organisms, except certain parasites, and can participate as intermediate in the synthesis of many other chemical compounds, including glutathione, spermine and spermidine, ethylene, N-acyl homoserine lactone, coenzyme Q10, creatine, melatonin, phosphatidylcholine, methylcobalamin, norepinephrine and carnitine. It has been used to treat inflammation, depression, liver disease, Alzheimer’s disease, liver disease, fibromyalgia, osteoarthritis, and colon cancer. Its many pharmacological applications have led to its continuously growing demand which means that without improved and sustainable production, the demand may not be satisfied. SAM is generated by the substrate L-methionine and ATP under the catalysis of SAM synthase. Heterologous gene expression has popularly been implemented in the improvement of microbial production of a variety of fine chemicals in industry. Whole-cell biotransformation is a promising alternative for the microbial production of important chemicals in contrast to classical fermentation. Whereas classical fermentation might be flawed with the demerit that while being hopeful of high productivity due to successful channelling of pathways and overexpression of key enzyme(s), often, the microbial chassis might convert some substrate into biomass if they are allowed to continue growing. This limitation could be overcome if cells are employed in the conversion of substrates to product (bioconversion) in their resting state. Bioconversion using whole-cells has attracted attention as an eco-friendly and sustainable method for the production of valuable compounds. Its implementation however is still garnering popularity in synthetic biology. While it offers a useful channel for the conversion of substrates to product without diversion to growth and cell maintenance, it also provides the advantage of minimising unwanted side reactions, easier products purification, and is less environmentally detrimental. The application of whole-cells in resting phase for biotransformation or bioconversion circumvents the need of intracellular enzyme purification for catalysis while also allowing enzymes function in the more natural intracellular environment. Ironically, despite these advantages, this method remains largely unreported for the production of SAM as there is scarcity of data showing its implementation. It has however been employed successfully in the production of other chemicals, and was therefore explored in this study to investigate its applicability for the production of SAM by cells expressing heterologous SAM synthase. E. coli is an organism of choice for the expression of many heterologous proteins. It is well studied, easy to manipulate, and its use as microbial chassis for biomanufacture is well established. It offers the advantages of fast growth, stability, and flexible respiration. In a bid select SAM synthase encoding genes that could demonstrate good performance in the production of SAM in E. coli, the genes from Bacillus cereus and Corynebacterium glutamicum were employed. A major constraint for SAM synthases is their susceptibly to product inhibition. The activity of most SAM synthases is often inhibited when the product accumulates beyond a certain threshold. Therefore, a sustainable production platform would require amelioration of this constraint. In this study sodium p-toluenesulfonate (pTsONa) was used for the relief of product inhibition and the results demonstrated its effectiveness when applied in high concentrations. This study described a simple, cheap and effective strategy based on synthetic biology for improving the production of SAM in E. coli. The SAM synthase-encoding genes BcmetK and CgmetK employed in this study were amplified from the genomes of B. cereus and C. glutamicum as templates, the plasmid pETDuet1 was used as the vector, and E. coli BL21(DE3) was used for expression. The SAM synthetic strains E. coli BL21/pETDuet1-BcmetK and E. coli BL21/pETDuet1-CgmetK were constructed. Secondly, the conditions for the whole-cell biotransformation by the recombinant strains were optimized. The recombinant E. coli BL21/pETDuet1-BcmetK after 5 h produced 464 mg/L SAM under optimal conditions [50 mmol/L L-methionine, 50 mmol/L ATP,1.2% (by volume) biocatalyst from stock of OD₆₀₀ 9.0, 600 mmol/L pTsONa, 50 mmol/L MgSO₄, 100 mmol/L K₂SO₄, 45 ℃, and pH 8.0]. The recombinant E. coli BL21/pETDuet1-CgmetK after 5 h produced 528 mg/L SAM under optimal conditions [50 mmol/L L-methionine, 40 mmol/L ATP, 0.8% (by volume) biocatalyst from stock of OD₆₀₀ 9.0, 800 mmol/L pTsONa, 50 mmol/L MgSO₄, 100 mmol/L K₂SO₄, 45 ℃, and pH 8.5]. This study demonstrates that the catalytic activity of SAM synthase derived from C. glutamicum is better than that of B. cereus, implying that it is more favourable for the synthesis of SAM by whole-cell biotransformation. In this study, a SAM-producing strain was successfully constructed, although the theoretical yield was not significantly high, it however provides an important reference for the sustainable biosynthesis of SAM. Furthermore, the strategies detailed in the study could be exploited for the production of similar natural compounds.