Sheng Yuhua, Wu Yaokang, Zhang Linpei, Lv Xueqin, Li Jianghua, Liu Long, Du Guocheng, Chen Jian, Liu Yanfeng
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Science Center for Future Foods, Jiangnan University, Wuxi, China.
Microb Biotechnol. 2025 May;18(5):e70145. doi: 10.1111/1751-7915.70145.
Creatine (CR) is a naturally occurring amino acid derivative that plays a key role in cellular energy homeostasis, which has wide-ranging applications in food and medicine. Currently, the lack of green and sustainable CR biomanufacturing methods has led to reliance on chemical methods for industrial CR synthesis. This study presents a biological approach to synthesising CR using whole-cell catalysis by engineered Escherichia coli. First, through screening of critical enzymes from different sources and dual-enzyme co-expression strategies, arginine: glycine amidinotransferase (AGAT) from Amycolatopsis kentuckyensis and guanidinoacetate N-methyltransferase (GAMT) from Mus caroli were introduced to construct the CR biosynthesis pathway, yielding 0.83 g/L CR production. Then, the expression level of GAMT, the critical rate-limiting enzyme, was optimised by screening the ribosome binding site and N-terminal coding sequences, resulting in a 92% enhancement of CR production, reaching 1.59 g/L. Next, the endogenous ornithine and methionine cycles were further engineered to boost the synthesis of the precursor guanidinoacetate (GAA) and methyl donor S-adenosylmethionine (SAM), leading to a 68% increase in CR production, reaching 2.67 g/L. Finally, considering adenosine triphosphate (ATP) is required as a cofactor for SAM biosynthesis, we integrated the reconstitution methionine cycle with a polyphosphate kinase-based ATP regeneration system, achieving a CR titre of 5.27 g/L with a productivity of 0.22 g/L/h, and the molar conversion of substrate arginine was 71 mol% over 24 h following the engineering process. This study is the first report achieving whole-cell catalysis of CR production in engineered E. coli with a dual enzyme cascade using arginine as substrate, providing a new platform for CR production and insights into the biosynthesis of high-value metabolites that rely on ATP consumption.
肌酸（CR）是一种天然存在的氨基酸衍生物，在细胞能量稳态中起关键作用，在食品和医药领域有广泛应用。目前，缺乏绿色可持续的CR生物制造方法导致工业CR合成依赖化学方法。本研究提出了一种利用工程化大肠杆菌全细胞催化合成CR的生物学方法。首先，通过筛选不同来源的关键酶和双酶共表达策略，引入来自肯塔基拟无枝酸菌的精氨酸：甘氨酸脒基转移酶（AGAT）和来自小家鼠的胍基乙酸N-甲基转移酶（GAMT）构建CR生物合成途径，CR产量为0.83 g/L。然后，通过筛选核糖体结合位点和N端编码序列优化限速关键酶GAMT的表达水平，使CR产量提高92%，达到1.59 g/L。接下来，进一步改造内源性鸟氨酸和蛋氨酸循环以促进前体胍基乙酸（GAA）和甲基供体S-腺苷甲硫氨酸（SAM）的合成，使CR产量增加68%，达到2.67 g/L。最后，考虑到SAM生物合成需要三磷酸腺苷（ATP）作为辅因子，我们将重组蛋氨酸循环与基于多磷酸激酶的ATP再生系统整合，在工程改造后24小时内实现了5.27 g/L的CR滴度，生产率为0.22 g/L/h，底物精氨酸的摩尔转化率为71 mol%。本研究是首次报道利用精氨酸作为底物通过双酶级联在工程化大肠杆菌中实现CR生产的全细胞催化，为CR生产提供了新平台，并为依赖ATP消耗的高价值代谢物生物合成提供了见解。
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