Har Jie Ren Gerald, Agee Alec, Bennett R Kyle, Papoutsakis Eleftherios T, Antoniewicz Maciek R
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St, Newark, DE, 19716, USA.
The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
Appl Microbiol Biotechnol. 2021 Jan;105(2):869-876. doi: 10.1007/s00253-020-11058-0. Epub 2021 Jan 6.
Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed, and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. The evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source. KEY POINTS: • Adaptive evolution of E. coli enables biosynthesis of all amino acids from methanol. • Overexpression of threonine biosynthesis pathway improves methanol assimilation. • Methanol-dependent growth is seen in minimal media with homoserine as co-substrate.
最近尝试创建合成甲基营养型大肠杆菌的研究发现，在甲醇存在的情况下，从头合成氨基酸对实现自主甲基营养生长提出了重大挑战。先前构建的依赖甲醇的菌株需要化学计量的共底物和甲醇共同利用。因此，这些菌株无法进化到仅以甲醇为碳源生长。在这项工作中，我们探索了一种替代方法，以在无化学计量耦合的基本培养基中实现从甲醇衍生的碳合成所有氨基酸。首先，我们确定苏氨酸的生物合成限制了我们的甲基营养型大肠杆菌的生长。为了解决这个问题，我们进行了适应性实验室进化，以产生一种能在含有甲醇和苏氨酸的基本培养基中高效生长的菌株。分析了进化菌株的甲醇同化和生长情况，有趣的是，我们发现进化菌株能从甲醇衍生的碳合成所有氨基酸，包括苏氨酸。然后通过优化的苏氨酸生物合成途径的过表达对进化菌株进行进一步改造。我们表明，所得的甲基营养型大肠杆菌菌株具有以高丝氨酸作为共底物的依赖甲醇的生长表型。与先前依赖甲醇的菌株不同，高丝氨酸的共同利用与甲醇同化在化学计量上没有联系。因此，对该菌株的进一步工程改造和连续的适应性进化可能使它能够以甲醇作为唯一碳源进行自主生长。要点：• 大肠杆菌的适应性进化能够从甲醇合成所有氨基酸。• 苏氨酸生物合成途径的过表达提高了甲醇同化能力。• 在以高丝氨酸作为共底物的基本培养基中观察到依赖甲醇的生长。
