Abstract

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.

中文翻译：

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甲基营养型大肠杆菌的适应性实验室进化能够从甲醇衍生的碳中合成所有氨基酸

摘要

最近创建合成大肠杆菌甲基营养型生物的尝试发现，在甲醇存在下从头进行氨基酸的从头生物合成对实现自主甲基营养型生物提出了重大挑战。以前设计的依赖于甲醇的菌株需要共同利用化学计量量的共底物和甲醇。因此，这些菌株不能进化为仅在甲醇上生长。在这项工作中，我们探索了一种替代方法，可以在最小的介质中以化学计量方式从甲醇衍生的碳生物合成所有氨基酸。首先，我们发现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，以产生一种菌株，该菌株可以在含有甲醇和苏氨酸的基本培养基中高效生长。分析了进化菌株的甲醇同化和生长，有趣的是，我们发现进化菌株从甲醇衍生的碳中合成了所有氨基酸，包括苏氨酸。然后通过优化苏氨酸生物合成途径的过表达来进一步改造进化后的菌株。我们证明了产生的甲基营养型大肠杆菌该菌株具有以高丝氨酸为共底物的甲醇依赖性生长表型。与以前的甲醇依赖性菌株相反，高丝氨酸的共同利用与化学同化没有化学计量联系。这样，该菌株的未来工程设计和连续的适应性进化可以使甲醇作为唯一碳源实现自主生长。

关键点

•大肠杆菌的适应性进化能够从甲醇生物合成所有氨基酸。

•苏氨酸生物合成途径的过表达改善了甲醇的同化作用。

•在以高丝氨酸为共底物的基本培养基中，可以看到甲醇依赖性生长。