Genetic diversity is a result of evolution, enabling multiple ways for one particular physiological activity. Here, we introduce this strategy into bioengineering. We design two hydroxytyrosol biosynthetic pathways using tyrosine as substrate. We show that the synthetic capacity is significantly improved when two pathways work simultaneously comparing to each individual pathway. Next, we engineer flavin-dependent monooxygenase HpaBC for tyrosol hydroxylase, tyramine hydroxylase, and promiscuous hydroxylase active on both tyrosol and tyramine using directed divergent evolution strategy. Then, the mutant HpaBCs are employed to catalyze two missing steps in the hydroxytyrosol biosynthetic pathways designed above. Our results demonstrate that the promiscuous tyrosol/tyramine hydroxylase can minimize the cell metabolic burden induced by protein overexpression and allow the biosynthetic carbon flow to be divided between two pathways. Thus, the efficiency of the hydroxytyrosol biosynthesis is significantly improved by rearranging the metabolic flux among multiple pathways.

中文翻译：

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羟基酪醇生物合成中代谢通量重排的混杂酶活性辅助多途径网络设计。

遗传多样性是进化的结果，为一种特定的生理活动提供多种方式。在这里，我们将这一策略引入生物工程中。我们以酪氨酸为底物设计了两种羟基酪醇生物合成途径。我们表明，与每个单独的途径相比，当两条途径同时工作时，合成能力显着提高。接下来，我们使用定向发散进化策略，将黄素依赖性单加氧酶 HpaBC 设计为酪醇羟化酶、酪胺羟化酶以及对酪醇和酪胺均具有活性的混杂羟化酶。然后，突变的 HpaBC 用于催化上面设计的羟基酪醇生物合成途径中缺失的两个步骤。我们的结果表明，混杂的酪醇/酪胺羟化酶可以最大限度地减少蛋白质过度表达引起的细胞代谢负担，并允许生物合成碳流在两条途径之间分配。因此，通过重新安排多个途径之间的代谢通量，可以显着提高羟基酪醇生物合成的效率。