The yeast Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilize its metabolism as a basis for bio-based production. To illustrate the synthetic biology strategies to be followed and showcase its potential, we describe a comprehensive approach to rationally engineer a metabolic pathway in K. marxianus. We use the phenylalanine biosynthetic pathway both as a prototype and because phenylalanine is a precursor for commercially valuable secondary metabolites. First, we modify and overexpress the pathway to be resistant to feedback inhibition so as to overproduce phenylalanine de novo from synthetic minimal medium. Second, we assess native and heterologous means to increase precursor supply to the biosynthetic pathway. Finally, we eliminate branch points and competing reactions in the pathway and rebalance precursors to redirect metabolic flux to a specific product, 2-phenylethanol (2-PE). As a result, we are able to construct robust strains capable of producing over 800 mg L⁻¹ 2-PE from minimal medium. The strains we constructed are a promising platform for the production of aromatic amino acid-based biochemicals, and our results illustrate challenges with attempting to combine individually beneficial modifications in an integrated platform.

中文翻译：

---

合理设计马克斯克鲁维酵母以创建用于生产芳香产品的底盘

马尔克斯克鲁维酵母酵母因其快速生长，耐热性和广泛的底物范围等有用特性，为工业生物技术提供了独特的潜力。作为新兴的替代平台，马克斯克鲁维酵母需要开发和验证代谢工程策略，以最好地利用其代谢作为生物基生产的基础。为了说明要遵循的合成生物学策略并展示其潜力，我们描述了一种合理设计马克斯克鲁维酵母代谢途径的综合方法。。我们将苯丙氨酸的生物合成途径用作原型，因为苯丙氨酸是具有商业价值的次级代谢产物的前体。首先，我们修改和过表达途径具有抗性的反馈抑制，从而过量的苯丙氨酸从头从合成的基本培养基中。第二，我们评估原生和异源途径，以增加生物合成途径的前体供应。最后，我们消除了路径中的分支点和竞争反应，并重新平衡了前体，以将代谢流重新定向到特定的产物2-苯基乙醇（2-PE）。结果，我们能够构建能够产生超过800 mg L ^-1的坚固菌株2-PE（基本培养基）。我们构建的菌株是生产基于芳香族氨基酸的生化试剂的有前途的平台，我们的结果说明了尝试在集成平台中组合各个有益修饰的挑战。