Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products
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icrobial Cell Factories Open Access
RESEARCH
Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products Arun S. Rajkumar and John P. Morrissey*
Abstract Background: 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 utilise its metabolism as a basis for bio-based production. Results: 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. Conclusions: 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. Keywords: 2-phenylethanol, Aromatic amino acid, Shikimate pathway, Pentose phosphate pathway, Phosphoketolase, PEP synthase Introduction Microbial cell factories are an important part of an emerging sustainable bioeconomy. By metabolically engineering microbial hosts, it is possible to synthesise chemical compounds that are currently sourced from non-renewable resources or from renewable resources that may not meet the growing demands of the chemical, food and pharmaceutical industries. The process typically *Correspondence: [email protected] School of Microbiology, Centre for Synthetic Biology and Biotechnology, Environmental Research Institute, APC Microbiome Institute, University College Cork, Cork T12 K8AF, Ireland
involves cloning heterologous pathways for such compounds into host microbes and altering native pathways to ensure that metabolism is optimised for the products of interest. The budding yeast Saccharomyces cerevisiae has proved to be an excellent host for de novo synthesis of valuable secondary metabolites in the flavonoid, stilbenoid and alkaloid families [1]. Several of these are directly derived from aromatic amino acids: flavonoids, stilbenoids and aroma compounds from phenylalanine [2]; benzylisquinoline alkaloids from tyrosine [3]; and monoterpene alkaloids from trypto
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