Abstract
BackgroundThe 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.ResultsTo 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.ConclusionsThe 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.
Highlights
Microbial cell factories are an important part of an emerging sustainable bioeconomy
Establishing feedback‐resistant phenylalanine biosynthesis in Kluyveromyces marxianus The aromatic amino acid biosynthetic and degradative pathways in K. marxianus share all steps in common with S. cerevisiae (Fig. 1; Additional file 1: Table S1), though there are some differences in numbers of paralogous genes
Besides homologues of the two aminotransferases found in S. cerevisiae (ARO8 and ARO9), sequenced K. marxianus genomes contain a third gene annotated as another putative aminotransferase [25]
Summary
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 metabolic engineering of S. cerevisiae to overproduce aromatic amino acids (AAAs) has been the subject of considerable research and development to produce diverse secondary metabolites of commercial value [1, 7]. Phenylalanine and tyrosine are commercial products in themselves; at present they are among the few amino acids that are not predominantly industrially produced by bacterial fermentation on an industrial scale [8]. 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
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