Wednesday, 01 July 2015 01:00

Engineering a carnitine producing strain of the industrial yeast Saccharomyces cerevisiae

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S cerevisiaeL-carnitine, a medically relevant, amino acid-derived molecule is a valuable target for biotechnological production. Researchers at the Institute for Wine Biotechnology, Stellenbosch University has recently provided the first report of a metabolically engineered carnitine producing strain of the industrial yeast, Saccharomyces cerevisiae, an organism that does not natively produce its own carnitine. This was achieved by cloning and reconstructing the Neurospora crassa L-carnitine biosynthesis pathway in the baker’s yeast to create an L-carnitine producing strain. The engineered yeast strains are able to catalyze the synthesis of L-carnitine from the pathway’s precursor, trimethyllysine, as well as from intermediates. Several native S. cerevisiae genes were identified that contribute to, or interfere with, the heterologous pathway. This includes (i) the threonine aldolase Gly1p which effectively catalyzed the second step of the pathway, fulfilling the role of a serine hydroxymethyltransferase, (ii) the arginine transporter Can1p which was identified as the yeast transporter for trimethyllysine, and (iii) the two serine hydroxymethyltransferases, Shm1p and Shm2p, which reduced the flux through the heterologous pathway. The work opens opportunities for using an engineered, L-carnitine producing S. cerevisiae strain in various industrial applications.

L-Carnitine is a vitamin-like molecule with GRASS (generally recognized as safe) status that plays an essential and conserved shuttling function in eukaryotes by transferring activated acyl residues across intracellular membranes. This function is supported by the activities of various carnitine acyl-transferases and transporters, whose activity is collectively referred to as the carnitine shuttle. The importance of this compound and of the carnitine shuttle in eukaryotic energy metabolism has been highlighted by many studies linking diverse metabolic diseases to a dysfunction of carnitine related metabolism. These disorders are in many cases effectively treated through dietary carnitine supplementation. Supplementation studies have revealed that carnitine has a broad range of beneficial and potentially therapeutic effects. To cater for the growing L-carnitine market, current industrial processes are producing the compound from chemical industry waste products, such as D-carnitine, γ-butyrobetaїne and cronobetaїne, using whole cell biotransformations by Escherichia coli and Proteus spp. strains.

The initial characterisation of the eukaryotic carnitine biosynthesis pathway was carried out in the fungus, Neurospora crassa, but the conserved enzymatic activities and most of the corresponding genes have now been characterised in various higher eukaryotes. The eukaryotic carnitine biosynthesis pathway catalyses the conversion of the pathway’s precursor, trimthyllysine, through four hydroxylation and dehydrogenation reactions to form L-carnitine (Fig. 1). In contrast to most eukaryotes, the industrial yeast, S. cerevisiae, is unable to neo-synthesize its own carnitine and is solely dependent on carnitine uptake from the extracellular environment. The wide range of genetic tools available for the introduction and heterologous expression of genes in this model organism provides an opportunity of genetically engineering a carnitine producing yeast with a variety of possible industrial applications. Yeast with the capacity to synthesise carnitine can provide an alternative to the existing industrial synthesis processes and also open additional doors to fermented or single cell protein products enriched with L-carnitine. This study describes the cloning and expression of the four genes encoding the enzymes from the N. crassa carnitine biosynthesis pathway in S. cerevisiae. The data indicates that the entire pathway is functional in yeast and provides the first description of S. cerevisiae strains capable of synthesising L-carnitine from TML. Especially encouraging, was the modified strains ability to convert 58% of the intermediate gamma-butyrobetaїne to L-carnitine in laboratory conditions. Standard optimisation protocols is expected to raise this yield close to what is observed in current bacterial production systems.

The carnitine biosynthesis pathway

The introduced pathway is shown to interact with, and to make use of, several native yeast proteins and these interactions shed new light on yeast metabolic enzymes, including several serine hydroxymethyltransferases that either contribute to, or negatively interfere with, the pathway. In addition, the yeast threonine aldolase, Gly1p is shown to functionally compensate for the N. crassa SHMT. In addition, Can1p is identified the TML transporter in S. cerevisiae. These interactions provide various avenues for the possible targeted improvement of the efficiency of the heterologous carnitine biosynthesis pathway in S. cerevisiae.

Journal Reference: Franken, J., Burger, A., Swiegers, J. H., & Bauer, F. F. (2015). Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 1-13.

AuthorsAuthors: Jaco Franken, Florian Bauer
Institute for Wine Biotechnology
Stellenbosch University

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