Rewiring central sulphur metabolism in Saccharomyces cerevisiae

Abstract

Of the 20 proteinogenic amino acids used by life, only two contain a sulphur atom: methionine and cysteine. While most bacteria, fungi and plants possess the ability to synthesise sulphur amino acids from simple carbon, nitrogen and sulphur sources, methionine and cysteine are essential to all animals, including humans, and need to be taken up with their diet. Since most common feedstock crops are relatively low in sulphur amino acid content, methionine becomes the first growth limiting for many farm animals, especially young piglets, poultry and a variety of farmed fish and crustaceans, and needs to be added externally to animal feeds. Annually, more than one million tonnes of DL-methionine are produced, mostly by chemical synthesis, but chemical synthesis of methionine uses non-renewable resources and toxic intermediates, and produces a racemic mixture of methionine. The D-enantiomer has to be converted into the L-form either in the body of farm animals, reducing its nutritional value, or by enzymatic conversion, increasing the costs of pure L-methionine. These hurdles in methionine synthesis call for a more sustainable production method of L-methionine. Several studies investigated the fermentative production of L-methionine in Escherichia coli or Corynebacterium glutamicum, but no commercial process has been established to date. Other approaches aimed at improving the nutritional value of plants by increasing the methionine production and/or overexpressing a methionine storage protein, but most studies increased the total sulphur amino acid content only slightly. This thesis aims to develop Saccharomyces cerevisiae strains with high amounts of sulphur amino acids that could be added directly to animal feeds. S. cerevisiae (baker's yeast) is a generally recognised as safe (GRAS) organism, which is genetically tractable, has a well described sulphur metabolism and is widely used in food production and animal feedstocks. Chapter 3 describes a Design of Experiments (DOE) approach to identify key genetic and environmental factors influencing methionine production in S. cerevisiae. Despite the inability to generate all designed strains, the approach was able to increase the methionine titre more than 5-fold by deleting SAM2, MET30 & MET32 and inserting a strong promoter in front of the open reading frame of MET6 and STR3. Furthermore, the precursor homoserine was recognised as a possible bottleneck in the biosynthesis of methionine. In chapter 4, the native sequence encoding the S. cerevisiae aspartate kinase (Hom3p) was mutated to remove its feedback inhibition by threonine. The mutation drastically increased the amounts of homoserine inside and outside the cells and induced the accumulation of cysteine in the growth medium, but failed to increase methionine titres. In order to investigate additional bottlenecks in the pathway, two bacterial O-acetylhomoserine sulfhydrylases (OAH-SHLases) with reduced feedback inhibition by methionine were expressed in homoserine accumulating strains. The expression of one of the OAH-SHLases, RsMetZ, did not increase methionine titres but caused the accumulation of large amounts of cystathionine, which is a precursor for the biosynthesis of cysteine. However, the co-expression of RsMetZ and the gene encoding the cysteine synthase, CYS3, did not elevate the amount of cysteine. Finally, in chapter 5, three methionine storage proteins from plant seeds were overexpressed in S. cerevisiae, but only the 10-kDa δ-zein was able to be detected. However, the expression of 10-kDa δ-zein in methionine overproducing strains did not differ from the wild-type. In summary, this work achieved to rewire central sulphur metabolism and increase the accumulation of sulphur amino acids in S. cerevisiae. These results represent first steps towards engineering yeast as a sulphur rich food and animal feed additive.