In many ascomycetous yeasts, the cell wall is composed of two main types of macromolecules: (a) polysaccharides, with a high content of β-1,6-and β-1,3-linked glucan chains and minor amounts of chitin; and (b) cell wall proteins of different types. Synthesis and maintenance of these macromolecules respond to environmental changes, which are sensed by the cell wall integrity (CWI) signal transduction pathway. We here present a first systematic analysis of the cell wall composition of the milk yeast, Kluyveromyces lactis. Electron microscopic analyses revealed that exponentially growing cells of K. lactis supplied with glucose as a carbon source have a wall thickness of 64 nm, as compared to 105 nm when growing on 3% ethanol. Despite their increased wall thickness, ethanol-grown cells were more sensitive to the presence of zymolyase in the growth medium. Mass spectrometric analysis identified 22 covalently linked cell wall proteins, including 19 GPI-modified proteins and two Pir wall proteins. Importantly, the composition of the cell wall glycoproteome depended on carbon source and growth phase. Our results clearly illustrate the dynamic nature of the cell wall of K. lactis and provide a firm base for studying its regulation.

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Non-ribosomal peptide synthetases (NRPSs) are key enzymes in microorganisms for the assembly of peptide backbones of biologically and pharmacologically active natural products. The monomodular NRPS-like enzymes comprise often an adenylation (A), a thiolation (T), and a thioesterase (TE) domain. In contrast to the NRPSs, they do not contain any condensation domain and usually catalyze a dimerization of α-keto carboxylic acids and thereby provide diverse scaffolds for further modifications. In this study, we established an expression system for NRPS-like genes in Saccharomyces cerevisiae. By expression of four known genes from Aspergillus terreus, their predicted function was confirmed and product yields of up to 35 mg per liter culture were achieved. Furthermore, expression of ATEG_03090 from the same fungus, encoding for the last uncharacterized NRPS-like enzyme with an A-T-TE domain structure, led to the formation of the benzoquinone derivative atromentin. All the accumulated products were isolated and their structures were elucidated by NMR and MS analyses. This study provides a convenient system for proof of gene function as well as a basis for synthetic biology, since additional genes encoding modification enzymes can be introduced.

The tryptophan derivative 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA) is present in many plants and foods including fermentation products of the baker's yeast Saccharomyces cerevisiae. MTCA is formed from tryptophan and acetaldehyde via a Pictet-Spengler reaction. In this study, up to 9 mg/L of MTCA were detected as a mixture of (1S,3S) and (1R,3S) isomers in a ratio of 2.2:1 in Saccharomyces cerevisiae cultures. To the best of our knowledge, this is the first report on the presence of MTCA in laboratory baker's yeast cultures. Expression of three fungal tryptophan prenyltransferase genes, fgaPT2, 5-dmats, and 7-dmats in S. cerevisiae resulted in the formation of MTCA derivatives with prenyl moieties at different positions of the indole ring. Expression of these genes in dimethylallyl diphosphate and tryptophan overproducing strains led to generation of up to 400 mg/L of prenylated MTCAs as mixtures of (1S,3S) and (1R,3S) diastereomers in ratios similar to that of unprenylated MTCA. The structures of the described substances including their stereochemistry were unequivocally elucidated by mass spectrometry as well as one- and two-dimensional NMR spectroscopy. The results of this study provide a convenient system for the production of high amounts of designed prenylated MTCAs in S. cerevisiae. Furthermore, our work can be considered as an excellent example for the construction of more complex molecules by introducing just one key gene.