The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g ؊1 (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.

Systems for easily controlled, conditional induction or repression of gene expression are indispensable tools in fundamental research and industrial-scale biotechnological applications. Both native and rationally designed inducible promoters have been widely used for this purpose. However, inherent regulation modalities or toxic, expensive or inconvenient inducers can impose limitations on their use. Tailored promoters with user-specified regulatory properties would permit sophisticated manipulations of gene expression. Here, we report a generally applicable strategy for the directed evolution of promoter regulation. Specifically, we applied random mutagenesis and a multi-stage flow cytometry screen to isolate mutants of the oxygen-responsive Saccharomyces cerevisiae DAN1 promoter. Two mutants were isolated which were induced under less-stringent anaerobiosis than the wild-type promoter enabling induction of gene expression in yeast fermentations simply by oxygen depletion during cell growth. Moreover, the engineered promoters showed a markedly higher maximal expression than the unmutated DAN1 promoter, under both fastidious anaerobiosis and microaerobisois.

Nine potential (fatty) alcohol dehydrogenase genes and one alcohol oxidase gene were identified in Yarrowia lipolytica by comparative sequence analysis. All relevant genes were deleted in Y. lipolytica H222ΔP which is lacking β-oxidation. Resulting transformants were tested for their ability to accumulate ω-hydroxy fatty acids and dicarboxylic acids in the culture medium. The deletion of eight alcohol dehydrogenase genes (FADH, ADH1-7), which may be involved in ω-oxidation, led only to a slightly increased accumulation of ω-hydroxy fatty acids, whereas the deletion of the fatty alcohol oxidase gene (FAO1), which has not been described yet in Y. lipolytica, exhibited a considerably higher effect. The combined deletion of the eight (fatty) alcohol dehydrogenase genes and the alcohol oxidase gene further reduced the formation of dicarboxylic acids. These results indicate that both (fatty) alcohol dehydrogenases and an alcohol oxidase are involved in ω-oxidation of long-chain fatty acids whereby latter plays the major role. This insight marks the first step toward the biotechnological production of long-chain ω-hydroxy fatty acids with the help of the nonconventional yeast Y. lipolytica. The overexpression of FAO1 can be further used to improve existing strains for the production of dicarboxylic acids.

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The sequencing of the genomes of several organisms was the first step in understanding genome organisation, gene function and of course life itself. Until now very much information could be gained by searching for similarities between genes or structures and finding homologies or direct orthologous genes. But with the sequencing data, more and more questions arise, which cannot be answered by simply looking the data. Although the yeast Saccharomyces cerevisiae was the first eukaryotic organism, the genome of which was sequenced, almost 14 years later there are still 900 uncharacterised and 800 dubious genes (26 % at all). The unconventional yeast Yarrowia lipolytica also harbours a high number of genes with unknown function-ca. 2,300 (35 %). One hundred seventy out of 220 Yarrowia lipolytica specific genes with no homology to other yeasts are also uncharacterised, which should carry the "differences" between the yeast species. So far only 50 industrial useful genes were characterised (proteases, lipases, esterases, etc.).Genes or gene products that do not have any known function have to be analysed in a more classical, genetical way. Especially the functions of membrane proteins which are resistant to a lot of investigation steps are only rarely elucidated. The Gpr1 protein of Yarrowia lipolytica is such an example. The function cannot be estimated by finding similarities to other proteins with known function. The more the information and phenotypic effects are available, the more complex the interacting network appears. This chapter will show the ongoing discovery of the function of the Gpr1/FUN34/YaaH protein family. This is especially shown for the Gpr1 protein from Yarrowia lipolytica and its orthologues in Saccharomyces cerevisiae. Here the different and also controversial facts are reviewed and discussed.

The synthesis of complete genes is becoming a more and more popular approach in heterologous gene expression. Reasons for this are the decreasing prices and the numerous advantages in comparison to classic molecular cloning methods. Two of these advantages are the possibility to adapt the codon usage to the host organism and the option to introduce restriction enzyme target sites of choice. C.U.R.R.F. (Codon Usage regarding Restriction Finder) is a free Java(®)-based software program which is able to detect possible restriction sites in both coding and non-coding DNA sequences by introducing multiple silent or non-silent mutations, respectively. The deviation of an alternative sequence containing a desired restriction motive from the sequence with the optimal codon usage is considered during the search of potential restriction sites in coding DNA and mRNA sequences as well as protein sequences. C.U.R.R.F is available at http://www.zvm.tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/mikrobiologie/allgemeine_mikrobiologie/currf.

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