The ascomycetous yeast Yarrowia lipolytica has been established as model system for studies of several research topics as well as for biotechnological processes in the last two decades. However, frequency of heterologous recombination is high in this yeast species, and so knockouts of genes are laborious to achieve. Therefore, the aim of this study was to check whether a reduction of non-homologous end-joining (NHEJ) of double strand breaks (DSB) results in a strong increase of proportion of homologous recombinants. The Ku70-Ku80 heterodimer is known as an essential protein complex of the NHEJ. We show that deletion of YlKU70 and/or YlKU80 results in an increase of the rate of transformants with homologous recombination (HR) up to 85 % in each case. However, it never reaches near 100 % of HR in any case as described for some other yeast. Furthermore, we demonstrated that growth of Δylku strains was similar to that of the wild-type strain. In addition, no differences were detected between the Δylku strains and the parent strain in respect to sensitivity to the mutagenic agent EMS as well as to the antibiotics hygromycin, bleomycin and nourseothricin. However, Δylku70 and Δylku80 strain showed a slightly higher sensitivity against UV rays. Thus, the new constructed Δylku strains are attractive recipient strains for homologous integration of DNA fragments and a valuable tool for directed knockouts of genes. Nevertheless, our data suggest the existence of another system of non-homologous recombination what may be subject of further investigation.
This mini-review presents a summary of research results of biotechnological production of alpha-ketoglutaric acid (KGA) by bacteria and yeasts. KGA is of particular industrial interest due to its broad application scope, e.g., as building block chemical for the chemical synthesis of heterocycles, dietary supplement, component of infusion solutions and wound healing compounds, or as main component of new elastomers with a wide range of interesting mechanical and chemical properties. Currently KGA is produced via different chemical pathways, which have a lot of disadvantages. As an alternative several bacteria and yeasts have already been studied for their ability to produce KGA as well as for conditions of overproduction and secretion of this intermediate of the tricarboxylic acid cycle. The aim of this mini-review was to summarize the known data and to discuss the potentials of biotechnological processes of KGA production.
Coordination failure among owners of heterogeneous debt types increases distress costs. Covenants reduce expected distress costs by lowering the probability of liquidity shortages, increasing liquidation values, and incentivizing creditor monitoring. We predict and find that new debt contracts include more covenants when borrowers’ existing debt structures are more heterogeneous. Our findings suggest that covenants are not only used to address creditor–shareholder conflicts but also to reduce the expected costs of coordination failure among creditors. Furthermore, our results indicate a dynamic component missing from static debt structure models: Debt heterogeneity entails additional covenants (i.e., constraints) when raising future debt. This paper was accepted by Lauren Cohen, finance.
Oxo- and hydroxy-carboxylic acids are of special interest in organic synthesis. However, their introduction by chemical reactions tends to be troublesome especially with regard to stereoselectivity. We describe herein the biotechnological preparation of selected oxo- and hydroxycarboxylic acids under "green" conditions and their use as promising new building blocks. Thereby, our biotechnological goal was the development of process fundamentals regarding the variable use of renewable raw materials, the development of a multi purpose bioreactor and application of a pilot plant with standard equipment for organic acid production to minimize the technological effort. Furthermore the development of new product isolation procedures, with the aim of direct product recovery, capture of products or single step operation, was necessary. The application of robust and approved microorganisms, also genetically modified, capable of using a wide range of substrates as well as producing a large spectrum of products, was of special importance. Microbiologically produced acids, like 2-oxo-glutaric acid and 2-oxo-D-gluconic acid, are useful educts for the chemical synthesis of hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. The chiral intermediate of the tricarboxylic acid cycle, (2R,3S)-isocitric acid, is another promising compound. For the first time our process provides large quantities of enantiopure trimethyl (2R,3S)-isocitrate which was used in subsequent chemical transformations to provide new chiral entities for further usage in total synthesis and pharmaceutical research.Oxo- and hydroxy-carboxylic acids are of special interest in organic synthesis. However, their introduction by chemical reactions tends to be troublesome especially with regard to stereoselectivity. We describe herein the biotechnological preparation of selected oxo- and hydroxycarboxylic acids under "green" conditions and their use as promising new building blocks. Thereby, our biotechnological goal was the development of process fundamentals regarding the variable use of renewable raw materials, the development of a multi purpose bioreactor and application of a pilot plant with standard equipment for organic acid production to minimize the technological effort. Furthermore the development of new product isolation procedures, with the aim of direct product recovery, capture of products or single step operation, was necessary. The application of robust and approved microorganisms, also genetically modified, capable of using a wide range of substrates as well as producing a large spectrum of products, was of special importance. Microbiologically produced acids, like 2-oxo-glutaric acid and 2-oxo-D-gluconic acid, are useful educts for the chemical synthesis of hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. The chiral intermediate of the tricarboxylic acid cycle, (2R,3S)-isocitric acid, is another promising compound. For the first time our process provides large qua...
To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP(+)-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19% more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95% producing up to 186 g l(-1) KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.
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