The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10-15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0-6.8 and resulted in production of 127-140 g l(-1) CA with a yield Y (CA) of 0.75-0.82 g g(-1), whereas at pH 5.0, 87 g l (-1) with a yield Y (CA) of 0.51 g g(-1) were produced. The CA-productivity Q (CA) increased from 0.40 g l (-1) h(-1) at pH 5.0 up to 0.73 g l (-1) h(-1) at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene-dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.

Citric acid (CA) is mainly produced in a biotechnological process using Aspergillus niger. In this process, large amounts of wastes have to be removed. Since the use of Yarrowia lipolytica for CA production is an environmental compatible alternative method, the CA production was optimized in regard to growth temperature and pH as well as substrate and product inhibition. The highest value of the maximum specific growth rate at pH 6.5 was found to be μmax = 0.192 h–1, whereas the largest amount of CA of 24.91 g/L as well as the highest selectivity of the bioprocess (89.9 % CA) and the maximum yield (0.22 gCA/gGlucose) were obtained at pH 6.0. During the growth phase, the temperature optimum was found to be in the range of 30–34 °C (μmax = 0.132 h–1). Nevertheless, the highest concentration of CA during the production phase was obtained at 30 °C (41 g/L CA, 93.1 % CA, 0.55 gCA/gglucose). In studying the substrate inhibition of the process, a clear tendency of decrease in the maximum specific growth rate was detected when the initial glucose concentration was increased from 50 g/L (μmax = 0.17 h–1) to 200 g/L (μmax = 0.055 h–1). The addition of 120 g/L CA to the culture broth at the start of the production phase reduced the production of CA from 32.1 g/L to 7.4 g/L.

Functionalized compounds, which are difficult to produce by classical chemical synthesis, are of special interest as biotechnologically available targets. They represent useful building blocks for subsequent organic syntheses, wherein they can undergo stereoselective or regioselective reactions. "White Biotechnology" (as defined by the European Chemical Industry [ http://www.europabio.org/white_biotech.htm ], as part of a sustainable "Green Chemistry,") supports new applications of chemicals produced via biotechnology. Environmental aspects of this interdisciplinary combination include: Use of renewable feedstock Optimization of biotechnological processes by means of: New "high performance" microorganisms On-line measurement of substrates and products in bioreactors Alternative product isolation, resulting in higher yields, and lower energy demand In this overview we describe biotechnologically produced pyruvic, 2-oxopentaric and 2-oxohexaric acids as promising new building blocks for synthetic chemistry. In the first part, the microbial formation of 2-oxocarboxylic acids (2-OCAs) in general, and optimization of the fermentation steps required to form pyruvic acid, 2-oxoglutaric acid, and 2-oxo-D-gluconic acid are described, highlighting the fundamental advantages in comparison to chemical syntheses. In the second part, a set of chemical formula schemes demonstrate that 2-OCAs are applicable as building blocks in the chemical synthesis of, e.g., hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. Finally, some perspectives are discussed.

No longer just analytical: Previously, (2R,3S)‐isocitric acid (1), a component of the citric acid cycle, had not been available on a preparative scale. A new route to this acid on a kilogram scale combines a biotechnological formation through fermentation from sunflower oil with a chemical separation process. In a variety of transformations into further chiral derivatives, 1 is established as a valuable new member of the chiral pool (see scheme).

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