Andreas Aurich

White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses

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 feedstockOptimization of biotechnological processes by means of: New “high performance” microorganismsOn-line measurement of substrates and products in bioreactorsAlternative 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.

Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric, isocitric, pyruvic (PA), and α-ketoglutaric (KGA) acids, triggered by growth limitation and excess of carbon source. This is leading to an increased interest in this non-conventional yeast for biotechnological applications. To improve the KGA production by Y. lipolytica for an industrial application, it is necessary to reduce the amounts of by-products, e.g., fumarate (FU) and PA, because production of by-products is a main disadvantage of the KGA production by this yeast. We have examined whether the concentration of secreted organic acids (main product KGA and PA as major by-product and FU, malate (MA), and succinate (SU) as minor by-products) can be influenced by a gene–dose-dependent overexpression of fumarase (FUM) or pyruvate carboxylase (PYC) genes under KGA production conditions. Recombinant Y. lipolytica strains were constructed, which harbor multiple copies of the respective FUM1, PYC1 or FUM1, and PYC1 genes. Overexpression of the genes FUM1 and PYC1 resulted in strongly increased specific enzyme activities during cultivation of these strains on raw glycerol as carbon source in bioreactors. The recombinant Y. lipolytica strains showed different product selectivity of the secreted organic acids KGA, PA, FU, MA, and SU. Concentrations of the by-products FU, MA, SU, and PA decreased significantly at overproduction of FUM and increased at overproduction of PYC and also of FUM and PYC simultaneously. In contrast, the production of KGA with the multicopy strains H355A(FUM1) and H355A(FUM1-PYC1) was comparable with the wild-type strain H355 or slightly lower in case of H355(PYC1). KGA productivity was not changed significantly compared with strain H355 whereas product selectivity of the main product KGA was increased in H355A(FUM1).

Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids

The yeast Yarrowia lipolytica is one of the most intensively studied “non-conventional” yeast species. Its ability to secrete various organic acids, like pyruvic (PA), citric, isocitric, and alpha-ketoglutaric (KGA) acid, in large amounts is of interest for biotechnological applications. We have studied the effect of the alpha-ketoglutarate dehydrogenase (KGDH) complex on the production process of KGA. Being well studied in Saccharomyces cerevisiae this enzyme complex consists of three subunits: alpha-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and lipoamide dehydrogenase. Here we report the effect of overexpression of these subunits encoding genes and resulting increase of specific KGDH activity on organic acid production under several conditions of growth limitation and an excess of carbon source in Y. lipolytica. The constructed strain containing multiple copies of all three KGDH genes showed a reduced production of KGA and an elevated production of PA under conditions of KGA production. However, an increased activity of the KGDH complex had no influence on organic acid production under citric acid production conditions.

Syntheses with a Chiral Building Block from the Citric Acid Cycle: (2R,3S)‐Isocitric Acid by Fermentation of Sunflower Oil

The citric acid cycle constitutes a main metabolic process. Since its discovery in 1937 by H. A. Krebs, all of its intermediates have been prepared in multigramm amounts—with one exception: (2R,3S)-isocitric acid (1, dSthreo-isocitric acid). As a new member of the chiral pool it would be an interesting starting material for organic synthesis. This chiral a-hydroxy tricarboxylic acid is mainly accompanied by its constitutional isomer, citric acid (2). However, attempts to separate 1 from 2 have so far been successful only on an analytical scale. Though experiments have been carried out to achieve synthetic access to ent-isocitric acid (ent-1), again only milligram quantities were obtained. As a result of the scarce availability of 1 there is virtually no application known for it in synthesis. In databases only (2R,3S)-isocitric acid trimethyl ester (3), (2R,3S)-isocitric acid lactone-2,3-dicarboxylic acid dimethyl ester (5), (2R,3S)-isocitric acid lactone2,3-dicarboxylic acid (6), and (2R,3S)-isocitric acid lactone2,3-dicarboxylic acid anhydride (7) are listed superficially. Surprisingly, no attempts have been made to obtain 1 by fermentation, although a large number of yeasts are known to produce and excrete citric acid and (2R,3S)-isocitric acid in varying ratios when grown on long-chain n-alkanes or glucose. So far, these fermentations have been optimized for high levels of citric acid excretion. Herein we describe a combination of ecologically desirable biotechnological and chemical methods yielding enantiopure (2R,3S)-isocitric acid (1) and its derivatives in kilogram amounts, thus representing an unadulterated application of the “white biotechnology for green chemistry” concept. We discovered that the thiamine auxotrophic yeast Yarrowia lipolytica excretes organic acids in high percentage when it is grown on vegetable oils with an excess of thiamine under nitrogen-limited, aerobic conditions. Our aim was to achieve the highest possible ratio of isocitric to citric acid and concomitant high isocitric acid concentration. We succeeded in producing isocitric acid concentrations of 93 gL 1 and 1/2 ratios of 1.14:1 on the pilot-plant scale in the cultivation of wild-type Y. lipolytica EH59 on refined sun flower oil—a hitherto unrivalled achievement especially with regard to the use of renewable vegetable raw materials. After filtration of the biomass, electrodialysis was performed to convert the obtained trisodium salts into the free acids, before the removal of water was accomplished under reduced pressure. Then, it was time to search for an adequate process to separate the two isomers. Esterification of the highly viscous concentrated solution yielded the corresponding triesters of both tricarboxylic acids. From this mixture citric acid trimethyl ester 4 crystallized as a colorless solid, while (2R,3S)-isocitric acid trimethyl ester 3 did not, as it is a liquid under standard conditions. Utilizing this formerly unknown fact, separation of the isomeric esters 3 and 4 could be carried out simply by filtration of 4 from 3 (Scheme 1). In view of the intended application of 1 in

Repeated fed-batch fermentation using biosensor online control for citric acid production by Yarrowia lipolytica

Biosensor-controlled substrate feeding was used in a citric acid production process with the yeast strain Yarrowia lipolytica H222 with glucose as the carbon source. The application of an online glucose biosensor measurement facilitated the performance of long-time repeated fed-batch process with automated bioprocess control. Ten cycles of repeated fed-batch fermentation were carried out in order to validate both the stability of the microorganism for citric acid production and the robustness of the glucose biosensor in a long-time experiment. In the course of this fermentation with a duration of 553 h, a slight loss of productivity from 1.4 g/(L×h) to 1.1 g/(L×h) and of selectivity for citric acid from 91% to 88% was observed. The glucose biosensor provided 6,227 measurements without any loss of activity.

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.