Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

BACKGROUND: Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. RESULTS: For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. CONCLUSIONS: Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes

Acid Production by Oral Strains of Candida albicans and Lactobacilli

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42–66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Predicting ground reaction forces of human gait using a simple bipedal spring-mass model

Aircraft design must be lightweight and cost-efficient on the condition of aircraft certification. In addition to standard load cases, human-induced loads can occur in the aircraft interior. These are crucial for optimal design but difficult to estimate. In this study, a simple bipedal spring-mass model with roller feet predicted human-induced loads caused by human gait for use within an end-to-end design process. The prediction needed no further experimental data. Gait movement and ground reaction force (GRF) were simulated by means of two parameter constraints with easily estimable input variables (gait speed, body mass, body height). To calibrate and validate the prediction model, experiments were conducted in which 12 test persons walked in an aircraft mock-up under different conditions. Additional statistical regression models helped to compensate for bipedal model limitations. Direct regression models predicted single GRF parameters as a reference without a bipedal model. The parameter constraint with equal gait speed in experiment and simulation yielded good estimates of force maxima (error 5.3%), while equal initial GRF gave a more reliable prediction. Both parameter constraints predicted contact time very well (error 0.9%). Predictions with the bipedal model including full GRF curves were overall as reliable as the reference

Improved Isolation of Microbiologically Produced (2<i>R</i>,3<i>S</i>)‑Isocitric Acid by Adsorption on Activated Carbon and Recovery with Methanol

A new, efficient method for the isolation of (2<i>R</i>,3<i>S</i>)-isocitric acid (ICA) from its fermentation solution was developed. It is noteworthy that this method is based on selective adsorption directly from the fermentation solution on activated carbon, followed by the release of both ICA and citric acid by means of elution with methanol and their final separation by known methods. Thereby, several disadvantages were overcome: Electrodialysis is no longer necessary to remove cations such as Na⁺ from the fermentation solution. Also, several hitherto accompanying dyestuffs were not observed with this method. Furthermore, removal of water by distillation is expendable. Eventually, the new crude product is of a quality that also avoids the use of a tedious slide vane rotary vacuum pump distillation of the trimethyl esters of both acids, which hitherto was the basis for the separation of ICA. In summary, the new method distinctly spares energy as well as time