Investigation of the co-metabolic transformation of 4-chlorostyrene into 4-chlorophenylacetic acid in Pseudomonas fluorescens ST.

The side-chain oxygenation of styrene is able to yield substituted phenylacetic acids from corresponding styrenes by co-metabolic transformation. This co-metabolization was investigated in Pseudomonas fluorescens ST using 4-chlorostyrene as co-substrate. It was shown that non-substituted styrene is necessary to ensure the co-metabolic process. Furthermore, aspects affecting the co-transformation were studied, e.g. cell density, amount of inducer, pH, effects of co-substrate/co-product. It was demonstrated that 4-chlorophenylacetic acid and 4-chlorostyrene are able to inhibit the reaction. But, these inhibitions are influenced by salt and trace elements. Finally, a protocol was established which considers all findings. Therewith, about 6.7 g L-1 co-product were obtained after 451 h. Compared to previous studies, the co-product concentration was improved by the factor 1.4 while the reaction time was decreased by the factor 18.5. The study offers also aspects for prospective improvements in order to establish an efficient way to gain substituted acids without genetic manipulation.

Heterologous production of different styrene oxide isomerases for the highly efficient synthesis of phenylacetaldehyde.

The styrene oxide isomerase (SOI, StyC) represents a key enzyme of the styrene-degrading pathway and has been discussed as promising biocatalyst during recent studies. The enzyme enables the synthesis of pure phenylacetaldehyde from styrene oxide. In this study the native as well as the corresponding codon-optimized genes of three different SOIs from Rhodococcus opacus 1CP (StyC-1CP), Sphingopyxis fribergensis Kp5.2 (StyC-Kp5.2), and Pseudomonas fluorescens ST (StyC-ST) were investigated for the expression in Escherichia coli BL21(DE3)pLysS. Specific enzyme activities of 61.9±7.5Umg-1, 23.2±2.8Umg-1, and 10.9±1.2Umg-1 were achieved after 6-9h for the codon-optimized gene of strain 1CP and the native genes of Kp5.2 and ST, respectively. Afterwards, these enzymes were enriched and applied for biotransformation studies. A complete conversion of 150mM styrene oxide to phenylacetaldehyde was observed for the enzyme StyC-Kp5.2 indicating a significantly improved stability towards product inactivation. Remarkably, more than 300mM product (>36gL-1, yield of about 80%) were finally synthesized from 400mM substrate with 150U of this enzyme within 60-120min. This represents the highest product concentration which has been reached with this type of enzymes, so far.

Reconstruction of the Metabolic Potential of Acidophilic Sideroxydans Strains from the Metagenome of an Microaerophilic Enrichment Culture of Acidophilic Iron-Oxidizing Bacteria from a Pilot Plant for the Treatment of Acid Mine Drainage Reveals Metabolic Versatility and Adaptation to Life at Low pH.

Bacterial community analyses of samples from a pilot plant for the treatment of acid mine drainage (AMD) from the lignite-mining district in Lusatia (East Germany) had previously demonstrated the dominance of two groups of acidophilic iron oxidizers: the novel candidate genus "Ferrovum" and a group comprising Gallionella-like strains. Since pure culture had proven difficult, previous studies have used genome analyses of co-cultures consisting of "Ferrovum" and a strain of the heterotrophic acidophile Acidiphilium in order to obtain insight into the life style of these novel bacteria. Here we report on attempts to undertake a similar study on Gallionella-like acidophiles from AMD. Isolates belonging to the family Gallionellaceae are still restricted to the microaerophilic and neutrophilic iron oxidizers Sideroxydans and Gallionella. Availability of genomic or metagenomic sequence data of acidophilic strains of these genera should, therefore, be relevant for defining adaptive strategies in pH homeostasis. This is particularly the case since complete genome sequences of the neutrophilic strains G. capsiferriformans ES-2 and S. lithotrophicus ES-1 permit the direct comparison of the metabolic capacity of neutrophilic and acidophilic members of the same genus and, thus, the detection of biochemical features that are specific to acidophilic strains to support life under acidic conditions. Isolation attempts undertaken in this study resulted in the microaerophilic enrichment culture ADE-12-1 which, based on 16S rRNA gene sequence analysis, consisted of at least three to four distinct Gallionellaceae strains that appear to be closely related to the neutrophilic iron oxidizer S. lithotrophicus ES-1. Key hypotheses inferred from the metabolic reconstruction of the metagenomic sequence data of these acidophilic Sideroxydans strains include the putative role of urea hydrolysis, formate oxidation and cyanophycin decarboxylation in pH homeostasis.