Time-Optimized Isotope Ratio LC-MS/MS for High-Throughput Quantification of Primary Metabolites.

Cellular metabolite concentrations hold information on the function and regulation of metabolic networks. However, current methods to measure metabolites are either low-throughput or not quantitative. Here we optimized conditions for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for quantitative measurements of primary metabolites in 2 min runs. In addition, we tested hundreds of multiple reaction monitoring (MRM) assays for isotope ratio mass spectrometry of most metabolites in amino acid, nucleotide, cofactor, and central metabolism. To systematically score the quality of LC-MS/MS data, we used the correlation between signals in the 12C and 13C channel of a metabolite. Applying two optimized LC methods to bacterial cell extracts detected more than 200 metabolites with less than 20% variation between replicates. An exhaustive spike-in experiment with 79 metabolite standards demonstrated the high selectivity of the methods and revealed a few confounding effects such as in-source fragments. Generally, the methods are suited for samples that contain metabolites at final concentrations between 1 nM and 10 µM, and they are sufficiently robust to analyze samples with a high salt content.

Oxidation of fatty aldehydes to fatty acids by Escherichia coli cells expressing the Vibrio harveyi fatty aldehyde dehydrogenase (FALDH).

Fatty acids represent an important renewable feedstock for the chemical industry. To enable biotechnological one carbon truncations of fatty acids, the enzymes α-dioxygenase and fatty aldehyde dehydrogenase (FALDH) have to be combined in a two-step process. We expressed an FALDH from V. harveyi in E. coli and characterized its substrate spectrum with a focus on the number and position of double bonds in the fatty aldehyde molecules. Synthesis of the expected fatty acid products was proven by analysis of whole cell biotransformation products. Coexpression of a H(2)O-forming NADPH oxidase (NOX) from Lactobacillus sanfranciscensis led to the implementation of a cofactor regeneration cycle in in vitro oxidation experiments. The presence of NOX in whole cell biotransformations improved reaction velocity but did not result in higher product yields. We could further demonstrate that at least part of the endogenous NAD(P)(+) regeneration capacity in the resting cells results from the respiratory chain. The whole cell catalyst with the high broad range FALDH activity described here is an important biotechnological module for lipid biotransformation processes, especially the shortening of fatty acids.

Forward genetic in planta screen for identification of plant-protective traits of Sphingomonas sp. strain Fr1 against Pseudomonas syringae DC3000.

Sphingomonas sp. strain Fr1 has recently been shown to protect Arabidopsis thaliana against the bacterial leaf pathogen Pseudomonas syringae DC3000. Here, we describe a forward genetic in planta screen to identify genes in Sphingomonas sp. Fr1 necessary for this effect. About 5,000 Sphingomonas sp. Fr1 mini-Tn5 mutants were assayed for a defect in plant protection against a luxCDABE-tagged P. syringae DC3000 derivative in a space-saving 24-well plate system. The bioluminescence of the pathogen was used as the indicator of pathogen proliferation and allowed for the identification of Sphingomonas sp. Fr1 mutants that had lost the ability to restrict pathogen growth before disease symptoms were visible. Potential candidates were validated using the same miniaturized experimental system. Of these mutants, 10 were confirmed as plant protection defective yet colonization competent. The mutants were subsequently evaluated in a previously described standard microbox system, and plants showed enhanced disease phenotypes after pathogen infection relative to those inoculated with the parental strain as a control. However, the disease severities were lower than those observed for control plants that were grown axenically prior to pathogen challenge, which suggests that several traits may contribute to plant protection. Transposon insertion sites of validated mutants with defects in plant protection were determined and mapped to 7 distinct genomic regions. In conclusion, the established screening protocol allowed us to identify mutations that affect plant protection, and it opens the possibility to uncover traits important for in planta microbe-microbe interactions.

Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase.

Green notes are substances that characterize the aroma of freshly cut grass, cucumbers, green apples, and foliage. In plants, they are synthesized by conversion of linolenic or linoleic acid via the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL) to short-chained aldehydes. Current processes for production of natural green notes rely on plant homogenates as enzyme sources but are limited by low enzyme concentration and low specificity. In an alternative approach, soybean LOX2 and watermelon HPL were overexpressed in Saccharomyces cerevisiae. After optimization of the expression constructs, a yeast strain coexpressing LOX and HPL was applied in whole cell biotransformation experiments. Whereas addition of linolenic acid to growing cultures of this strain yielded no products, we were able to identify high green note concentrations when resting cells were used. The primary biotransformation product was 3(Z)-hexenal, a small amount of which isomerized to 2(E)-hexenal. Furthermore, both aldehydes were reduced to the corresponding green note alcohols by endogenous yeast alcohol dehydrogenase to some extent. As the cosolvent ethanol was the source of reducing equivalents for green note alcohol formation, the hexenal/hexenol ratio could be influenced by the use of alternative cosolvents. Further investigations to identify the underlying mechanism of the rather low biocatalyst stability revealed a high toxicity of linolenic acid to yeast cells. The whole cell catalyst containing LOX and HPL enzyme activity described here can be a promising approach towards a highly efficient microbial green note synthesis process.

Over-expression of chloroperoxidase in Caldariomyces fumago.

The filamentous fungus Caldariomyces fumago secretes a chloroperoxidase (CPO). To increase its production, we integrated a CPO-expression cassette into the non-transcribed spacer regions of the rDNA in C. fumago. One strain was obtained that had twice the CPO activity when grown in shake-flask and bioreactor compared to the wild-type. The highest CPO activity from the bioreactor cultivation was 3,236 U ml(-1). This is the highest value reported so far.