Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state

Abstract

Novel enzymes that are stable in diverse conditions are intensively sought because they offer major potential advantages in industrial biotechnology, and microorganisms in extreme environments are key sources of such enzymes. However, most potentially valuable enzymes are currently inaccessible due to the pure culturing problem of microorganisms. Novel metagenomic and metaproteomic techniques that circumvent the need for pure cultures have theoretically provided possibilities to identify all genes and all proteins in microbial communities, but these techniques have not been widely used to directly identify specific enzymes because they generate vast amounts of extraneous data.In a first step towards developing a metaproteomic approach to pinpoint targeted extracellular hydrolytic enzymes of choice in microbial communities, we have generated and analyzed the necessary conditions for such an approach by the use of a methanogenic microbial community maintained on a chemically defined medium. The results show that a metabolic steady state of the microbial community could be reached, at which the expression of the targeted hydrolytic enzymes were suppressed, and that upon enzyme induction a distinct increase in the targeted enzyme expression was obtained. Furthermore, no cross talk in expression was detected between the two focal types of enzyme activities under their respective inductive conditions. Thus, the described approach should be useful to generate ideal samples, collected before and after selective induction, in controlled microbial communities to clearly discriminate between constituently expressed proteins and extracellular hydrolytic enzymes that are specifically induced, thereby reducing the analysis to only those proteins that are distinctively up-regulated.

Metaproteogenomics-guided enzyme discovery: Targeted identification of novel proteases in microbial communities

“…The recipe was modified a number of times until a final composition was found that enabled the microbial communities to reach metabolic steady state. The reactors and the defined medium is describe in detail in paper I [125]. By changing the chemical composition of the medium over a time period of about five months it allowed the microbial communities to adapt to the changes and maintain a complete and viable biogas producing community.…”

“…This makes them highly interesting as targets for enzyme discovery. In paper I [125], it was shown that the conditions for an efficient and targeted enzyme discovery by metaproteomics are conceivable. In particular it was shown that it is possible to establish a viable microbial community using a chemically defined medium, which is necessary for the downstream analysis.…”

Methods development for metaproteomics-guided bioprospecting of novel enzymes

“…What is important to note is that a viable microbial community was established, which was maintained at a metabolic steady state, which in turn produced an environment with very low background of extracellular hydrolytic enzymeactivity. By exchanging the substrate during culturing, the community responded measurably to the change by expression of extracellular, hydrolytic enzymes to specifically break down the substrate added, without interference of contaminating substances and proteins that originate from the original habitat (paper I [118]). This is of outstanding importance, because it is usually very difficult in meta-omics projects to identify relevant enzymes or target the production of specific enzymes against the large background of all proteins that are collected.…”

“…It contains over 40 individual components and was refined several times during this study to allow for the microbial communities to reach a metabolic steady state. Detailed information on the final recipe is given in paper I [118]. The gradual exchange of culturing medium during the experiments resulted in a microbial community in a constructed environment that was eventually free of components and impurities from the original biogas reactor habitats after an operation time of approximately five months.…”

“…This suppression was expected, because if chemically simple nutrients are available, the energy and nutrient consuming task of producing hydrolytic enzymes will not be necessary and therefore not undertaken by the microorganisms of the microbial community by using evolutionary principles of microbial metabolism [124]. From this enzyme suppressed steady state, it was possible to control and induce targeted hydrolytic activity by adding the complex substrate during a starvation period [124] and to identify the microbial enzymes against a clear background (see paper I) [118]. For the induction of cellulolytic enzyme activity, cellulose in form of filter paper (Whatman no.1) was chosen as the sole complex carbon source instead of the simple monomeric sugar glucose.…”