Systems Biology of Clostridium

The following sections are included:

• TABLE OF CONTENTS
• PREFACE

Solvent production from clostridia began 100 years ago and evolved into one of the largest global biotechnological processes in the first half of the 20th century. Despite its rather low level of productivity, this process remained feasible until it was replaced by more economic crude oil-based production. In reviving the process, sparked by rising crude oil prices, optimization of yield and productivity is essential. Modern scientific tools, integrating methods from molecular biology to mathematical modeling and process control, are employed to gain a thorough understanding of interactions during the process at molecular, cellular and bioreactor levels. A systematic approach with which to implement this interdisciplinary research is to construct a metabolic network from the sequenced genome. Such a genome-wide metabolic network is the basis for the set-up of mathematical equations as well as for studying regulatory interactions between its components. This first step, accompanied by suggestions for further work, is presented in this chapter.

In recent years the genus Clostridium has assumed greater prominence in terms of both the diseases individual species cause and as a consequence of the renewed importance of certain strains in the production of chemical commodities from renewal biomass, and in particular biofuels. This has precipitated the determination of a plethora of genome sequences, and, more importantly, an acceleration in efforts directed at the development of genetic systems to facilitate the exploitation of the data being generated. As a consequence, new methods have been formulated with which directed mutants can be made to assist reverse genetic studies, most notably insertional systems based on retargeting group II introns, e.g. the ClosTron. In parallel to this, inroads have been made towards the development of efficient, random mutagens for forward genetic approaches, in the form of exemplification of transposome technology and a mariner-based transposon in Clostridium perfringens and Clostridium difficile, respectively. Still to be implemented are rapid and reliable methods for creating in-frame deletions as well as more effective methods for delivering large segments of DNA into the genome. The formulation of such methods has already reached an advanced stage of development in our laboratory. In the meantime, there is still some way to go before clostridial researchers can boast the degree of sophistication available to those researchers studying Bacillus.

Clostridium acetobutylicum is a strictly anaerobic, Gram-positive bacterium and is characterized by its fermentation products acetone and butanol. Its potential for the biotechnological production of these bulk chemicals initiated intensive research activities and C. acetobutylicum became a model organism for apathogenic clostridia. Thus, it is not surprising that this organism is also an attractive candidate for systems biology studies. Several of the necessary “-omic” technologies, including proteomics, have been applied to C. acetobutylicum. However, in relation to other microorganisms, the knowledge of the proteome of C. acetobutylicum is limited…

Among the numerous solventogenic clostridial strains that have been isolated, two species have emerged as model organisms for studying butanol production, viz. Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052. The metabolic switch from acid production to solvent production which is typical for these organisms is coupled to spore formation but also general stress response. By the availability of the genome sequences of both model organisms, we were able to compare the genetic organization of their stress response. High similarities existed regarding the presence of genes encoding heat shock proteins, their classification into established Bacillus subtilis hsp gene categories as well as their organization within operons and their regulatory structures. C. acetobutylicum and C. beijerinckii possess class I and III hsp genes, with class III hsp genes including members of the clpC operon. Both organisms also possess genes for potential class IV hsp genes, however, they do not appear to contain class II type hsp genes. Similarities and differences in the organization of the genes and gene clusters are discussed.

The Gram-positive bacterium Clostridium acetobutylicum responds to variations of the external pH value by changing its metabolic profile. Since it is unable to maintain a constant internal pH value it shifts its metabolism between two distinct phases differing in metabolome, proteome, and transcriptome. During acidogenesis (high pH value) C. acetobutylicum primarily produces the acids acetate and butyrate. In contrast, the bacterium enters solventogenesis for low pH levels during which the main products are the industrially relevant solvents acetone and butanol…

In an industrial surrounding, process models are useful for process evaluation, prediction, and control. The abstraction of a process time course into an empirical equation may be sufficient under these conditions, yet this type of model cannot be extrapolated and does not provide further insight into the process. The development of mechanistic process models, based on transport phenomena and the corresponding balance equations, yields an improved process description and additionally a set of interpretable model parameters. Extending this concept to its limits, using the availability of molecular data on various levels of intracellular activities, may lead to the ultimate mathematical model of a cell interacting with its environment. This model will reflect the actions of molecules within the cell, creating the complex systems behavior that is observed in cellular processes. It will accommodate all data created when working with cells and allow prediction of events within the cell in interrelation with its environment. From a long history of interest in and applications of clostridia, model development now evolves from simple descriptions towards systems biology and the in silico Clostridium.

Many bacteria are known to regulate genes in concert with cell density, a phenomenon termed ‘quorum sensing’. Stochastic and deterministic modelling approaches have revealed previously unknown properties of quorum sensing systems and generated novel, experimentally testable hypotheses. This chapter focuses on agr-type quorum sensing systems, which rely on the production of an autoinducing cyclic peptide signal that is sensed via a classical two-component system. agr-dependent quorum sensing is currently best understood in staphylococcal species, but homologous systems are present in a wide range of Gram-positive bacteria, particularly members of the class Clostridia. Deterministic modelling approaches for the staphylococcal agr systems are presented. Using Clostridium acetobutylicum as an example, it is investigated how existing models can be adapted to investigate agr systems present in other species.

Solvent-producing clostridia have attracted renewed scientific interest because of their potential to produce biofuels. Clostridium acetobutylicum ATCC824 and Clostridium beijerinckii NCIMB 8052 are the best-studied solventogenic species, and the availability of their entire genome sequences offered the possibility to compare both solventogenic species at the gene level. General genome features as well as COG (clusters of orthologous groups of proteins) categorizations were summarized. All genes coding for the various proteins of the main catabolic pathway were discussed and compared, including paralogous and orthologous genes. Whenever possible, the most likely catabolic gene was identified, based on available experimental data. Overall, for most enzymatic steps comparable genes were found in both genomes, although C. beijerinckii in general harbors more paralogs, resulting also in a relatively large genome size. Essential differences were found as well. C. acetobutylicum contains a pyruvate decarboxylase, while C. beijerinckii contains an Rnf cluster and a trimeric bifurcating hydrogenase. Moreover, in C. acetobutylicum most of the solventogenic genes are located on the pSOL1 megaplasmid, which is absent in C. beijerinckii. The physiological background of the observed similarities and differences was discussed.

During the first half of the 20th century the industrial acetone-butanol-ethanol (ABE) fermentation was second only in importance to the industrial ethanol fermentation. The strategic importance of the fermentation process for the United Kingdom, France and the United States during World War I, and the subsequent expansion of commercial fermentation in a number of countries in the Western world, has been extensively documented. More recently information regarding the industrial processes that operated in Russia and China has also been published. The butanol fermentation is also known to have been operated in Taiwan and Japan before, during and after World War II. It is apparent that the large-scale industrial fermentation process that was established in Taiwan and Japan was of significant strategic importance during the war. Commercial butanol fermentation processes were also in operation in both Japan and Taiwan after the war. However, there is very little information available regarding what was undoubtedly a major industrial fermentation industry in the Far East. This review attempts to draw together disparate information from a variety of sources to provide an overview of this largely overlooked facet of the industrial butanol fermentation process. The strategic importance of this fermentation during World War II, which formed the basis of the subsequent establishment of commercial processes, provides an interesting case study with some relevance for the current renewed interest in the production of biobutanol for use as biofuel and chemical feedstock.

The following section is included:

• INDEX