Bernhard Eikmanns prefers not to get involved in research that will end up on bookshelves. So it was an easy decision for the biologist to drop the research he was doing into bacteria that are difficult to cultivate during his doctoral studies and concentrate instead on Corynebacterium glutamicum, a bacterial species that is much easier to cultivate. Corynebacterium glutamicum is an excellent object for scientific research and industrial application. Industrial concerns use this irregularly rod-shaped soil bacterium to produce amino acids such as glutamic acid and lysine by fermentation at scales of several million tons at a time. Like the majority of the 20 amino acids required for the synthesis of proteins, L-glutamic acid and lysine are produced from intermediates of the glucose degradation pathway and the citric acid cycle.
During a research stay at the Massachusetts Institute of Technology in Tony Sinskey’s laboratory in 1987, the microbiologist, who is now 53 years old, learned how to manipulate Corynebacterium glutamicum using genetic engineering tools and methods. C. glutamicum was discovered in Japan in 1957 as a natural and effective producer of glutamic acid and has since become an important object of applied molecular biology research.
It took around five years before it was possible to genetically manipulate the soil bacterium in the same kind of ways as the biotechnologists’ most popular research object, Escherichia coli. Eikmanns’ research at the Jülich Research Centre, University of Düsseldorf, where he did his habilitation, and at the Ulm-based Institute of Microbiology and Biotechnology (Director: Peter Dürre), involves trying to gain insights into the metabolism of C. glutamicum, which consists of around 3,300 genes, and then optimising it for technical purposes. He has been working with partners from industry, notably researchers from Evonik Industries, for many years, and both partners benefit from this cooperation. Since the late 1980s, Eikmanns has been focusing on research that is nowadays referred to as “white or industrial biotechnology”. Like many researchers around the world, Eikmanns, who is a member of European consortia such as ERA-IB, is looking for sustainable solutions for a biobased, post-fossil economy involving “his” bacterium. The findings obtained from research into C. glutamicum could potentially be transferred to the pathogen Mycobacterium tuberculosis and used for the benefit of humans. A project undertaken in cooperation with a colleague from Ulm, Steffen Stenger (Institute of Medical Microbiology and Hygiene), and colleagues from Hanover (Hanover Medical School) has led to promising results, which Eikmanns now plans to analyse in greater detail.
After several decades of successful Corynebacterium research, Eikmanns is still surprised by this bacterium: it is very robust and able to fight off many environmental influences, it does not regulate its metabolism as much as other bacteria, and it possesses many proteins that transport metabolites out of the cell. Eikmanns explains that losing these intermediary products does not really make sense for the cells as they lack energy and substrate. However, the researchers have not yet been able to satisfactorily explain this phenomenon. Biotechnologists can manipulate the metabolism of the amino acid producer C. glutamicum at four locations: 1) the uptake of substrate (glucose), 2) the central metabolism, 3) the biosynthesis pathways and 4) the transport of the amino acids out of the cell. Eikmann’s research approach (“Many people are not interested in things that are obvious – but we are”) has provided his research group with high lysine quantities produced by C. glutamicum. The sale of the related patent enabled Eikmanns to purchase the equipment required to analyse amino acids and proteins.
Eikmanns explained that all research involving C. glutamicum and other organisms up until 1998/1999 focused on the central biosynthesis pathways. Little consideration was given to the transport of compounds out of the cell, and Eikmanns was the only researcher to look at the precursors of these compounds.Eikmann was interested in the intermediates that were required by a certain secondary metabolic pathway and how the production of a certain intermediate could be manipulated and regulated. He discovered that the accumulation of intermediates led to the inhibition of the pathway involved in their production and postulated the need to look at the “secondary pathways” involved in the supply of amino acids, lysine for example. Eikmanns refers to this as the “biochemistry of intermediate supply”. This “secondary pathway” led him to discover a number of previously unknown enzymes and he found that C. glutamicum was simpler than other organisms and was able to use parallel biosynthesis pathways.
Compounds need to reach a specific concentration inside cells before transport proteins take action and excrete the metabolites into the nutrition medium. Summarising the results of 10 years of research into transport molecules, Eikmanns explains that a particular number of molecules must be present to activate such transport molecules. “Compounds that need to be removed from the cell have to be present in a quantity that is high enough for the compounds to make contact with the transport protein.” Before they are able to increase the concentration of transporter, the researchers need to identify around a dozen potential transporter candidates. This is being done by comparing the sequences of the 150 or so known membrane proteins with yeast and E. coli proteins. Wild-type bacterial strains do not react adversely to being enriched with transport proteins, and their transport rate speeds up considerably. In addition to working on aspects of applied white biotechnology, Eikmanns’ team of 12 researchers also focuses on knowledge-oriented research. The scientists discovered by accident that C. glutamicum also metabolises arabitol, a pentavalent sugar alcohol that occurs in nature. The researchers have tested and confirmed their finding and discovered three C. glutamicum genes that convert arabitol into a key intermediate, which is also known to be present in other organisms. They have not yet found out whether the genes of the three enzymes that are involved in the arabitol pathway can be used for biotechnological applications. The first thing on the researchers’ agenda was to identify and characterise the enzymes biochemically. This led to the discovery that the enzymes involved in the arabitol pathway are regulated by a protein. Despite their huge industrial importance, only 1,500 metabolic reactions of C. glutamicum are known. Eikmanns estimates that there are still around 2,000 reactions that need to be investigated, which represents a hefty workload for the relatively small number of Corynebacterium researchers worldwide (around 100). Eikmanns believes that the scientific community will not have a transparent C. glutamicum available within the next 25 years and pointed out that this is not all regrettable as he believes that the bacterium most likely manages to survive in the laboratory with around half of its genes.
In addition to being used in academic research, bacteria such as C. glutamicum also play a major role as producers of chemical compounds and hence in the switch to a petrol-free economy. C. glutamicum can also be engineered to produce biofuel like B10 (a blend of petrodiesel with 10% biodiesel) which has created some controversy in Germany. Eikmanns’ solution to the problem is the use of isobutanol rather than ethanol. Engineered bacteria such as C. glutamicum are effective producers of isobutanol by fermentation. Once produced, isobutanol could then be used as fuel or as a precursor for the production of isobutene, a compound that is traditionally produced from petrol and used for the production of plastics.
Eikmanns and many other biotechnologists are intensively focused on expanding the substrate range of C. glutamicum in order to be able to counteract the “tank-plate dilemma”. C. glutamicum lives on glucose, a major food product, which explains why alternatives are being sought. Cellulose, which consists of C5 sugars (xylose and arabinose) that are not currently used for the production of food, might be an alternative substrate. Eikmanns and his colleagues are trying to use C. glutamicum for something US researchers have already succeeded doing with metabolically engineered Clostridium cellulolyticum bacteria, which are able to produce isobutanol from cellulose (W. Higashide, Y. Li, Y. Yang, J. C. Liao. Metabolic Engineering of Clostridium Cellulolyticum for Isobutanol Production from Cellulose. Applied and Environmental Microbiology, 2011; DOI: 10.1128/AEM.02454-10).
Eikmanns is working with a number of European colleagues on a project entitled BioProChemBB, which focuses on assessing the suitability of C. glutamicum as a producer of platform chemicals such as succinate, fumarate, malate, aspartate and itaconate. Eikmanns describes the initial successes: one of the substances (succinate) is being produced with fungi and some big companies have started to establish production facilities. It will still be a number of years before the effective production of other substances is implemented.While researchers like Eikmanns have plans to produce succinate using Corynebacteria, other researchers are attempting to do the same thing with yeasts, which, in contrast to bacteria, are able to tolerate lower pH values. Succinic acid is released into the medium where it increases its acidity, which is why two of the nine partners of the EU project are attempting to gain an in-depth understanding of the bacteria’s pH sensitivity. Future plans include the development of production strains that are able to tolerate dicarbonic acid and an investigation into the feasibility of a biobased production method (upscaling).
A cooperative project with colleagues from Ulm and Hanover might be the key to medical application that the Corynebacterium expert is seeking. Eikmanns believes that his expertise can be applied to Mycobacterium tuberculosis, a bacterium that is distantly related to C. glutamicum. While the pathogenicity mechanisms of M. tuberculosis, a highly efficient pathogen that kills around two million people annually, are well known, further research is necessary to gain detailed insights in the bacterium’s metabolism.M. tuberculosis bacteria are able to undergo some kind of hibernation in macrophages where they feed on fatty acids rather than sugar. Eikmanns' research group has intensively investigated the degradation and regulation of short-chain fatty acids in C. glutamicum and discovered a global regulator of the bacterium’s central metabolism. Eikmanns’ cooperation partners identified the same regulator in mycobacteria. The researchers then integrated the C. glutamicum regulator gene into the M. tuberculosis genome and vice versa and found that the mycobacterial gene fulfilled its original function when integrated in the C. glutamicum genome. The same was true for the C. glutamicum gene when it was integrated into the M. tuberculosis genome. As this fatty acid metabolic pathway is not found in humans, the researchers’ finding might open up new pharmaceutical strategies for the abatement of M. tuberculosis by specifically targeting the bacterial pathway. Eikmanns hopes that he will soon be able to identify and characterise the proteins that enable the pathogen to survive. These investigations would then provide the basis for the treatment of M. tuberculosis infections, which currently mainly involves therapies that target bacterial virulence factors.
Eikmanns believes that the traditional use of Corynebacteria for the industrial production of amino acids will gradually reduce their application in scientific research. He further believes that applied research will gradually shift towards synthetic biology. For example, C. glutamicum is unable to produce isobutanol naturally, but is able to do so when equipped with a metabolic pathway from other organisms. Eikmanns can well understand the criticism that is leveled at engineered products of this kind, but he also believes that the synthesis of organisms is in principle safe due to the fact that it originates from compounds that are also found in human and plant metabolisms.