Metabolic Engineering in Corynebacterium glutamicum

Corynebacterium glutamicum is a nonpathogenic, gram-positive, food-grade microorganism with a long fermentation history, and thus is potentially useful as a host strain for producing a number of recombinant DNA products. We have developed fundamental genetic and genomic tools that enable us to manipulate and redirect pathways involved in central carbon metabolism and amino acid production so that we can understand gene organization, structure and regulation at the molecular level. A better understanding of metabolic signal processes involved in the glucose response could have potential applications to clinical problems such as diabetes.

Corynebacteria are Gram-positive, aerobic Actinomycetes. Corynebacterium glutamicum comes from the same genus as the causative agents of diphtheria and tetanus. However, the strains we work with in the lab have no pathogenic properties. In fact they are used in the commercial production of food grade nutrient supplements for humans and animals.

The primary industrial interest in corynebacteria and brevibacteria relates to the production of amino acids by fermentation. In 1956, a researcher at Kyowa Hakko Kogyo Co. in Japan isolated soil bacteria that were capable of producing large amounts of glutamic acid, a popular flavoring in Japanese cuisine. The discovery of these bacteria proved to be a major innovation in the industry at the time, and today these bacteria account for worldwide production of more than 800,000 metric tons of glutamic acid (a.k.a. MSG, primarily for use as a flavor enhancer) and 330,000 metric tons of lysine (for feed additives) annually.

Current focus in Corynebacterium research is the use of metabolic engineering to develop strains with improved isoleucine production. Metabolic engineering is an interdisciplinary approach that integrates flux analysis, metabolic modeling, gene discovery, pathway engineering and biochemical and genomic analyses for strain improvement.

All of a cell's lysine, methionine, threonine and isoleucine are produced from aspartate. Corynebacterium modulates the partitioning of precursors into pathways specific for the individual amino acids in this family at several levels including feed-back sensitivity of several key enzymes; differences in the specific activities of competing enzymes at nodes where the pathways for two amino acids diverge; and genetic repression. Modifying the expression of enzymes downstream of aspartate makes it possible to divert carbon away from lysine and toward other amino acids, e.g. threonine or isoleucine.

We showed that overexpression of native threonine dehydratase is sufficient to divert carbon from lysine production to isoleucine production. Expression of a heterologous threonine dehydratase that is not subject to feedback inhibition resulted in even greater improvements in isoleucine production. Physiological data from this engineered strain show both a shift in the carbon balance and an increase in the total amount of carbon entering the pathway.

The lab developed the first Corynebacterium DNA microarrays, which are tools for analysis of differences in gene expression between two samples. To understand the global impact of the physiological shift we carried out transcriptional profiling of the engineered Corynebacterium glutamicum strain in which the carbon flux has been shifted from lysine to isoleucine. DNA microarray experiments can be used to anticipate and interpret cell-wide effects of perturbation and generate better awareness of the global effects of metabolic intervention.

The gene expression studies helped to elucidate the physiology involved in this process and to identify non-apparent physiological targets for investigation. We found that the changes in gene expression were not static across the timeframe we examined, which demonstrates the importance of temporal data in any gene expression study. In ongoing research we are using transcriptional reporters to study the regulation of the target genes that were identified using DNA microarrays. Integrating data from genetic analysis, transcriptional profiling and metabolic profiling allows us to better anticipate and understand biological systems and perturbations to biological systems.