Metabolic engineering, bioinformatics, evolutionary engineering

Mattheos A.G. Koffas, Assistant Professor

Metabolic Engineering

Metabolic Engineering is a relatively new area within the context of bioprocess engineering, which has been defined as the manipulation of intermediary metabolism through the use of genetic engineering techniques. Another term also used to describe the field is cellular engineering, which has emerged as an inclusive term that refers to the modification of cellular properties.

From an environmental and evolutionary point of view, Metabolic Engineering can be seen as a general method of in vitro evolution and rational strain design and can be divided into two basic categories:

• Modification of genes endogenous to the host organism to alter metabolic flux.
• Introduction of novel pathways by the introduction of new genes into the organism (heterologous expression).

In the second case, such introduction can create new metabolic pathways leading to modified cell properties including but not limited to synthesis of known compounds not normally made by the host cell, production of novel compounds (e.g., polymers, antibiotics, etc.) and the ability to utilize new nutrient sources.

Bailey (Science 252: 1668-1674 (1991)) describes the application of metabolic engineering as the recruitment of heterologous genes for the improvement of a strain, with the caveat that such introduction can result in new compounds that may subsequently undergo further reactions.

In a broader approach, Stephanopoulos et al. (Trends. Biotechnol. 11: 392-396 (1993)) first point to the fact that attempts to improve productivity of cellular systems or effect radical alteration of the flux through primary metabolic pathways is extremely difficult due to the control architectures at key branch points that have evolved to resist flux changes. Because of that, the conclusion is (Stephanopoulos, Curr. Opin. Biotech. 5:196-200 (1994)) that rather than modifying individual genes such as the "rate limiting step", in metabolic engineering it is necessary to systematically elucidate the control architecture of bioreaction networks. Because of that, multiple gene manipulation may be required for strain improvement. This leads to placing the main emphasis of current metabolic engineering, which is on integration (Metabolic Eng. 1: 1-10 (1999)). The focus is no longer on the analysis of individual, isolated reactions, but on integrated networks of metabolic pathways.

Within this context, in our group we are interested in the construction of production platforms of high-value commodity chemicals that can be used in nutrition, medicine and agriculture.

Transcription profiling

The progress of genomic research, with the availability of whole genome sequences, has allowed a more robust and global understanding of metabolic networks and their regulation, through the application of high-throughput tools such as DNA microarrays, a quantitative high-throughput method of screening using isolated genetic material as target. As a result, there is a growing number of successful applications of this method for the generation of (mostly) prokaryotic recombinant of mutant strains with specific genetic traits. There are two major challenges related to global genome profiling being addressed in our lab.

The first relates to the growing need for global transcription data that rely on in vivo rather than in vitro measurements. The significant achievement is to examine cellular responses within living cells, that is, within the immensely complex environment that even the simplest bacterial species present. The second challenge relates to the need for genome-wide transcription data for species whose genome sequence is still not available.

Our group is interested in addressing both these issues with the use of cellular arrays of reporter gene fusions. The group's long-term goals are:

• Identification of targets for the development of antibacterial agents, and
• Biotechnological processes optimization by identifying targets for metabolic pathway modification and eventually biocatalyst improvement.