One of the major aims in medium engineering is to make maximal conversion of the designed medium into useful product through cellular metabolic network (Fig. 3.4).
In more recent years based on microbial biochemistry pathways of metabolism attempts have been laid to set up metabolic network stoichiometric models.
For example, based on a review of the Penicillium chrysogenum biochemical pathways Jorgensen (1995) and his associates have set up its stoichiometric models.
Biochemical engineering analysis of these stoichiometric pathway models providing measures of individual step rates and metabolic fluxes have been defined by the name metabolic engineering. Recombinant methods have further enhanced the study of metabolic engineering. Application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway distribution and rates, cell functions can also be modified through precisely targeted alterations in normal cellular activities.
Recruitment of heterologous proteins enables extension of existing pathways to obtain new chemical products, alter post translational protein processing, and degrade recalcitrant wastes. The experimental and mathematical tools, required for rational metabolic engineering are becoming available more and more. However, complex cellular responses to genetic perturbations can complicate predictive designs.
Thus, metabolic engineering naturally acquired design engineering (NADE)/redesigning of DNA that deals with metabolic pathway network in a way that changes metabolic process of the cell. The cell manufactures more of the desired product as a result of the changes in several metabolic reactions.
Advantages of metabolic engineering have been noticed from cloning and expression of heterologous genes. These can serve several useful purpose, including extending an existing pathway to obtain a new product, creating arrays of enzymatic activities that synthesize a novel structure, shifting metabolic flow towards a desired product, and accelerating a rate determining step.
So far the usefulness of metabolic engineering includes the following:
1. In recruiting heterologous activities for strain improvement:
In such recruitment activities metabolic engineering is concerned with the following:
1. Synthesis of new products is enabled by completion of partial pathways.
2. Transferring multistep pathways: Hybrid metabolic networks.
3. Creating new products and new reactants.
4. Transfer of promising natural motifs.
2. Redirecting metabolic flow:
The routes of reactions in a metabolic pathway are directed towards maximizing useful product formation by the following means:
1. Directing traffic towards the desired branch.
2. Reducing competition for a limiting resource.
3. Revising metabolic regulation.
Thus consequence of completing metabolic engineering cycle may exhibit potentials and perils of rational design through:
1. Cloning in industrial strains.
2. Dissecting physiological responses.
3. Design principles and cell models; coping with complexity and coupling.
Metabolic Engineering Case Examples:
1. Penicillin fermentation:
Through ages production of penicillin by Penicillium chrysogenum has been improved through the development of modified strains by repeated rounds of mutations and selections on the same parental strain P. chrysogenum NRRL 1951. The current estimated report of production of penicillin from mutant strain from NRRL 1951 is around 40-50 g/L penicillin V after 200 h of cultivation.
It is hoped that the present production strains of P. chrysogenum could be further improved with respect to penicillin production, and with the development of recombinant DNA technology it has become possible to use the more rational approach i.e. metabolic engineering to strain improvement than traditional mutation and selection. In order to use metabolic engineering successfully the primary requirement of a map of the rate controlling step(s) in the metabolic pathway leading to penicillin production has been fulfilled.
2. Lysine fermentation:
Lysine is an important essential amino acid. It’s over production has been achieved using metabolic engineering principles. This was done by significant flux alterations in the primary metabolism of Corynebacterium glutamicum ATCC 21253 in its metabolic networks. In the new biotechnology area, chemical and biochemical engineers are more likely to find newer roles in quantification of fermentation processes based on metabolic engineering.
It will enable them to alter genetic character of the amino acid or other product forming normal catabolic pathways of a cell so that new substrates can be used or new or enhanced chemical products can be formed by that cell.
3. Plant cell culture fermentation:
In more recent years metabolic engineering has provided a new promising avenue for enhancing product formation in a plant or plant cell culture. Metabolic engineering of plant secondary metabolite pathways in the production of fine chemicals has been reported to be a possible approach to increase yields. Several ways of application of metabolic engineering to improve yields have been reported. Compartmentation strategy used for alkaloid biosynthesis in Cantharanthus roseus is one of the classical examples of the same.
4. Solventogenic clostridial fermentation:
By developing suitable software appropriate experiments of Clostridium acetobutylicum ATCC 824 fermentation was designed for metabolic flux distribution analysis in the biochemical pathway of acetone-butanol solvent production. This software development served, therefore, as a tool for the metabolic engineering of solventogenic, Clostridial fermentation and was useful in stoichiometric modeling of the fermentation process.
Engineering Science Foundations of Metabolic Engineering:
1. Molecular stoichiometric model:
For such a model knowledge of the in vivo fluxes involved in associated reactions in metabolic pathway is essential. This is because metabolic engineering seeks to replace the shotgun random mutagenic approach with a more rational effort. It is an integrative procedure involving identification of metabolic bottlenecks or limitations followed by manipulation to alter metabolic fluxes.
Many powerful molecular biology techniques have been developed in more recent years to manipulate the genetics of an organism. Also, these techniques enabled cloning and sequencing of genes for enzymes associated with the desired product.
The resultant genetic repertoire served as an useful tool in redesigning of an organism to enhance product yields. The task of engineering science here is to devise process conditions so as to maintain stable cultures and to realize the full genetic potential of the used culture.
Thus, thorough mapping of biosynthetic pathways is a prerequisite for any metabolic engineering programme. Such mapping is necessary to develop a so called fermentation equation to verify fermentation process data consistency, to develop gateway sensors and to predict maximum theoretical yields.
2. Appropriate computational software development:
In metabolic engineering for assessing in vivo fluxes, various strategies are now available to resolve singularities. Many investigators have incorporated optimality concepts to promote linear programming methods of resolving singularities. This was done for general utility of reformulating any stoichiometric model as a nonlinear constrained minimization system. It is therefore, essential that appropriate computer software be constructed.
Also, it should be able to process and/or act on data and make calculations which are beyond human comprehension. In metabolic engineering the calculation of in vivo fluxes becomes a nontrivial issue due to interactions of fermentation pathways.