Metabolic engineering
Encyclopedia
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
and regulatory processes within cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
to increase the cells' production of a certain substance. These processes are chemical networks that use a series of biochemical reactions and enzymes that allow cells to convert raw materials into molecules necessary for the cell’s survival. Metabolic Engineering specifically seeks to mathematically model these networks, calculate a yield of useful products, and pin point parts of the network that constrain the production of these products. Genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
techniques can then be used to modify the network in order to relieve these constraints. Once again this modified network can be modeled to calculate the new product yield.
The ultimate goal of metabolic engineering is to be able to use these organisms to produce valuable substances on an industrial scale in a cost effective manor. Current examples include producing beer
Beer
Beer is the world's most widely consumed andprobably oldest alcoholic beverage; it is the third most popular drink overall, after water and tea. It is produced by the brewing and fermentation of sugars, mainly derived from malted cereal grains, most commonly malted barley and malted wheat...
, wine
Wine
Wine is an alcoholic beverage, made of fermented fruit juice, usually from grapes. The natural chemical balance of grapes lets them ferment without the addition of sugars, acids, enzymes, or other nutrients. Grape wine is produced by fermenting crushed grapes using various types of yeast. Yeast...
, cheese
Cheese
Cheese is a generic term for a diverse group of milk-based food products. Cheese is produced throughout the world in wide-ranging flavors, textures, and forms....
, pharmaceuticals, and other biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
products.
Since cells use these metabolic networks for their survival, changes can have drastic effects on the cells'ability to survive. Therefore, trade-offs in metabolic engineering arise between the cells ability to produce the desired substance and its natural survival needs.
In addition to directly deleting and/or overexpressing the genes that encode for metabolic enzymes, the current focus is to target the regulatory networks in a cell to efficiently engineer the metabolism.
History and Application of metabolic engineering
In the past, the productivity of a desired metabolite in a microorganismMicroorganism
A microorganism or microbe is a microscopic organism that comprises either a single cell , cell clusters, or no cell at all...
was improved successively through chemically induced mutagenesis and subsequent selection of the best-suited phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
for the production. However, this technique began to stagnate because enzymes that should be modified to increase the productivity were unknown.
The term 'metabolic engineering' emerged in the 1990’s to describe bacteria that had been genetically engineered using the strategy of removing, one by one, the most restrictive bottlenecks in a metabolic pathway
Metabolic pathway
In biochemistry, metabolic pathways are series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by a series of chemical reactions. Enzymes catalyze these reactions, and often require dietary minerals, vitamins, and other cofactors in order to function...
and the recombinant DNA technology. The development of the strategy for manipulating metabolic pathways in an organism and the actual implementation of such strategy was described by Stephanopoulos using Corynebacterium
Corynebacterium
Corynebacterium is a genus of Gram-positive rod-shaped bacteria. They are widely distributed in nature and are mostly innocuous. Some are useful in industrial settings such as C. glutamicum. Others can cause human disease. C...
glutamicum and the lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
production was improved significantly upon removing some bottlenecks in Corynebacterium
Corynebacterium
Corynebacterium is a genus of Gram-positive rod-shaped bacteria. They are widely distributed in nature and are mostly innocuous. Some are useful in industrial settings such as C. glutamicum. Others can cause human disease. C...
glutamicum metabolic networks (Stephanopoulos et al, 1993).By knocking out some genes and over expressing other genes, Dellomonaco was also able to reverse the direction of the endogenous beta oxidation
Beta oxidation
Beta oxidation is the process by which fatty acids, in the form of Acyl-CoA molecules, are broken down in mitochondria and/or in peroxisomes to generate Acetyl-CoA, the entry molecule for the Citric Acid cycle....
cycle of fatty acids in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
and use it to efficiently synthesize alcohols and carboxylic acids with various chain lengths and functionalities which both have the potential to create biofuels and chemicals(Dellomanoco et al,2011). At the industrial scale, the engineering of microorganisms is very fruitful, providing the most convenient and cost-effective method for large-scale production. According to the Biotechnology Industry Organization
Biotechnology Industry Organization
Biotechnology Industry Organization is an industry lobby group founded 1993 in Washington, D.C. Carl B. Feldbaum was the president until he retired in 2004, and was succeeded by James C. Greenwood.-External links:* *...
, "more than 50 biorefinery
Biorefinery
A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power, heat, and value-added chemicals from biomass...
facilities are being built across North America to test and refine these strategies to produce biofuels and chemicals from renewable "biomass
Biomass
Biomass, as a renewable energy source, is biological material from living, or recently living organisms. As an energy source, biomass can either be used directly, or converted into other energy products such as biofuel....
", which can help reduce greenhouse gas emissions". Potential advanced biofuels that could replace gasoline
Gasoline
Gasoline , or petrol , is a toxic, translucent, petroleum-derived liquid that is primarily used as a fuel in internal combustion engines. It consists mostly of organic compounds obtained by the fractional distillation of petroleum, enhanced with a variety of additives. Some gasolines also contain...
include short-chain alcohols and alkanes, replace diesel
Diesel
Diesel fuel in general is any liquid fuel used in diesel engines. The most common is a specific fractional distillate of petroleum fuel oil, but alternatives that are not derived from petroleum, such as biodiesel, biomass to liquid or gas to liquid diesel, are increasingly being developed and...
include fatty acid methyl esters (FAMEs, biodiesel) and fatty alcohols and jet fuel include fatty acid
Fatty acid
In chemistry, especially biochemistry, a fatty acid is a carboxylic acid with a long unbranched aliphatic tail , which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from...
-and isoprenoid-based biofuels [Keasling,2010]. The food industry relies heavily on preservatives such as citric acid, which is currently produced by microbes at a rate of several hundred thousand tons per year (Eberhard O. Voit et al, 2002).
As the world population is growing, oil, food and energy prices are likely to climb. The challenge is not to stop using tires, eating or buying oil and clothes . Rather, it’s about developing alternative, more sustainable ingredients from which to produce the products we rely on. Recent advances in metabolic engineering are helping us to meet these challenges.
Metabolic pathway manipulation
Cameron and Tong (1993), classified the metabolic pathway manipulation in five groups, i.e., application aiming at- Yield and productivity improvement of products made by microorganisms.
- Expansion of the range of the substrate that can be metabolized by an organism.
- Formation of new and novel products.
- General improvements in cellular properties.
- XenobioticXenobioticA xenobiotic is a chemical which is found in an organism but which is not normally produced or expected to be present in it. It can also cover substances which are present in much higher concentrations than are usual...
degradation.
Example of Metabolic Pathway Manipulation
Consider the production DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate) pictured to the right. DAHP is made from the combination of the glycolysis and pentose phosphate pathway. Assume you have that 7 moles of glucose(glu) enter the cell. The goal of metabolic analysis would be to determine the maximum yield of DAHP one could get. For more complicated systems, methods involving flux balances or extreme pathway analysis would be employed to find this result. Due to the simplicity of this example, the maximum yield can be determined by inspection.In this pathway before PEP syntase(PPS) is added the only metabolites leaving the cell are carbon dioxide(CO2), Pyruvate(PYR) and DAHP with only glucose entering. Pyruvate has three carbons and carbon dioxide has one carbon. If only seven moles of glucose enter the cell, DAHP is left with only three moles. This means the theoretical yield is 3/7. This seems pretty low and one could wonder if more of the glucose could be utilized. This is one of the goals of metabolic engineering. The production of CO2 is unavoidable, but if the reaction of PEP to PYR was reversed then no carbon would be lost to the exit of pyruvate. This is exactly what the enzyme PPS does. Now since PYR no longer leaves the cell, DAHP gets 6 moles for every 7 moles of glucose that enters the cell. This gives a theoretical of 6/7, a 50% increase!
If a company produced DAHP, they would almost double their production, saving a lot money. Hopefully this example serves to show one reason why Metabolic Engineering is currently so attractive.
Metabolic Flux Analysis
An analysis of metabolic flux can be found at Flux balance analysisFlux balance analysis
Flux balance analysis is a mathematical method for analysing metabolism. It does not require knowledge of metabolite concentration or details of the enzyme kinetics of the system...
Setting up a Metabolic Pathway for Analysis
The first step in the process is to identify a desired product. Reactions and metabolic pathways that are able to create this product are researched using databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG). These databases contain copious genomic and chemical information including pathways for metabolism and other cellular processes. Using this research, an organism is chosen that will be used to create the desired product. Considerations that are taken into account when making this decision are how close the organism's metabolic pathway is to the desired pathway, the maintenance costs associated with the organism, and how easy it is to modify the pathway of the organism. Escherichia coli (E. coli) is widely used in metabolic engineering to synthesize a wide variety of products such as amino acids because it is relatively easy to maintain and modify. If the organism does not contain the complete pathway for the desired product, then genes that produce the missing enzymes must be incorporated into the organism. Most genes that are incorporated in this way are taken from a different species. This is a type of horizontal gene transferHorizontal gene transfer
Horizontal gene transfer , also lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism...
.
Analyzing a Metabolic Pathway
The completed metabolic pathway is modeled mathematically using linear algebra to find the theoretical yield of the product. Simple metabolic pathway analysis can be done by hand, but most require the use of software such as Constraint-Based Reconstruction and Analysis (COBRA) or the ETH Zurich Elementary Flux Mode Tool. These programs use the formula for specified systems shown above as well as extreme pathway analysis to determine the fluxes within the metabolic pathway. A flux is the rate at which a given reaction in the network occurs. To solve a network by hand using the equation for determined systems, the chemical balances associated with the known fluxes are placed in the matrix Gm and the known flux values are placed in the vector Vm. The known fluxes are typically those fluxes which are easy to control or measure such as the rate of a chemical introduced into an organism by the researcher. The chemical balances associated with the unknown fluxes are likewise placed in the matrix Gc, and the unknown flux variables are placed in the vector Vc. When solved, the equation yields the values of all the unknown fluxes.Determining the Optimal Genetic Manipulations
After solving for the fluxes of reactions in the network, it is necessary to determine which reactions may be altered in order to maximize the yield of the desired product. This can be accomplished through genetic engineering to modulate the expression of certain relevant proteins and enzymes. In order to determine, however, what specific genetic manipulations to perform, it is necessary to use computational algorithms, such as OptGene or OptFlux (Rocha et al. 2010). These tools maximize a certain objective when the reaction network and relevant fluxes are provided as inputs. They provide recommendations for which genes should be overexpressed, knocked out, or introduced in a cell to allow increased production of the desired product.The necessary genetic manipulations can be performed using standard molecular biology techniques. For example, plasmid vectors are often used to introduce genes for overexpression (Work et al. 1980) of certain proteins. Also, repressors for these genes may be inhibited to allow increased gene expression. On the other hand, if the computational software recommends the deletion of a specific gene, that gene may be knocked out in an organism. Often, this requires the deletion of the functional form of the gene from the host genome. Different techniques exist for preparing gene knockouts in different organisms (Work et. al 1980).