Metabolic Engineering

Summary

Metabolic engineering is a science associated with biology and chemistry that emerged in the 1990s. Metabolic engineering allows the design of biochemical pathways that do not exist in the natural world and the redesign of existing biochemical pathways, often using genetic engineering. Metabolic engineers modify biochemical pathways by reducing cellular energy use or waste production, changing the nutrient flow to the cells, or improving the productivity and yield of a particular pathway. Additionally, metabolic engineers have the potential to design new organisms tailor-made for the desired chemicals and production processes. Many novel compounds of industrial and medical interest can be produced by metabolic engineering. In the twenty-first century, the main efforts of metabolic engineers are concentrated on biofuels and pharmaceuticals.

Definition and Basic Principles

Metabolic engineering is a relatively new field that deals with the modification and optimization of metabolic pathways, mainly in microorganisms, by altering genes, nutrient uptake, or metabolic flow to allow the production of novel compounds that are of industrial and medical interest. Metabolic pathways of living organisms are not optimal for specific practical applications, but they can be modified using the tools of modern biotechnology, such as genetic engineering. Redesigning existing, natural metabolic pathways for useful purposes is a main objective of metabolic engineering. Metabolic engineering usually includes two phases—careful analysis of the metabolic pathway and genes involved in the pathway (analytical phase) and its modification (synthesis phase). Pathway analysis often includes metabolic control analysis, which involves the determination of which compounds can control the productivity and yield of a particular pathway. Metabolic engineering tasks include improvements in productivity and yield of a particular pathway, expansion of substrate range, elimination of waste, improvement of process performance, improvements of cellular activities, and extension of product array. Metabolic engineering is becoming one of the principal fields of biotechnology.

Production of many chemicals and fuels uses nonrenewable resources or limited natural resources. Metabolic engineering creates many alternatives to replace dangerous chemicals and petroleum-based transportation fuels with clean, green, and renewable chemicals and biofuels.

Background and History

The term “metabolic engineering” first appeared in the early 1990s. Since that time, the range of products that can be generated has increased significantly, partly because of remarkable advances in other fields related to metabolic engineering, such as DNA sequencing and genetic engineering. With DNA sequencing, scientists were able to identify the majority of metabolic genes and enzymes in many organisms. In the post-sequencing era, the obtained information is used for the practical construction of biochemical pathways or whole organisms with optimized functions through metabolic engineering.

In the 1990s, scientists developed new genetic tools that gave metabolic engineers more precise control over metabolic pathways. They also created analytical tools that allowed the metabolic engineer to track metabolites in a cell to identify new biochemical pathways more precisely.

Earlier in the twenty-first century, metabolic engineers joined other scientists in their quest for alternative fuels, which are in high demand because of fluctuating oil prices and concern about climate change.

How It Works

Metabolic engineering is based mainly on microbial metabolism. Microbes produce different kinds of substances that they use for the growth and maintenance of their cells. These substances can be useful for humans. The goal of metabolic engineering is to enhance the microbial production of useful substances. To achieve this goal, metabolic engineers must follow a particular route. They need to choose a friendly organism (host) for their metabolic manipulations. They need to find cheap and available substrates to use for modified metabolic pathways. Finally, metabolic engineers must be able to perform genetic manipulations of metabolic routes. Metabolic engineers can also alter nutrient uptake or metabolic flow. All these steps are dependent on each other. For example, genes cannot be manipulated in every organism; products or metabolic intermediates may be toxic to its host.

Host and Host Design. Generation of products by metabolic engineering has been achieved by transferring product-specific enzymes or entire metabolic pathways into so-called user-friendly microorganism hosts, which were used traditionally in industry. These industrial microorganisms grow rapidly on inexpensive culture media available in bulk quantities, are open to genetic manipulation (and genetic manipulation tools are available), and are nonpathogenic (do not cause disease). In addition, it is important that the host can survive (and thrive) under the desired process conditions (ambient versus extremes of temperature, pH). It is essential that the host is genetically stable (with the introduced pathway) and not susceptible to virus or another microbe's attack. Among the host microorganisms most widely used are Saccharomyces cerevisiae and Escherichia coli. S. cerevisiae, or baker's yeast, has been used for making bread and alcohol for thousands of years. It is one of the earliest domesticated organisms. This organism has come to be used in a large number of different processes within the biotechnological and pharmaceutical industries. Comprehensive knowledge of S. cerevisiae has been accumulated over a long period. In addition, the complete genome sequence of yeast is available, and yeast is nonpathogenic. The well-established fermentation and process technology for large-scale production with S. cerevisiae in bioreactors makes this organism very attractive for several industrial purposes.

Escherichia coli, commonly known is E. coli, is a bacterium that is widely used as a research (model) organism. It is easy to grow and genetically manipulate this bacterium, and its genome sequence is available. Several important products such as interferon (flu-fighting drug), insulin, and growth hormone are manufactured by genetically modified E. coli.

In addition to E. coli and S. cerevisiae, several other microorganisms are widely used as hosts for metabolic engineering manipulations, including bacteria Bacillus subtilis and Streptomyces coelicolor.

Finally, in addition to redesigning particular metabolic processes, metabolic engineers may also design de novo artificial cells that will produce desired products.

Substrates. To make metabolically engineered products, chemical substrates are needed. To make these products economically viable, inexpensive sources of substrates are required. Substrates must contain different chemical components, such as carbon, nitrogen, oxygen, and hydrogen. For example, metabolic engineers are looking at sugars from cellulosic biomass as potential substrates for biofuel production. Cellulosic biomass is a very attractive biofuel feedstock because it is abundant and, as it cannot be digested by humans, is not in demand for food. On a global scale, terrestrial plants produce almost 100 billion tons of cellulose per year and aquatic plants produce another 50 billion tons, making it one of the most abundant organic compounds on Earth.

Genetic Manipulation of Metabolic Routes. Genetic manipulation of metabolic pathways by adding or deleting genes or modifying the expression of existing genes in the host can serve several useful purposes. It can extend the existing pathways, shift the metabolic route into a desired pathway, or increase the rate-determined step of the particular metabolic route. Adding genes into the host consists of the following steps.

• The gene for the desired pathway is obtained from the non-host organism.
• The gene is inserted into the host cell.
• Host cells are induced to express (to cause the gene to manifest its effects) this “foreign” gene to produce the desired product.

One example of how gene manipulation is used in areas relevant to metabolic engineering is as follows: In the mold Aspergillus terreus, the producer of cholesterol-lowering drug lovastatin, genes were modified to increase their expression levels to change its metabolism in terms of drug production.

Another example is the introduction of bovine lactic acid pathway into S. cerevisiae. As a part of this, a gene responsible for speeding up the removal of hydrogen, which participates in lactic acid production, was expressed in S. cerevisiae, and lactic acid was produced at a rate of eleven grams per liter per hour. Because it tolerates acid, yeast may serve as an alternative to bacteria, which is usually used in industry for lactic acid production. Lactic acid is widely used as a food preservative.

Altering Nutrient Uptake or Metabolic Flow. Alteration of nutrient uptake or metabolic flow can be done not only by genetic manipulation but also by using inhibitors—simple chemicals or physical factors such as light or temperature.

The alteration of molecular hydrogen (H₂) production in green algae using high-intensity light is an example of metabolic flow modification by physical factors. H₂ is one of the possible energy carriers of the future. Microscopic green algae produce H₂ in photosynthetic reactions from water using sunlight as an energy source, usually in anoxic (without oxygen) conditions. Oxygen (O₂) produced by photosynthesis in green algae is an inhibitor of H₂ production. Brief illumination of algal cells by high-intensity light was accompanied by rapid suppression of photosynthetic O₂ evolution. The decline in the rate of O₂ evolution was accompanied by stimulation of H₂ production in algal cells.

Production Systems. All of the above-mentioned considerations are very important in metabolic engineering, although it is also important to ensure that the production of desired compounds by modified cells can be reproduced. This can be achieved by using bioreactors, in which important parameters such as pH, temperature, substrate supply, and other variables are controlled. It is even possible to modify cell metabolism by using bioreactors.

Applications and Products

There is a wide range of metabolic engineering products and applications. A number of novel applications and products will arise in the future.

Pharmaceuticals. Metabolic engineering is most promising in the production of pharmaceuticals. These include pharmaceuticals from different classes of natural products: alkaloids, isoprenoids, and flavonoids. Biosynthesis of natural products is an emerging area of metabolic engineering that offers significant advantages over conventional chemical methods. Some pharmaceutical compounds are too complex to be chemically synthesized or extracted from biomass organisms inexpensively.

Alkaloids are mainly plant-derived compounds that have been used as drugs such as morphine. Alkaloids are produced by simple extraction from plants. Studies show that alkaloids can be synthesized from amino acids by metabolic engineering in E. coli and S. cerevisiae.

Isoprenoids, organic compounds composed of two or more hydrocarbons, have a range of functions: pigments, fragrances, and vitamins. Isoprenoids are also the precursors to sex hormones. Many isoprenoids have been produced using microorganisms, including carotenoids and various plant-derived terpenes. Metabolic engineers are using S. cerevisiae as a cell factory for the biosynthesis of isoprenoids. One metabolic-engineering success is the production of Taxol, which is used to treat breast cancer. It is an isoprenoid that was first isolated in the bark of the Pacific yew (Taxus brevifolia). The demand for Taxol greatly exceeds the supply that can be obtained from its natural source. A partial Taxol biosynthetic pathway has been engineered in S. cerevisiae.

Another metabolic engineering success is the production of isoprenoids-carotenoids. Carotenoids are naturally occurring yellow, orange, and red pigments commonly found in plants such as carrots as well as in bacteria, algae, and fungi, and play an important role in fighting disease. Metabolic engineers have successfully introduced carotenoid biochemical pathways into nonproducing carotenoid microbes such as E. coli and S. cerevisiae.

Flavonoids are a group of secondary plant metabolites. These compounds can be used as antioxidants or antiviral, antibacterial, and anticancer drugs. Many flavonoid biosynthetic pathways are known, and a wide array of flavonoid compounds from S. cerevisiae are expected to be produced by metabolic engineering in the near future.

Chemicals. Numerous chemicals, such as amino acids, organic acids, vitamins, flavors, fragrances, and nutraceuticals, can be manufactured by metabolic engineering.

Glycerol (or glycerin) is a chemical produced by metabolic engineering. Glycerol is used to synthesize many products, ranging from cosmetics to lubricants to food preservatives. It is a by-product of soap or biodiesel manufacturing. About 10 pounds of crude glycerol are created for every 100 pounds of biodiesel produced. Purification of this glycerol is expensive, but it can also be used as fuel. Metabolically engineered S. cerevisiae strain produced more than 200 grams of glycerol per liter of liquid medium.

Another example of chemicals produced with help of metabolic engineering are sterols. The most well-known sterol is cholesterol. Sterols are important for living organisms as they are a part of the cellular membrane, participate in the synthesis of several hormones, and are also nutrient supplements. Several sterols are being produced from metabolically engineered S. cerevisiae.

Fuels. Metabolic engineering can be used in the production of biofuels. Several scientific laboratories demonstrated the feasibility of manipulating microorganisms to produce molecules similar to oil-derived products, although the yield was very low. Adjusting the metabolic pathways of microbes to produce fuels similar to gasoline has the potential to save an enormous amount of money. These fuels can be used in existing engines, unlike other biofuels that require modified engines or fueling stations.

Several research groups metabolically engineered microorganisms to produce ethanol fuel using cellulose as substrate. Another example of the work of metabolic engineers is biodiesel production. Biodiesel is a diesel substitute primarily obtained from vegetable oils such as soybean. However, the production of this fuel is limited by the absence of sufficient vegetable oil feedstocks. Another problem is that to produce biodiesel, oils should be modified by transesterification, a chemical reaction with methanol, catalyzed by acids or bases (such as sodium hydroxide). Researchers have investigated a number of biofuel processing techniques, including hydrotreating, biological sugar upgrading, catalytic conversion of sugars, gasification, pyrolysis, and hydrothermal processing. E. coli has been metabolically engineered to produce biodiesel directly using low-cost materials.

Careers and Course Work

Biotechnology, pharmaceutical, and biofuel companies are the biggest employers of metabolic engineers. As new biology-based products move from research into production, more metabolic engineers will be needed in industry, universities, and government laboratories.

Metabolic engineering is an interdisciplinary science. Coursework includes biochemistry, molecular biology, chemistry, genetic engineering, analytical chemistry, biochemistry, biochemical and bioprocess engineering, and microbiology. Most metabolic engineers have a bachelor of science in biology, biochemistry, genetic engineering, microbiology, or biotechnology. Advanced degrees (master's and doctorate) in molecular biology, biochemistry, biotechnology, or genetics are necessary for research and teaching positions. Some governmental institutions, such as the Joint BioEnergy Institute, help students develop educational paths in metabolic engineering by providing internship opportunities.

Social Context and Future Prospects

Redesigning life forms to benefit humankind is an exciting career. However, metabolic engineers must pay particular attention to ethical, legal, and political issues. The field requires sustained support from the public and government to continue this work. In the early twenty-first century, metabolic engineering was more of a collection of successful experiments than an established science. As interest in biochemicals, human health, and sustainability increased, the development of metabolic engineering techniques was needed to expand the range of products. Data science aided by artificial intelligence propelled metabolic engineering in the twenty-first century as well as advancements in peripheral fields like systems and synthetic biology.

The CRISPR-Cas9 genome-editing tool enables metabolic engineers to insert, delete, mutate, and knockout genes in microbial hosts faster and optimize the combinations of alterations. Further evolution of the technology found applications in gene regulation, epigenetic engineering, imaging, and chromatin engineering. Modern applications of metabolic engineering include improving health through pharmaceuticals and the reduction of environmental stressors. Positive environmental impacts include the development of biofuels, sustainable textiles, and alternatives to meat. The field aims to continually improve molecules to further impact the agricultural, chemical, pharmaceutical, and clean energy fields.

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