Industrial fermentation using bacteria, yeast and other microbes to produce specific products has the potential to reduce future dependence on fossil-fuel-derived chemicals. To further the economic viability of industrial fermentation processes, engineers have turned to genetic modification to stimulate production of a specific metabolic product, increase tolerance of the microbe to particular process conditions and increase product yields. This one-page reference outlines several microbial species that have undergone genetic modification most commonly, and describes some of the microbiology tools that are used to engineer microbial strains for industrial processes.
Microbes
The following microbes are common in industrial microbiology.
Escherichia coli (E. coli) occurs naturally in the gut of animals and humans (Figure 1). In addition to its status as a biological research model system, E. coli has been heavily used in industry due to its ease of culturing and handling, as well as its amenability to genetic manipulation. Enzymes produced by recombinant E.coli are used to catalyze specific fermentation reactions in industrial processes. Bioproducts such as ethanol, n-butanol, isoprene, biodiesel, 1,3-propanediol, as well as insulin and other recombinant proteins have been made with E. coli.
FIGURE 1. E. coli bacteria have been heavily used in industry for making products
Bacillus subtilis is a gram-positive soil bacterium that has proven to be a versatile model for studies of metabolism, as well as a platform for producing medicinal proteins and industrial enzymes.
Bacillus thuringiensis (bt) is a soil-dwelling, gram-positive bacterium that is used as a biological pesticide to control insects in agricultural settings. Bt produces proteins that are toxic to specific insects, making it an environmentally friendly alternative to chemical insecticides. Major crops, such as corn, cotton and potatoes, have been genetically engineered to produce Bt proteins to increase insect pest resistance.
Saccharomyces cerevisiae, also known as brewer’s yeast, is integral for making beer and wine, but is also used for making food additives, recombinant proteins and biofuels, such as ethanol. This species has proven to be efficient in generating products, and amenable to genetic engineering. Effective processes with S. cerevisiae need strict control of fermentation conditions.
Genetic modification techniques
Microbes naturally produce a variety of products in low concentrations that have been used as antibiotics, drugs, vitamins, enzymes, bulk organic compounds, polymers, amino acids, biofuels and so on. Because economical bioprocesses require microbial strains to produce high levels of a desired product that may not be an inherent property of that organism, modifications to their genetic material are made to boost production levels or increase other desired microbial properties or features. The following are some of the approaches and technologies that can be used to make these modifications.
Mutagenesis and strain selection. Microbial strains are exposed to chemical agents or X-rays that induce mutations in the DNA, then evaluated for the desired properties. Those exhibiting the desired characteristics are separated and isolated for further propagation and directed evolution.
Plasmids. Plasmids are small, (typically) circular DNA segments that serve as vectors to transport and introduce genes for desired traits into the cells of a host organism. They are found outside the chromosome of microbes in bacteria and archaea. As genetic engineering tools, they can be used to introduce, modify and remove target genes. Plasmids are transmissible via a process called conjugation, which involves the transfer of genetic material from one bacterial cell to another through direct contact. Engineered plasmids can be introduced into bacterial cells by transformation, a process whereby bacteria take up free DNA from their surroundings. Recombinant plasmids can be introduced to the cell’s surroundings and taken up by cells. If strains cannot take up free DNA from their surroundings, electroporation techniques can be used, where an electric pulse creates a temporary pore in the cell membrane of a bacterium for introducing new DNA.
Homologous recombination is a natural cellular process that enables the exchange of genetic material between similar DNA sequences. In nature, it repairs DNA damage and introduces genetic diversity, but in genetic engineering, it can be used to accomplish targeted gene editing by inserting or deleting specific DNA sequences in microbial genomes.
CRISPR-Cas9. Clustered regularly interspersed short palindromic repeats (CRISPR) technology precisely cleaves DNA, and has fast become a powerful genetic engineering tool. In bacteria, CRISPR functions as an adaptive immune system to protect bacteria against viral infections. In gene editing, a guide RNA segment is designed to match a specific DNA sequence target. The guide RNA directs the Cas9 enzyme to a specific area of the DNA sequence, where the Cas9 enzyme cleaves both strands of the DNA. The technology then takes advantage of the cell’s natural DNA repair mechanisms to edit genetic material.
References
Mahdizade-Ari, M., Dadgar, L. and others, Genetically Engineered Microorganisms and their Impact on Human Health, Int. J. Clin. Pract., March 9, 2024.
Phillips, E., Using Microbial Genetics to Engineer the Future, American Society for Microbiology (ASM) article, www.asm.org, Nov. 8, 2024.
Mustafa, M.G., Khan, M.G., Nguyen, D. and others, Techniques in Biotechnology, chapter found in “Omics Technologies and Bioengineering,” Academic Press, 2018.
Keller, W. Gezork, K., Popp, N. and others, Modern Fermentation and Fermenter Design, Chem. Eng., April 2024, pp. 35–39.