Chapter 16: Microbial Biotechnology

Green Biotechnology

FIGURE B16.10 Process Diagram: Agrobacterium-mediated transformation After their formation, calli are transferred to media that promote the growth of shoots and roots.

-Controlling growth rates gives a chance to obtain desired metabolites. •Primary metabolite = A product of metabolic processes required for growth of the microbe •Secondary metabolite = Not required for microbial growth, often produced during stationary phase FIGURE 16.5 Primary and secondary metabolite production a. The production of primary metabolites, such as ethanol in a yeast anaerobic culture, mirrors the increase in biomass. b. The production of secondary metabolites, such as antibiotics, is usually induced in stationary phase.

For yeast growing under anoxic conditions, ethanol represents a primary metabolite, a product of metabolic processes required for anaerobic growth of yeast under anoxic conditions (Figure 16.5). Secondary metabolites, such as antibiotics, are not required for growth and are often produced during the stationary phase of the growth curve.

Vitamins and Amino Acids •Vitamins and amino acids -Fortified food products rely on large amounts of these compounds being produced. -Microbial synthesis of B vitamins is one example. •Can be cost-competitive with chemical synthesis Make sure you understand vitamins are NOT RED biotech but WHITE

To explore the role of biotechnology in vitamin production, let's first look at vitamin B12. Plants do not synthesize or require this compound. Conversely, animals need vitamin B12, which is also known as cobalamin. Although the active forms of this essential enzyme cofactor are adenosylcobalamin and methylcobalamin, the ingested form is usually cyanocobalamin, which can be converted to the active forms by the body. Normally, we obtain a sufficient supply of this vitamin through our food intake and the activities of our gut microbiota. However, a deficiency of vitamin B12 in humans can lead to an illness called pernicious anemia. Unlike vitamin B12, vitamin B2 (or riboflavin) can be chemically synthesized relatively easily

White Biotechnology •What role do microbes play in industrial biotechnology? •The basic principle in this field is to use microbial conversion of low-cost biomass to products with a higher value and industrial use. •Examples include -Biofuel production -Bioplastics -Industrial enzyme production -Production of vitamins and amino acids

White biotech encompasses the many and diverse applications of biotechnology for industrial purposes—from the production of household cleaners, to cosmetics, to fuel for our cars. Currently, the production of many of these products requires the use of fossil fuels. However, fossil fuel reserves are limited and the burning of fossil fuels has environmental impacts. The increased use of microbes in these processes may alleviate some of these obvious problems. Bacteria, archaea, yeast, and algae are being tapped to produce biofuels that can power transportation or generate electricity and be used in the production of various chemicals. Perhaps microorganisms can contribute to an energy economy that is more environmentally friendly than the current fossil fuel-based system.

Conclusion:

•Biotechnology is still a relatively new field. •Even in a short time, dramatic and life-improving advancements have been made. •We should appreciate the chances to make things better with these technologies but remember to be cautious of possible drawbacks along the way.

16.4 Fact Check

1. What is a biorefinery, and how can it be used? 2. Describe how waste biomass can be used as feedstock. 3. Explain why butanol may be a better biofuel than ethanol. 5. What are PHAs, and why is there interest in them? 7. What are the advantages of using microbial enzymes for commercial applications?

•The biorefinery concept -Renewable biomass feedstock put in. -Microbial activities act on feedstock. Useful materials can be harvested out FIGURE 16.17 The biorefinery The biorefinery concept is modeled on the petroleum refinery; biomass is processed into a number of usable products. In the biorefinery, crude biomass feedstock would be converted to biomaterials, bioenergy, and biochemicals.

A biorefinery converts biomass, living or recently living biological substance, into a number of products, including chemicals, energy, and materials (Figure 16.17). Unlike crude petroleum, the use of biological materials as a feedstock, or raw starting material, is virtually limitless, providing us with a renewable source of starting materials. Crop plants that are specifically grown as biomass feedstock to support the biorefinery industry include the perennial grasses (such as Miscanthus), switchgrass, and fast-growing trees like poplar.

•Biofuels: Butanol and acetone -Ethanol may not be the best option. -Butanol (4 carbons) can also be used in internal combustion engines and has properties more similar to gasoline than ethanol. FIGURE 16.20 Lignocellulose biomass Lignocellulose is usually subjected to pretreatment by enzymatic, chemical, or physical methods to release the sugars. These sugars can then be used as feedstock for bioproduction. why is butanol a better choice than ethanol?

Butanol and Acetone Although the development of ethanol as a broadly used transportation fuel is promising, ethanol may not be the best possible biofuel. It only contains about 70 percent of the energy as gasoline and is more corrosive than gasoline, making long-term storage difficult. The four-carbon alcohol, butanol, may be a better and more economically viable transportation biofuel. It has similar physical properties to gasoline and can be used directly as a substitute for gasoline in current internal combustion engines. Butanol currently has a $7 billion to $8 billion per year annual market worldwide, being used primarily for latex, enamels, and lacquers, as an additive to plastics to keep them flexible, and as a solvent in the manufacture of antibiotics, vitamins, and hormones.

•Biotech applications can be broadly grouped into -Red: Medical applications -White: Industrial applications -Green: Agricultural sector

Current applications of biotechnology can be grouped broadly into red biotechnology related to medical applications, white biotechnology related to industrial applications, and green biotechnology related to the agricultural sector (Figure 16.1). FIGURE 16.1 Red, white, and green biotechnology The applications of biotechnology are very diverse, including the agricultural (green, represented by soybeans, an important crop), medical and pharmaceutical (red, represented by antibiotic capsules), and industrial (white, represented by ethanol fuel) sectors.

Ethanol as a biofuel •Biofuels: Ethanol -Microbes (yeast) ferment sugars to produce 2-carbon ethanol. -Ethanol can be used in internal combustion engines with little modification. FIGURE 16.18 Ethanol still a. In an ethanol still, heat is used to evaporate the ethanol from the fermented solution, which is then condensed around cooling coils and collected. b. Large stills are used for the distillation of industrial ethanol following fermentation.

Ethanol, a 2-carbon organic alcohol, has many uses in the industrial sector and has garnered much attention in recent years as a biofuel to replace or supplement gasoline as a transportation fuel. Strains of yeast like Saccharomyces cerevisiae generate ethanol during the fermentation of sugars during anaerobic growth. Because internal combustion engines can burn ethanol with little or no modification, this molecule may be a reasonable alternative to gasoline. The cost of commercial ethanol production depends in large part on the cost of the biomass feedstock that is used (Figure 16.19)

•Production of recombinant proteins -Expression vectors can be used to mass-produce recombinant proteins (20% or more of total cell protein produced!). -For example, insert a eukaryotic DNA sequence for a product of interest into a plasmid; put the plasmid into bacterial cells, grow, and harvest.

FIGURE 16.11 Expression vector Expression vectors have customized promoters to drive a high level of transcription. An operator, such as Olac of the lac operon, regulates the level of transcription. Expression vectors also contain an optimized ribosome binding site (RBS) including a Shine-Dalgarno (SD) sequence and a start codon ATG. The coding sequence of the protein of interest is inserted 6-10 bases downstream of the SD to maximize initiation of translation. The coding sequence for the protein follows, including the start codon ATG and stop codon TAA, TGA, or TAG. Transcriptional terminator sequences end transcription. Expression plasmids also contain a selectable marker gene, shown here as an ampicillin resistance gene, and an origin of replication for the expression host, shown here as oriV. Most cloned protein-coding sequences can be transcribed and translated, including those from eukaryal genes. Note that this figure is not to scale.

•Biofuels: Ethanol -Not without its own problems, however •Commercial scaling and cost of feedstock use can be difficult to overcome. Biofuels: Ethanol •Some lignin/cellulose feedstocks are difficult to break down. •These feedstocks are usually waste products, so figuring out a way to use them would be very cost-effective and environmentally friendly.

FIGURE 16.19 Commercial ethanol production using different feedstocks Commercial ethanol is commonly produced from sugar and starch. Ethanol production from cellulosic biomass is under development, with some demonstration plants in operation. Pretreatment results in the efficient conversion of the cellulose to a form that can be fermented by alcohol-producing organisms and is central to the successful production of ethanol from biomass

•Biofuels: Butanol and acetone -Currently, these products are used in latex, enamels, lacquers, and plastic additives. -Production via fermentation is possible. •Petrochemical production became cheaper in the 19 50s. •With oil becoming more expensive, fermentation production of butanol may see a resurgence.

FIGURE 16.21 Production of acetone, butanol, and ethanol by Clostridium acetobutylicum The initial fermentation phase produces butyrate and acetate (brown boxes) that can be used by Clostridium acetobutylicum in metabolic pathways that lead to the formation of acetone, butanol, and ethanol (pink boxes).

•Bioplastics -Plastic use and buildup (as it is usually nonbiodegradable) is tremendous. -Microbes can produce natural polyesters: •Polyhydroxybutyrate (P H B) from B. megaterium •Polyhydroxyalkanoates (P H A s) Imagine if naturally biodegradable plastics could replace these non-degradable plastics. Microbes may help us obtain this ideal. In the mid-1920s, researchers discovered that the bacterium Bacillus megaterium produces polyhydroxybutyrate (PHB), a natural polyester. We now know various microbes can produce an assortment of polyesters, collectively known as polyhydroxyalkanoates (PHAs), that are similar in many ways to the synthetic polyester used today in everything from clothes to plastic bottles to sleeping bags. PHB is the simplest and most common of these polymers formed by microbes. Know what bioplastics are

FIGURE 16.22 Non-biodegradable plastics washed up on a beach The increasing use of plastic and its resistance to degradation has led to a growing problem with plastics waste. The increased production and use of biodegradable plastics would go a long way toward solving this problem.

-PHAs = Microbial food source in times of starvation •Plastics made from PHAs are biodegradable. -Natural production of PHB/PHA requires an enzymatic process with an input of sugars. •The process can even be carried out in photosynthetic organisms (eco-friendly!).

FIGURE 16.23 Structures of bacterial polyhydroxyalkanoates (PHAs) PHA deposits are accumulated within bacterial cells as carbon storage polymers. Diverse structures of the PHAs result in a broad range of physical properties that are similar to those of existing petroleum-based plastics.

•Bioplastics -Researchers are currently working on ways to increase the yield of PHAs from plants. •Introducing bacterial PHA synthesis genes into different types of plants. •Right now, levels are too low to be commercially viable. •Goals include localizing PHA production to leaves or stems and production of PHA in food crops such as corn (think about why these might be valuable properties...).

FIGURE 16.24 The poly-3-hydroxybutyrate (PHB) synthesis cycle Knowledge of the genetics of PHA biosynthesis is important for the production of polymers with desired properties. This figure shows the pathway for synthesis of the most common PHA, poly-3-hydroxybutyrate (PHB). The enzymes ketothiolase and acetoacetyl-CoA reductase are responsible for building the CoA-activated form of the monomer; synthase carries out the polymerization reaction. The substrate specificity of the PHB (or PHA) synthase enzyme influences the final form of the polymer. The polymer within the cell can be broken down and used as a source of carbon and energy

•Vitamins and amino acids -Millions of tons of amino acids are produced by microbial synthesis every year. •Used for nutritional supplementation for humans and animals •Used for flavor enhancement products Overproduction of lysine is a prime example of tricking cells into overproducing a compound for human use

FIGURE 16.29 Worldwide production of amino acids The production of amino acids is dominated by l-glutamic acid, l-lysine, and dl-methionine, which are used mostly as additives for food and animal feed.

Applications of Transgenic Plants Once it had been demonstrated that transgenic plants could be generated, researchers then considered which traits to introduce into the major crop plants such as corn, soybean, cotton, canola, and rice. Two traits targeted for engineering into crop plants were tolerance to nontoxic herbicides and resistance to insect pests. In both cases, scientists turned to microbes to find useful genes for these traits. Here, we will briefly examine both of these examples -Herbicide resistance •Roundup poisons plants but not mammals. •Genetically engineering Roundup-resistant plants means farmers can use it year-round. •A plasmid containing a gene for a resistant form of the enzyme EPSP was introduced into plants using biolistics by Monsanto scientists.

FIGURE 16.32 Glyphosate Glyphosate is a specific inhibitor of the EPSP synthase enzyme, which catalyzes a key step in the synthesis of aromatic amino acids. It is a very effective herbicide. Humans and other mammals do not contain the aromatic amino acid synthesis pathway because they do not produce these amino acids. This greatly reduces the likelihood of human toxicity.

•Applications of transgenic plants -Insect resistance •Bt toxin is produced by the bacterium Bacillus thuringiensis. •Bt toxin is highly specific and lethal to certain insect larvae that might feed on crops, but it isn't naturally in plants!

FIGURE 16.34 Process Diagram: Bacillus thuringiensis crystals and mode of action a. Bt crystals are commercially produced and widely marketed for insecticide use. b. This electron micrograph shows intracellular protein crystals that are produced during sporulation and have insecticidal activity that is only activated after ingestion by the larvae. c. After the toxin is activated, it binds to cadherin receptors in the membranes of gut epithelial cells, where it forms a pore that increases permeability to water, cations, and ATP, resulting in cell lysis.

•Applications of transgenic plants -Insect resistance •Sprays of Bt were initially used, but transgenic plants carrying Bt were a logical next step. •Transgenic tobacco expressing Bt shows far less insect feeding damage on valuable leaves. •Resistance may arise in the insects, however, as they evolve under this new selective pressure.

FIGURE 16.35 Transgenic tobacco expressing Bt toxin The expression of Bt toxin in transgenic plants such as transgenic tobacco (left) showing resistance to tobacco hornworm larvae, compared to the non-transgenic leaf (right), can be very effective at preventing major crop losses due to insect predation while avoiding the use of chemical insecticides.

•Secondary metabolites as therapeutics -Antibiotics •Penicillin •Streptomycin/actinomycin -Statins, inhibitors of cholesterol synthesis (produced by several fungi). -Improvements of strains and fermentation techniques have dramatically lowered production costs for many of these drugs.

First, a number of other secondary metabolites from microbes, besides antibiotics, have been discovered to have therapeutic effects. However, microbes are not the only original source of useful secondary metabolites. The antimalarial compound artemisinin is extracted traditionally from the plant Artemisia annua, but as we will see at the end of the chapter, it can now be produced by recombinant microorganisms. Not all drugs based on secondary metabolites are antimicrobial in nature. One of the most profitable classes of drugs on the market today is a secondary metabolite that inhibits the activity of hydroxymethylglutaryl (HMG)-CoA reductase, a key enzyme in the cholesterol synthesis pathway. These HMG-CoA reductase inhibitors, called "statins," were introduced in 1987 as a treatment to lower cholesterol linked to cardiovascular disease. They act by binding to the HMG-CoA reductase binding site, thus blocking interaction with the substrate.

-Fermentation = Controlled and regulated aerobic/anaerobic culture of microbes to produce desired substances •Bioreactors •Fed-batch reactors •Chemostats FIGURE 16.4 Types of bioreactors In the fed-batch reactor (left side), concentrated nutrient (feed) is added in a controlled manner until the maximum concentration of cells is reached, and then the biomass is harvested. In the chemostat (right side), some amount of biomass is continually removed (effluent) as the same amount of nutrient solution is added (feed). The addition of nutrient solution to the chemostat can continue indefinitely. Bioreactor This bioreactor at the Iogen Corporation, a biotechnology company located in Ottawa, Canada, has a capacity of 200,000 L.

In industrial microbiology, fermentation refers to any industrial process involving the culture of microorganisms, either aerobic or anaerobic, for the production of desired substances. Industrial fermentations take place in large-culture vessels called bioreactors or fermenters that are designed to maximize cell density and product yield (Figure 16.3). Bioreactors are specially designed so that environmental conditions, including nutrients, oxygen, pH, and temperature, can be controlled precisely. Generally, bioreactors are designed to support maximum production of the desired product. A fed-batch reactor (Figure 16.4) supports very high cell densities by providing the culture with a growth-limiting nutrient, such as a carbon source, over time. This process controls the growth rate and can often prevent the production of non-desired side products. In continuous bioreactors, like the chemostat shown in Figure 16.4, an equivalent amount of culture is removed as new medium is added.

•Agrobacterium—Nature's genetic engineer -A. tumefaciens causes crown gall tumors on plants. •It does so by carrying a tumor-producing plasmid. •Part of this plasmid is transferred into plant cells. •This makes it a good delivery system for gene insertion into plants (transgenic plant production). •This represents a cross-kingdom transfer of DNA (bacteria to plants). FIGURE 16.31 Crown gall a. Tumors can form at the crown of the plant, where the roots meet the stem, but they can also form elsewhere, even below ground on the root. In this photo, the tumor has formed higher up on the stem. These tumors result from infection by Agrobacterium tumefaciens. b. The pTi contains T-DNA that is transferred into the plant cell through the action of the products of the vir genes that act on the ends of the T-DNA. Once in the plant cell, the DNA inserts into the plant genome, and expression of the genes results in the production of phytohormones and opines. The opine catabolism genes confer the ability to use the opines as nutrients. c.Agrobacterium cells inoculated into a wound site results in transfer of the DNA into the nucleus, causing the formation of the crown gall tumor.

It represents one of the first demonstrated examples of trans-kingdom genetic transfer

How can molecular biology tools be used to improve microbial strains? •Mutagenesis •Production of recombinant proteins -Expression vectors can be used to mass-produce recombinant proteins (20% or more of total cell protein produced!). -Fusion proteins -Designer organisms: Synthetic biology What are the different manipulations we can do to bacteria or proteins to make better strains to use in biotechnology? Be able to explain recombination.

Mutagenesis How can we generate beneficial genetic alterations in naturally occurring microbes? We can rely on random mutagenesis. Site-directed mutagenesis (Toolbox 16.1) allows researchers to make specific mutations at specific known sites within a DNA molecule. In recent years, methods have been developed to precisely modify genomes directly, a process called genome editing. These methods were first developed using zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs)

CRISPR-Cas Genome Editing

Recently, another genome-editing tool has emerged that does not rely on DNA-binding protein motifs. This tool is based on a naturally occurring system that has evolved to protect bacterial cells from infecting bacteriophage. The CRISPR-Cas system includes both clustered regularly interspaced short palindromic repeats (CRISPR) and a CRISPR-associated (Cas) enzyme, together abbreviated as CRISPR-Cas. The repeat sequences were first observed during cloning of E. coli genes in 1987, but the function was not apparent at the time. Repeat sequences were subsequently observed in many other genomes as genome sequences became available. Although there was much speculation, there was no clue as to their true function. It was not until 2007 that Rodolphe Barrangou, Sylvain Moineau, and colleagues at Université Laval, in collaboration with the Danisco company, demonstrated that CRISPR provides resistance to bacteriophage in Streptococcus thermophilus and therefore is a type of adaptive immunity.

Biofuels: Biodiesel and Algae Where is the future?

Renewable forms of diesel, "biodiesel," are currently made from plant oils. Soybean, palm, and rapeseed (canola) oils lead the market in different parts of the world. However, the use of plant-derived oils for biodiesel is probably unsustainable using current practices. None of these crops can supply enough oil to significantly reduce global diesel consumption without displacing cropland currently devoted to food crops or inducing farmers to put more virgin land under cultivation.

Green Biotechnology •What role do microbes play in agricultural biotechnology? •Modern agriculture also benefits from biotechnology applications. -Pesticide/herbicide production -Synthetic fertilizers •Genes can also be inserted into plants to make them better. •These methods do have possible human health and environmental repercussions, however. know examples and the controversy with GMO

The importance of biotechnology in industrial and pharmaceutical settings. Biotechnology is also critically important to modern agriculture. Green biotech refers to the use of biotechnology in this setting. Today, modern agriculture involves the use of large amounts of insecticides, herbicides, and synthetic fertilizers. In addition to being economically costly, this type of intensive agriculture poses human health concerns and environmental repercussions

•Industrial enzymes -Many commercial products require enzymes, or biocatalysts, for production. •Enzymes for production of high fructose corn syrup •Enzymes in laundry detergents -Considerable effort is spent on either improving production amounts of these enzymes or improving the enzymes themselves. FIGURE 16.27 Common consumer products based on industrially produced enzymes HFCS is found in soft drinks and many other foods. A number of different enzymes are used in household detergents to enhance the stain removal properties. What are some of the products you see in White Biotech?

The production of many commercial products, including foods, detergents, textiles, and paper, requires the use of enzymes. Many of these enzymes, often referred to as biocatalysts, originate from microbes. In the production process, these microbial enzymes have several advantages over organic chemistry alternatives. The biocatalysts quite frequently exhibit high specificity and high efficiency. Additionally, they are biodegradable. reflect on the fact that it contains a sugary mix of glucose and fructose called high fructose corn syrup (HFCS). This HFCS is produced by the action of amylase enzymes on cornstarch to make corn syrup, followed by treatment with glucose isomerase to adjust the ratio of glucose to fructose. The amylase enzymes are purified from cultures of Bacillus sp., whereas glucose isomerase is purified from cultures of Streptomyces sp. The HFCS is much cheaper than sugar, and about 10 megatonnes of high fructose corn syrup is produced each year in the United States alone When you do your laundry, note that the detergent likely includes a potent mix of lipases, amylases, proteases, glycosidases, and oxidases that work together to remove dirt and stains at lower wash temperatures. In fact, the detergent industry represents the largest single market for microbial enzymes, with other key markets being baking, beverage, and dairy. Because of these applications, the enzymes themselves are valuable products

Sources of Microbes •What microbes and genetic materials are available for biotechnology applications? -Scientists build and maintain culture collections. •Preserved specimens that other scientists can obtain at minimal cost •Help promote confirmation of findings through repeatable independent research -Bioprospecting = Searching for useful new microbes to cultivate and add to collections .

To facilitate the widespread availability of microbial strains, microbiologists throughout the world deposit microbes that have been isolated and characterized in publicly available archives called culture collections (Figure 16.2). These collections, containing freeze-dried, frozen, or otherwise preserved living samples of the microbial cultures, allow scientists from around the world to obtain these organisms at minimal cost. As a result, experimental findings reported in the scientific literature can be confirmed by independent research, and those findings can be extended through additional studies. Termed bioprospecting, such searches for novel organisms, biological materials, or biological processes in nature, have fueled innovations in biotechnology (Perspective 16.1).

•Human proteins as therapeutics -Often produced in bacterial or yeast systems -Type I interferons (antitumor and antiviral capacities) -Human insulin (previously harvested from pigs or cattle, enormously expensive) FIGURE 16.16 Production of recombinant insulin Insulin was the first recombinant human protein to be produced and marketed. The coding sequences for each of the two chains were cloned to make translational fusions with the lacZ gene encoding β-galactosidase. Following expression in E. coli, the polypeptides were purified and separated, the β-galactosidase tag was removed, and the resulting peptides were combined to make active insulin.

Type I interferons with antitumor and antiviral properties. One of the earliest successes of recombinant DNA technology involved the production of a human hormone Prior to the advent of recombinant DNA technology, the insulin used to treat people with type 1 diabetes was extracted from pigs or cattle. In 1978, shortly after the first demonstrations of DNA cloning using restriction enzymes in 1973 (see Section 10.3), researchers at Genentech announced the cloning and expression of human insulin in E. coli (Figure 16.16). This achievement marked a milestone in molecular biotechnology. The recombinant human insulin was safer and more plentiful than the pig- and cattle-derived alternatives. Today, various insulin analogs, versions of human insulin experimentally modified to improve their characteristics, are available for people with type 1 diabetes.

biotechnology

refers to the use of biological processes or organisms for the production of goods such as antibiotics or for services such as food preservation, wastewater treatment (see Section 17.5), and bioremediation of polluted soils (see Section 14.4).

Red Biotechnology •What role do microbes play in pharmaceutical biotechnology?

•The two major uses of microbes in the pharmaceutical industry are -Producers of secondary metabolites with therapeutic properties -Hosts for the production of recombinant human proteins