Lec # 9 Commercial products from microorganisms and food biotechnology Dr. Shah Rukh Abbas 4-2-2015

Fermentation Products from microorganism metabolites Enzymes Antibiotics Bioconversion Fuel microbial plastics

1. By-products of Microorganism vs chemical synthesis antibiotics, organic compounds, and pharmaceuticals. 2. Advantages reduce risks and complexities reduce cost and pollution less toxic and hazardous 3. environmental cleanup

Products from microorganisms The production of antibiotics through fermentation was a biotechnological breakthrough after World War II Microbial cells can now produce interferons, interleukins, factor VIII, erythropoietin, human insulin,, nerve growth factor human growth hormone 1. Metabolites Primary metabolites-produced during the organism’s growth phase, those compounds are essential to an organism’s metabolites or end products. Secondary metabolites- are not essential to cell growth or function and are characteristically produced quite late in the growth cycle.

Examples of primary and secondary metabolites produced by microbes Primary metabolites Secondary metabolites Amino acids Antibiotics Vitamins Pigments Nucleotides Toxins Polysaccharides Alkaloids Ethanol Many active pharmacological compounds (e.g cyclosporin, dopastin) Acetone Butanol Lactic acid

2. Enzymes Examples of microbial enzymes and their uses Enzymes Uses Lipase Enhances flavour in cheese making Lactase Breaks down lactose to glucose and galactose: lactose free milk products Protenase Digests proteins α-amylase Production of high fructose corn syrup Tissue plasminogen activators (TPA) Dissolves blood clots

3. Antibiotics (i) Characteristics -small metabolites with antimicrobial activity -produced by Gram-positive, Gram-negative and fungi (ii) Action in different ways -disrupting the plasma membrane of microbial cells -inhibiting cell wall synthesis, -inhibiting synthesis of important metabolites such as proteins, nucleic acid, and folic acid. (iii) develop new antibiotics with recombinant DNA biotechnology - combining antibiotic biosynthetic pathways and the targeted genetic modification of pathways. - feeding unusual substrates to microorganism that contains a pathway from another organisms

4. Bioconversions (i) Definition: - Microorganisms modify a given compound to a structurally related compound. - Bioconversion are useful when a multistep chemical synthesis is more expensive or inefficient or is impossible to achieve e.g recovering products from a large animal source. Various steroid hormones such as Cortisone, used to treat pain and inflamation, and Progesterone, used to prevent miscariages, can be synthesised commercially by microorganisms. The process involves providing an appropriate microorganism with a commonly available sterol compound that it can convert to medically valuable hormone.

INFORM. vol. 2. no. 3 (March 1991)

‘Biochemicals from bacterial factories’, Materials. www. research ‘Biochemicals from bacterial factories’, Materials . www.research.bayer.com/renewables

(ii). The use of immobilized cells (immobilized enzymes) - technology has been developed for the immobilization of enzymes onto water-insoluble supports - minimized the cost of production by permitting repeated use of the enzymes - substantially increased the stability of the enzymes The use of whole cell systems obviates enzyme isolation and immobilization

5. Fuels Methane-a cleaner and renewable alternatives, a natural gas produced by anaerobic bacteria in swamps and landfills. (ii) Hydrogen-combustion produces only energy and water. Two hydrogen ion----------> hydrogen gas Hydrogenase in bacterial, such as Clostridium and eukaryotic algae Chlorella. Bacterial enzyme hydrogenase readily produce hydrogen gas from two hydrogen ions.

Atmospheric pollution or water pollution Advantages of H2 Much lighter than air Rapid diffusivity (3.8 times faster than natural gas) Non-flammable concentration (quickly) Non-toxic There are H2 fuel cell cars. Concerns: Its extremely combustible; there are concern for its storage High cost of its extraction Atmospheric pollution or water pollution Fuel CO2 released per 1000kJ energy H2 CH4 1.2 Petroleum 1.6 Coal 1.9 Ethanol Biomass 2.2

5. Plastics (i) a global industry providing more than 300 billion pounds per year of materials valued at over $150 billion. - extraction and processing of fossil carbon and increases of greenhouse gases accumulation (ii) Biodegradable microbial bioplastics- PHA (poly hydroxyalkanoate) - a group of microorganisms including Acinetobactor,Clostridium, Microcystis, Pseudomonas, Rhizobium, Spirulina, Streptomyces produces PHA for storage of energy. - the desired polymers are produced by supplying microbes defined nutrients in correct ratios.

- R can be hydrogen or hydrocarbon chains of up to around C13 in length, and x can range from 1 to 3 or more. Varying x and R effect hydrophobicity, Tg, Tm, and level of crystallinity. When R is a methyl group and x=1, the polymer is poly-3- hydroxybutyric acid (PHB), the base homopolymer in the PHA family. When R is a methyl group and x=0, the polymer is polylactic acid (PLA)

Food Biotechnology Food biotechnology is the application of technology to modify genes of animals, plants, and microorganisms to create new species which have desired production, marketing, or nutrition related properties Food biotechnology is and will continue to be an important area in science as the world’s human population continues to increase and the world’s agricultural lands continue to decrease.

Why genetically modify food?

Extended Shelf Life Milk The first steps in genetic modification were for food producers to ensure larger profits by keeping food fresher, longer. This allowed for further travel to and longer availability at markets, etc… Extended Shelf Life Milk

Example: Long Shelf Tomatoes These genetically modified tomatoes promise less waste and higher profits. Typically, tomatoes produce a protein that softens them after they have been picked. Scientists can now introduce a gene into a tomato plant that blocks synthesis of the softening protein. Without this protein, the genetically altered tomato softens more slowly than a regular tomato, enabling farmers to harvest it at its most flavorful and nutritious vine-ripe stage.

2) Efficient Food Processing By genetically modifying food producing organisms, the wait time and quantity of certain food processing necessities are optimized. Again this is a money saver.

Example: Rennin Production The protein rennin is used to coagulate milk in the production of cheese. Rennin has traditionally been made in the stomachs of calves which is a costly process. Now scientists can insert a copy of the rennin gene into bacteria and then use bacterial cultures to mass produce rennin. This saves time, money, space and animals. Also working on producing lactose-free or low lactose milk (have done this in mice, but not in cows yet)… also possibly looking at caffeine free coffee beans. Rennin in the top test tube… not there in the bottom one.

3) Better Nutrient Composition Some plants, during processing, lose some of the vital nutrients they once possessed. Others are grown in nutrient poor areas. Both these problems can be solved by introducing genes into plants to increase the amount or potency of nutrients. “Biofortification”

Example: Golden Rice Scientists have engineered "golden rice", which has received genes from a daffodil and a bacterium that enable it to make beta-carotene. This offers some promise in helping to correct a worldwide Vitamin A deficiency. β-Carotene is a strongly-colored red-orange pigment abundant in plants and fruits.

4) Efficient Drug Delivery Inserting genes into plants/animals to produce essential medicine or vaccines. “Biopharming” use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a genetically modified organism (GMO).

Many Unpatented Examples A cow with the genetic equipment to make a vaccine in its milk could provide both nourishment and immunization to a whole village of people now left unprotected because they lack food and medical help (in progress). Bananas and potatoes make hepatitis vaccines (done). Making AIDS drugs from tobacco leaves (done). Harvest vaccines by genetically altering hydroponically grown tomato plants to secrete protein through their root systems into the water (done).

Potential Problems??? With every technology there is an associated risk involved. The following are some examples of potential problems associated with food biotechnology.

1) Creating “Superbugs” Since many of the “vectors” used to introduce genes to plants and animals are bacteria and viruses, it is realistic to think there is a chance they could undergo a mutation and prove harmful or become recombinant like the H1N1 virus and thus more virulent. However, the bacteria and viruses used in these procedures are usually non-pathogenic. Viruses Bacteria

2) Negative Affects on Human Health Most of these food products undergo testing to see if any adverse health effects occur. However, allergies were not thought of in one case where a gene from a brazil nut was transferred to soy bean plants! Thankfully a food product was not pursued as someone came to their senses! Important to note that not all genes from a potential allergenic food will cause an allergy.

3) Ethics How many human genes would an organism have to have before you consider it human??? The following food types have a variety with human genes added: rice (immune system genes that prevent diarrhea), baby food (lactoferrin and lysozyme) and any farm animal (Human growth hormone). Lysozyme destroys bacterial cell walls and lactoferrin is an iron binding protein that also fights off viruses and bacteria.

Assignment 3 What is the best reason for genetically modifying food? What is a potential problem that you feel should be studied further before advancing farther into food biotechnology? What food item would you genetically engineer? Why? For what purpose? What genes would you transfer?