Lecture 11 - Biotechnology PDF

BS2002 Introductory Microbiology Week 12: Biotechnology Dr Sze Chun Chau School of Biological Sciences [email protected] Biotechnology Biotechnology = technology based on biology /biological entity ↑ harnesses cellular and biomolecular processes to develop t...

BS2002 Introductory Microbiology Week 12: Biotechnology Dr Sze Chun Chau School of Biological Sciences [email protected] Biotechnology Biotechnology = technology based on biology /biological entity ↑ harnesses cellular and biomolecular processes to develop technologies and products benefit larger humankind ~ application at industrial scale is implied research may be done to develop product but needs to scale up for application to reach the phase of "biotechnology" Biology/ biological entity may be plants, animals, fungi, bacteria, archaea = microorganisms Technology that harnesses… ~ main focus Microoorganisms’ biology DIRECTLY animals not microorganisms , plants & - Other living things’ biology INDIRECTLY by using recombinant DNA technologies in microorganisms used as tools - simple Synthetic biology using microorganisms as the “material” for “synthesis” & starting material Microbes in Food production (via Fermentation): traditional biotechnology Recap from Food Microbiology lecture Site of microbial activities: on/in food product isi site of microbial activity Microbes in Waste/Wastewater treatment: traditional biotechnology Composting for fertilizers food for unwanted Processing domestic wastewater - organisms spoiling as g alque pos e. toxin. , - high organic content in wastewater e ecosystem >> bacteria consumes organic content in wastewater putS released >> water & carbon dioxide after consumpt of - organic content - lower organic content wastewater can be released into the environment Site of microbial activities: in waste/wastewater (held in process tanks) Microbes in Bioactive Compound Industrial Production: traditional biotechnology Bioactive compounds known to be produced by some microorganisms: Antibiotics Amino acids Organic acids Vitamins emulsifiers ~ Biosurfactants Biopolymer e.g. polyesters like polyhydroxyalkanoates (PHA) Site of microbial activities: industrial-scale fermenters with specific culture media, growth conditions What is the common pattern for traditional microbial biotechnology here? Discovered that certain microorganisms have metabolic capabilities that can - Ferment food - Reduce organic content in waste - Generate useful compounds Grow them in large scale in the actual sites (food/waste) under controlled conditions to promote the growth of the microbe-of- interest OR in prescribed culture medium in controlled fermenters for poth of bioactive empd  fermenter Growing Microbes… Stirrer stirred fermenters ~ nutrients in ~ waste out liquid enteringa flowing continuous feed - a same rate out Leam betw nutrients & waste) continual addition of a critical nutrient so that microbes will not have excess substrate controlled growth available at any given time rate prevents production and accumulation of undesirable metabolic waste products a contain wa crobes Growing microbes at industrial scale are often >> more energy efficiency >> cheaper >> generate higher yields >> has less toxic wastes …than via chemical methods Traditional Biotechnology rely on the natural metabolic capabilities of microorganisms but… genetic manipulation of microorganisms through molecular techniques >> enhances /expands what microorganisms can do in their original functions >> allow the “cheap microbial factories” to be used to produce compounds that are NOT naturally from the original microorganism additi of foreign genes Enhance/Expand what microbes can do in their original functions e.g. better yields of penicillin & poed naturally Genetically modify genes so that e.g. - enzyme/active biomolecule structure is changed: more efficient catalysis of biochemical pathway OR greater stability - regulatory components are changed: gene expression becomes greatly enhanced OR more easily activated OR can operate under cheaper conditions, increasing the overall yields of bioactive compounds Methods may include: Random mutagenesis by UV, transposon, chemicals and then SCREEN for functionally desirable mutants Site-directed mutagenesis (the mutation sites are often determined based on research studies of genes’ functions) Allow the “cheap microbial factories” to be used to produce compounds that are NOT naturally from the original microorganism From other strains of microbes From plants From animals Recombinant DNA techniques to introduce the heterologous genes into the host microorganisms (which may have very established and economical culture conditions) that not from itself ~ carries are genes >> Transgenic microorganism Note: If the host organism receiving the heterologous genes are animals or plants, then the result is a transgenic animal or plant & biotech but not microbial biotech Combinatorial Biosynthesis/ Bioprocess – Pathway Engineering biochemical pathways transfer and expression of genes between different organisms can give rise to novel metabolic processes and products goal is to change an industrially important biochemical pathway for synthesis or biodegradation (e.g. pollutants) E.g. Pseudomonas cepacia can degrade phenols but cannot degrade methyl- and chlorophenols, which are toxic industrial pollutants 2 genes from Pseudomonas putida that express enzymes that can remove methyl- and chloro- groups from the phenol ring Ithose genes ~ >> recombinant DNA technique to incorporate in P. cepacia’s biodegradation pathway >> Pathway Engineering Pathway Engineering: The Indigo Synthesis Example -natural due ~ foreign but Pseudomonas has Napthalene oxygenase gene >> clone onto vector DNA (plasmid) Transform into E.coli indole ~ converts tryptophan to E. coli has Tryptophanase activity Process below will take place in E. coli & can now poce indigo Environmental Gene Mining What if we don’t know what genes can be used specifically to engineer a particular pathway? “Mine” for functional genes from the environmental samples (without cultivation of microbes from these samples) not all microbes are culturable as their culture coud" diff env samples instead of growingI microorganism , are unknown ~ cloning we extract their DNA automate can be used for screening  ~ heterologous gere from animal origin Animal gene >> expressed in transgenic bacteria e.g. cloning and expression of bovine somatotropin & others paced in a similar way growthhor a ~ recombinant but expressed in E. coli not everycananimal be gene Coli expressed in E. as somepost-translational modificate cannot be reproduced in bacteria Bacteria in producing transgenic plants e.g. Agrobacterium tumefaciens with herbicide resistance gene & bacteria A. tumifaciens - plant pathogen Ti plasmid – tumour inducing plasmid of A.tumifaciens Heterologous gene e.g. herbicide resistance gene carried on Ti plasmid can be transferred directly into plant cells >> insert into chromosome of plants tumour induct genes are - removed infects I plant w herbicide resistance gere herbicide resistant multiplied gene introduced into plant cloned into restor can replicateanisms -  Engineered Bacteria and Therapeutic Delivery 3 outcomes derived engineered Sacteria for from we wantI pathogens therapeutic purposes = property of invading (a) Pathogens that grow in the low-oxygen host environment of a tumor can be attenuated and genetically modified to release drugs directly inside tumors (b) Pathogens that elicit a strong immune response can be attenuated and engineered to release antigens normally found on tumor cells. This antigen release stimulates antibody targetin9 turn ~ immure production and immune system destruction of cells tumors als commensalism betw qut probiotic cellsa ~ (c) Probiotic strains normally found in the healthy gut can be engineered to release therapeutic molecules to treat various diseases as well as help train the immune system to natural-ls tolerate beneficial microbes. e exploitingbast & host to befor engineer dog delivery Microbial Energy Conversion world relies on petroleum to fuel almost all transportation, coal and natural gas generate power and heat finite resources contribute to greenhouse gases and global climate change microbial transformation provides renewable energy sources ~ microbes as source of fuel biofuels produced by microbial transformations ethanol and biodiesels for cars and machines I poled by microbes methane microbial fuel cells bioreactors that use microbes to change stored chemical energy in organic matter to electricity Microbial Biofuels ~ pot Ethanol from Caldicellulosiruptor bescii Ethanol added to gasoline is generated by corn traditionally A waste of corn, a food source (shortage of food supply) not sustainable practice Use crop residues with cellulose and hemicellulose instead genetically engineered Caldicellulosiruptor bescii hand to degrade a gram-positive anaerobic and thermophilic bacterium naturally produces cellulase and hemicellulase enzymes that can convert cellulose and hemicellulose to glucose precursor to ethanol path - naturally produce not much ethanol from glucose altered the terminal steps of the C. bescii glycolytic pathway >> replace genes encoding lactate dehydrogenase and other acidic fermentation products with a bifunctional acetaldehyde/alcohol dehydrogenase from another thermophile, Clostridium thermocellum shifted 70% of the C. bescii fermentation products to ethanol Microbial Biofuels Triacylglycerides (TAG) from cyanobacteria and microalgae precursor of biodiesels genetically engineered to enhance TAG production Anaerobic digester fed with manure~ from anaerobic digest" controlled production of methane (CH4) in archaea no net increase in CO2 production 50–100% efficiency of production can be applied to wastewater treatment to generate energy Methanogenic consortia microbes produce methane in oil fields  Microbial Fuel Cell battery has cathode a anode cathode/anode movement of ions to generates electricity in battery captures electrons to generate electricity microbes oxidize organic matter anaerobically continuously fed a rich diet of organic substrates generated by basteria protons diffuse across the ~ membrane and are deposited on anodes electrons flow to cathode generati electricity but there are challenges as waste movement poled have impact on ion  Microbial Biosensor Living microbial cells, enzymes or organelles act as “receptor” Receptor detects specific substances Detection generates biological reaction products that gets converted into electrical currents >> signal conversion in device  Synthetic Biology and Genome Editing Stitch together bits and pieces of DNA (“biobricks”) from various sources to create artificial genes, operons, or entire genomes building from scratch Can edit these new genetic constructs at will to change the properties of existing organisms Can even fabricate an entirely synthetic organism material to be as simple possible is > microorganisms perfect Harting - as Sample question Which of the following are “Pathway Engineering”? A. Indigo synthesis by E.coli B. Methane production by anaerobic digester C. Biodegredation of chlorophenol by Pseudomonas cepacia D. Yeast fermentation of ethanol E. Ethanol production by Caldicellulosiruptor bescii Ans: A, C, E