Lecture Summaries

1 Introduction and Overview Instructors and students will introduce themselves and discuss the course objectives and syllabus. We will also introduce the field of microbial biotechnology and genetic engineering. We will describe public paper databases like PubMed and talk about the general structure of scientific publications.
2 Wine and Beer: Ethanol The best example of using microorganisms to make useful products is in the production of alcoholic beverages like wine and beer. Brewing and winemaking have been practiced all over the world for centuries. With the advent of molecular biology and modern genetic techniques, we can optimize alcohol and flavor compound biosynthesis in the organisms that are typically used for alcoholic beverage production. The first paper details optimization of ethanol production in the brewer's yeast, Saccharomyces cerevisiae. The second paper discusses engineering of S. cerevisiae for low-alcohol wine production.
3 Food Additives The most commonly used food additive that is produced by microorganisms is the well-known monosodium glutamate (MSG). The annual worldwide demand is estimated at 2.5 Mt (megatonnes). For the production of MSG it is essential to modify organisms to reach higher productivity as well broadening their substrate spectrum, because microorganisms usually do not naturally possess a high enough productivity for a market application and often cannot grow on the inexpensive substrate media used in industrial production. The two papers show examples of how these goals can be achieved. Paper 1 highlights how it is possible to produce glutamate from starch, which previously had not been a usable carbon source for the organism. Paper 2 shows how the altered genetic regulation of a key enzyme for production can change product yield.
4 Amino Acids Amino acids have a huge market; they are especially used as feed additives, with lysine, threonine and methionine being the most important. Lysine alone has an estimated annual demand of 1.5 Mt (megatonnes). For many years lysine production strains were solely derived from random mutagenesis, but today modern manipulation techniques allow for rational strain design. Paper 1 presents how genome sequencing and comparisons led to a minimally mutated strain that is able to efficiently produce lysine. In paper 2, the authors use a computational model to develop a rationally designed production strain. The strain is the most efficient lysine producer currently available.
5 Enzymes Microbial enzymes (e.g. lipases and proteases, which break down lipids and proteins, respectively) are very useful in biotechnology, including for formulation of household products, such as laundry and other detergents. The first paper investigates the in vitro evolution of a thermostable lipase from the bacterium Bacillus subtilis. An increase in the temperature optimum of a lipase enzyme can broaden its range of applications (e.g. in detergent formulations that are effective at higher temperatures). The second paper describes a metagenomic approach (examination of all genomes in a community of microorganisms) for the discovery of a novel alkaline protease enzyme. This paper illustrates one of the methods used to discover biotechnologically important enzymes.
6 Specialty Chemicals The production of specialty chemicals by microorganisms is an exciting alternative to chemical synthesis, because of the possibility of building highly complex molecules without producing toxic wastes. Succinate (an intermediate of the TCA cycle) can be produced using bacteria and it can be used as replacement substrate (replacing the two chemicals acetylene and the toxic formaldehyde) in the synthesis of chemicals like butanediol and tetrahydrofuran, which are both used as solvents in industrial applications. Because of the huge existing market for these solvents, succinate also has a great market potential if used as alternative building block for these solvents. For this reason the U.S. Department of Energy identified biotechnologically derived succinate as one of the top 12 value-added chemicals. The first paper demonstrates how succinate can be produced efficiently using oxygen deprivation (paper 1), which generates metabolites of the TCA cycle. The second paper shows that it is also possible to produce succinate under aerobic conditions.
7 Field Trip: Microbia Field trip to a biotechnology company. Instead of regular class this week, we will visit Microbia in Cambridge, MA. Microbia is a company that utilizes recombinant bacteria to produce biopolymers (e.g. poly-hydroxyalkanoates), biofuel molecules, and other chemicals from carbon sources like refined sugars. We will hear an overview about the company and its mission and then take a tour of the facilities to see the production processes up close.
8 Biofuels I A global concern is the rapid use of petroleum fuels in the face of potentially dwindling supplies. Many alternative methods are being examined to produce fuels that will reduce our dependence on fossil fuels, and many novel bio-based efforts are being touted as renewable alternative fuels. Initially, ethanol production was considered a viable alternative. Given the poor energy density of ethanol compared to other biofuels (longer carbon chain compounds like butanol and octanol store more chemical energy per mole, compared to ethanol) and the "food v. fuel" controversy (i.e. using corn for fuel rather than for human consumption), microbial-based higher chain alcohol (e.g. butanol) production is more appealing as a source of renewable biofuels. The first paper illustrates the methods of production of branched-chain alcohol biofuels and the metabolic engineering needed to produce these compounds. The second paper describes an approach that reverses the fatty acid beta-oxidation cycle and the use of intermediates from this reversed cycle for biofuel and chemical biosynthesis.
9 Biofuels II Biofuels can also be derived from non-alcoholic substrates. One promising example is the use of triacylglycerols (TAGs) that are naturally produced in high amounts by some microorganisms. The TAGs represent a way of storing carbon under nitrogen limitation. TAGs can be extracted and turned into fatty acid methyl esters (FAMEs), which are a form of biodiesel that can directly be used as gasoline. Paper 1 describes an organism that is capable of producing high amounts of TAGs naturally, while paper 2 shows how to turn an organism that does not naturally produce biofuel into a production platform for various fuel types.
10 Biopolymers I Attractive alternatives to petroleum-based plastics are microbially-produced biopolymers. The most well-studied microbial biopolymers are polyhydroxyalkanoates (PHAs), which are carbon and energy storage molecules for many species of bacteria. The paradigm organism for polyhydroxyalkanoate production is the betaproteobacterium Ralstonia eutropha (also known as Cupriavidus necator). R. eutropha can synthesize up to 80% of its cell dry weight as PHA, specifically the homopolymer polyhydroxybutyrate (PHB). The first paper discusses the physiology of PHB production in wild-type R. eutropha cells. Understanding the conditions that induce PHA biosynthesis is the first and most important step in developing a PHA bioplastic production process. R. eutropha can be engineered to produce PHA copolymers that have favorable properties and greater range of application than PHB. The second paper describes a process of metabolic engineering to produce unique PHA polymers.
11 Biopolymers II There are many useful and valuable biopolymers in addition to PHAs. In recent years, production pathways for lactate-based polymers (e.g. polylactate; PLA) have been engineered in a variety of bacteria. Typically, PLA is synthesized chemically using a toxic tin catalyst. Microbial PLA production would offer the opportunity to produce renewable, bio-based PLA and lactate-based polymers. The first paper reports seminal studies of lactide polymerization in a microbial system. The polymer cyanophycin is a non-ribosomally produced polypeptide that has a myriad of applications, e.g. as a polyacrylate substitute, a precursor of dipeptides and conversion to other polypeptides for nutritional and pharmaceutical applications. The second paper discusses optimization of cyanophycin production in the biotechnologically important bacterium Ralstonia eutropha. R. eutropha is adept at carbon storage, so high cell density cultures and high cyanophycin-content cells are attainable through metabolic engineering.
12 Biopharmaceuticals and Biosensors Biopharmaceuticals such as antibiotics, vaccines and diagnostic tools like biosensors represent more exciting products that can be produced by microbes. Microorganisms are ideally suited for the production of such highly complex molecules, mostly because they are able to produce secondary metabolites that are already biologically active. This ability can be exploited to produce a broad variety of structurally similar molecules. The first paper used the production of bioplastic linked to a specific antigen protein to effectively deliver a tuberculosis protein vaccine. This simplifies the delivery of the tuberculosis protein and the immunological response is enhanced. Biosensors can provide an inexpensive solution to determine different substances in the body without the need of a surgery. The second paper shows the potential of using a bacterial strain as in vivo biosensor for the detection and read out of the antibiotic tetracycline in the rat intestine.
13 Oral Presentation A 15 minute summary on a paper in the biotechnology literature, picked by students.