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ADDICTING BACTERIA TO CAFFEINE FOR BIOTECHNOLOGICAL APPLICATIONS
Mani Subramanian, Ph.D
Center for Biotechnology & Bioprocessing
Professor, Chemical & Biochemical Engineering
University of Iowa
Iowa City, Iowa
The larger research philosophy in Subramanian research laboratory has been “biofeedstock to chemicals via enzymes and microorganisms”. The overarching technology developed in Subramanian laboratory that drives this philosophy is “spray-dried microbial cells for biocatalysis applications”. Spray drying kills cells; but enables efficient catalysis via easy passage of small molecules in and out, while retaining enzymes inside. Enzymes do not leach from the spray-dried cells. Spray dried cells are also stable at room temperature, easier to handle, scalable, recyclable, and robust with respect to biocatalysis. Applications of spray dried cells towards production of chemicals that have been developed or in development include:
- Production of pyruvate from lactate (technology licensed to a large company)
- Production of D-lactate and other R-hydroxyacids of pharmaceutical relevance
- Production of bioxylitol from corn stover hydrolysate (at pilot scale)
- Deriving value from caffeine or coffee/tea waste (in development)
On the fundamental side, Subramanian laboratory has focused on microbial degradation of purine alkaloids (1-6). Prominent members of this class of natural products are caffeine and theobromine. Two different pathways of microbial degradation of caffeine and theophylline have been elucidated. Novel enzymes (and genes) are involved in the catabolism of caffeine. The entire genetic map of N-demethylation pathway of caffeine has been discovered in Pseudomonas putida CBB5 (1-4). The genetic map of an alternate pathway for caffeine degradation via C-8 oxidation has also been established (5, 6). The genes and enzymes involved in caffeine degradation are of value in terms of producing fine chemicals, fuel and animal feed from caffeine/theophylline &/or coffee/tea waste. Another interesting application of the enzyme, caffeine dehydrogenase (6) that catalyzes the 8-oxidation of caffeine includes a “diagnostic test for detection of caffeine in pharmaceutical formulations, beverages and nursing mother’s milk”.
- E.M. Quandt, M.J. Hammerling, R.M. Summers, P.B Otoupal, B. Slater, R.N. Alnahhas, A. Dasgupta, J.L. Bachman, M.V. Subramanian and J.E. Barrick (2013). Decaffeination and measurement of caffeine content by addicted E. coli with refactored N-demethylation operon from Pseudomonas putida CBB5. Manuscript accepted for publication in ACS Synthetic Biology.
- R. M. Summers, T.M. Louie, C.L. Yu, L. Gakhar, K. C. Louie, and M. Subramanian (2012). Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. J. Bacteriol. 194: 2041-2049.
- R. Summers, T.M. Louie, C.L. Yu and M. Subramanian (2011). Characterization of a broad-specific non-heme iron N-demethylase from P. putida CBB5 capable of utilizing purine alkaloids as sole source of carbon and nitrogen. Microbiology 157: 583-592.
- C.L. Yu, M. Louie, R. Summers, S. Gopishetty and M.V. Subramanian (2009). Two distinct pathways for metabolism of theophylline and caffeine are co-expressed in Pseudomonas putida CBB5. J. Bacteriol. 191: 4624-32.
- S.K. Mohanty, C.L. Yu, S. Das, T.M. Louie, L. Gakhar and M. Subramanian (2012). Delineation of caffeine C-8-oxidation pathway in Pseudomonas sp. CBB1: Characterization of a new trimethyluric acid monooxygenase and genes involved in trimethyluric acid metabolism. J. Bacteriol. 194: 3872-3882.
- C.L. Yu, Y. Kale, S. Gopishetty, M. Louie and M.V. Subramanian (2008). A novel caffeine dehydrogenase in Pseudomonas sp. Strain CBB1 oxidizes caffeine to trimethyluric acid. J. Bacteriol 190: 772-776.
PROTEIN ENGINEERING IN BIOTECHNOLOGY
RECONSTRUCTING ANCESTRAL ENZYMES TO CREATE NEW CATALYTIC ACTIVITIES
Romas Kazlauskas, Ph.D.
Department of Biochemistry, Molecular Biology & Biophysics
University of Minnesota
St. Paul, Minnesota
One major branch of biotechnology is industrial enzymes with ~ $5 billion in sales, which is similar to ad sales on Facebook. The three main applications are detergents, food and drink, and other, which include the synthesis of drugs, chemical intermediates & biofuels. Using enzymes for these syntheses creates more efficient, less polluting syntheses since enzymes are more selective than chemical reagents.
To create industrial enzymes, protein engineers improve nature's enzymes to be more stable, to accept unnatural substrates and even to change the type of reaction that they catalyze. In one case, researchers replaced 15% of the amino acids in the enzyme to make it millions of times better than the starting enzyme. This change is the molecular equivalent to engineering a mouse into a human, since mouse proteins typically differ from human proteins by 15%.
A new evolution-based approach to engineering enzymes is to start with ancestral enzymes. Evolutionary biologists hypothesize that ancestral enzymes catalyzed several related reactions (they were generalists), then evolution created specialists for each reaction. We hypothesize that generalists are a better starting point for engineering industrial enzymes. I will describe recreating ancestral enzymes and testing their ability to catalyze unnatural reactions.
Probiotics for Animals - Direct Fed Microbials and The Avian Gut Microbiota
Gregory R. Siragusa, Ph.D.
Senior Principal Scientist
DuPont Nutrition and Health
Direct fed microbials (DFM - microorganisms which when fed exert beneficial effects on poultry performance, health, and immunity) routinely demonstrate efficacy in enhanced feed conversion and growth performance that is comparable to that obtained with subtherapeutic antibiotic usage. The mechanistic basis of the probiotic is largely unknown. Our laboratory has used a microbial ecology approach to understanding gut microbial communities, and host immune response from DFM feeding in broilers, layers and turkeys. A unique DFM strain selection and formulation process is presented which is based on an understanding of the genetic diversity and levels of Clostridium perfringens and avian pathogenic Escherichia coli. Changes in microbial diversity and profile or balance of the avian gut are associated with disease and poor performance; examples of this are the cases of clostridial dermatitis, and focal duodenal necrosis and the concomitant changes in gut microflora. Probiology is the study of probiotics and their interaction with the host. The probiotic concept is evolving and a new generation of DFMs, for different feedstuffs, climates and genetic lines of poultry is potentially on our horizon. We are currently in an era of explosive growth in this field. This new knowledge of microbial communities will lead to new generations of DFMs and probiotics and opportunities for the food and industrial microbiologist.