Title

Improving 1,3-butanediol production in E. coli using a protein engineering approach

Conference Dates

July 14-18, 2019

Abstract

Traditional chemical production processes have high yields but require harsh reaction conditions and use non-renewable feedstocks derived from petroleum [1, 2]. These processes have a negative impact on the environment, which motivates the development of more sustainable processes as replacements [2]. Advances in systems metabolic engineering over the past thirty years have given rise to bioprocesses where engineered microbes make chemicals from natural feedstocks under mild reaction conditions [1]. The promise of the field has also resulted in financial resources being made available to the development and commercialization of bioprocess. According to a recent report by Ontario Genomics [3], global investment in the field is projected to be at $38.7B in 2020, a 12-fold increase from what it was at in 2013. Recently, a novel aldolase-based pathway for producing 1,3-butanediol (BDO) in E. coli was reported by Nemr et. al [4, 5]. 1,3-BDO is a commercially viable product as it is used in formulations in cosmetics products, and as a precursor for pharmaceuticals [2]. This pathway involves the conversion of pyruvate to acetaldehyde via the EutE enzyme from E. coli, followed by the conversion of acetaldehyde to 3- hydroxybutanal via the enzyme BH1352 – a Deoxyribose-phosphate aldolase (DERA) – from Bacillus halodurans and subsequently by the conversion of 3-hydroxybutanal to 1,3-BDO via the enzyme PA1127 (an aldo-keto reductase) from Pseudomonas aeruginosa [5]. We examined the crystal structure of BH1352, which revealed key residues involved in catalytic activity in the substrate binding pocket. We show that two DERA mutants F160Y and F160Y/M173I improve the production of 1,3-BDO 5-fold and 6-fold respectively in bench-scale bioreactors [6]. References: 1. Bonk, B. M., Tarasova, Y., Hicks, M. A., Tidor, B., & Prather, K. L. (2018). “Rational design of thiolase substrate specificity for metabolic engineering applications”. Biotechnology and bioengineering, 115(9), 1–16. http://doi.org/10.1002/bit.26737 2. Burk, M. J. (2010). Sustainable production of industrial chemicals from sugars. International Sugar Journal, 112(1333), 30. 3. Ontario Genomics. (2017). Ontario Synthetic Biology Report 2016. Retrieved from: http://www.ontariogenomics.ca/syntheticbiology/Ontario_Synthetic_Biology_Report_2016.pdf 4. Nemr, K., Müller, J. E. N., Joo, J. C., Gawand, P., Choudhary, R., Mendonca, B., et al. (2018). Engineering a short, aldolase-based pathway for (R)-1,3-butanediol production in Escherichia coli. Metabolic Engineering, 48, 13–24. http://doi.org/10.1016/j.ymben.2018.04.013 5. Nemr, K. (2018). Metabolic Engineering of Lyase-Based Biosynthetic Pathways for Non-natural Chemical Production (Unpublished doctoral dissertation). University of Toronto, Toronto, ON, Canada. 6. Kim, T. (2019). Biochemical and Structural Studies of Microbial Enzymes for the Biosynthesis of 1,3-Butanediol (Unpublished doctoral dissertation). University of Toronto, Toronto, ON, Canada

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