ENGINEERING A MICROBIAL CHASSIS FOR NON-STANDARD AMINO ACID BIOSYNTHESIS AND INCORPORATCION
Date
2023-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Genetic code reprogramming augments protein chemistry with non-standard amino
acids (nsAAs), enabling the expansion of protein applications as therapeutics, sensors,
and biocatalytic tools. Despite this potential of nsAA-containing proteins, major
barriers arise for industrial applications of nsAAs: expensive nsAA synthesis and low
cellular uptake. One approach to mitigate both of these challenges is to program cells
to generate nsAAs intracellularly from inexpensive precursors. A recently
characterized class of enzymes, L-threonine transaldolase (TTAs), convert diverse
aldehydes to β-hydroxy non-standard amino acids (β-OH-nsAAs), which are nsAAs
that contain a hydroxyl substituent at the β-carbon. This study sought to improve β OH-nsAA yield in vivo by engineering a microbial platform in Escherichia coli to
stabilize substrate molecules, as well as coupling the TTA with an alcohol
dehydrogenase (ADH) and a phosphite dehydrogenase (PTDH), to shift reaction
equilibrium. In parallel, this work also assessed the predictive ability of a PyRosetta
computational model to design enzymes with high affinity for a β-OH-nsAA substrate.