We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of l-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst.
- Authors
Hou, Ying; Hossain, Gazi; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Liu, Long
- Abstract
In our previous study, we produced phenylpyruvic acid (PPA) in one step from l-phenylalanine by using an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase ( l-AAD) from Proteus mirabilis KCTC2566. However, the PPA titer was low due to the degradation of PPA and low substrate specificity of l-AAD. In this study, metabolic engineering of the l-phenylalanine degradation pathway in E. coli and protein engineering of l-AAD from P. mirabilis were performed to improve the PPA titer. First, three aminotransferase genes were knocked out to block PPA degradation, which increased the PPA titer from 3.3 ± 0.2 to 3.9 ± 0.1 g/L and the substrate conversion ratio to 97.5 %. Next, l-AAD was engineered via error-prone polymerase chain reaction, followed by site-saturation mutation to improve its catalytic performance. The triple mutant D165K/F263M/L336M produced the highest PPA titer of 10.0 ± 0.4 g/L, with a substrate conversion ratio of 100 %, which was 3.0 times that of wild-type l-AAD. Comparative kinetics analysis showed that compared with wild-type l-AAD, the triple mutant had higher substrate-binding affinity and catalytic efficiency. Finally, an optimal fed-batch biotransformation process was developed to achieve a maximal PPA titer of 21 ± 1.8 g/L within 8 h. This study developed a robust whole-cell E. coli biocatalyst for PPA production by integrating metabolic and protein engineering, strategies that may be useful for the construction of other biotransformation biocatalysts.
- Subjects
AMINO acids; BIOCATALYSIS; MUTAGENESIS; ESCHERICHIA coli; POLYMERASE chain reaction
- Publication
Applied Microbiology & Biotechnology, 2016, Vol 100, Issue 5, p2183
- ISSN
0175-7598
- Publication type
Academic Journal
- DOI
10.1007/s00253-015-7048-5