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.
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.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation of Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation of Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
authors.forename: Gazi Sakir
authors.initials: GS
authors.lastname: Hossain
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation of Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA.
authors.forename: Hyun-Dong
authors.initials: HD
authors.lastname: Shin
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. gcdu@jiangnan.edu.cn.|Synergetic Innovation of Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China. gcdu@jiangnan.edu.cn.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. longliu@jiangnan.edu.cn.|Synergetic Innovation of Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China. longliu@jiangnan.edu.cn.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
mesh_terms: D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D010667:Phenylpyruvic Acids / Q000378:metabolism*; D015202:Protein Engineering; D011513:Proteus mirabilis / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D050505:Mutant Proteins; D010667:Phenylpyruvic Acids; D011994:Recombinant Proteins; D010649:Phenylalanine; D000581:Amidohydrolases; C031606:phenylpyruvic acid
keywords: Error-prone PCR; Fed-batch biotransformation; L-Amino acid deaminase; Phenylpyruvic acid; Site-saturation mutagenesis; Whole-cell biocatalyst