Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of l-amino acid deaminase improves PPA production with an Escherichia coli whole-

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• 作者：Ying Hou ; Gazi Sakir Hossain ; Jianghua Li…
• 关键词：l ; Amino acid deaminase ; Phenylpyruvic acid ; Whole ; cell biocatalyst ; Error ; prone PCR ; Site ; saturation mutagenesis ; Fed ; batch biotransformation
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2016
• 出版时间：March 2016
• 年：2016
• 卷：100
• 期：5
• 页码：2183-2191
• 全文大小：704 KB
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• 作者单位：Ying Hou (1) (2)
  Gazi Sakir Hossain (1) (2)
  Jianghua Li (1) (2)
  Hyun-dong Shin (3)
  Guocheng Du (1) (2)
  Long Liu (1) (2)
  
  1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China
  2. Synergetic Innovation of Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China
  3. School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

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.