One-step biosynthesis of α-ketoisocaproate from l-leucine by an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase from Proteus vulgaris

Song, Yang; Li, Jianghua; Shin, Hyun‐Dong; Du, Guocheng; Liu, Long; Chen, Jian

doi:10.1038/srep12614

Cited by 33 publications

(39 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As an alternative, Corynebacterium glutamicum was genetically engineered to synthesize α‐KIC, yielding a titer of only 9.23 g L −1 . Recently, 69.1 g L −1 α‐KIC has been efficiently synthesized at a 50.3 % conversion rate by expressing L‐AAD derived from Proteus vulgaris in E. coli BL21, followed by an attempt to optimize protein expression levels; however, α‐KIC titer only increased to 86.55 g L −1 within 20 h . In addition, although the procedure for synthesizing α‐KMV using amino acid oxidase has been briefly outlined, the yield is very low (less than 10 g L −1 ) .…”

Section: Resultsmentioning

confidence: 99%

Production of α‐Ketoisocaproate and α‐Keto‐β‐Methylvalerate by Engineered L‐Amino Acid Deaminase

et al. 2019

View full text Add to dashboard Cite

This study aimed to develop an efficient enzymatic strategy for industrial production of α‐ketoisocaproate (α‐KIC) and α‐keto‐β‐methylvalerate (α‐KMV) from L‐leucine and L‐isoleucine, respectively. L‐amino acid deaminase from Proteus mirabilis (PmLAAD) was heterologously expressed in E. coli BL21(DE3) and modified to increase its catalytic efficiency by engineering the PmLAAD substrate‐binding cavity and entrance tunnel. Four essential residues (Q92, M440, T436, and W438) were identified from structural analysis and molecular dynamics simulations. Residue Q92 was mutated to alanine, and the volume of the binding cavity, enzyme activity, and the kcat/Km value of mutant PmLAAD Q92A increased to 994.2 Å3, 191.36 U mg−1, and 1.23 mM−1 min−1, respectively; consequently, the titer and conversion rate of α‐KIC from L‐leucine were 107.1 g L−1 and 98.1 %, respectively. For mutant PmLAADT436/W438A, the entrance tunnel, enzyme activity, and the kcat/Km value increased to 1.71 Å, 170.12 U mg−1, and 0.70 mM−1 min−1, respectively; consequently, the titer and conversion rate of α‐KMV from L‐isoleucine were 98.9 g L−1 and 99.7 %, respectively. Therefore, augmentation of the substrate‐binding cavity and entrance tunnel of PmLAAD can facilitate efficient industrial synthesis of α‐KIC and α‐KMV.

show abstract

Section: Resultsmentioning

confidence: 99%

Production of α‐Ketoisocaproate and α‐Keto‐β‐Methylvalerate by Engineered L‐Amino Acid Deaminase

et al. 2019

View full text Add to dashboard Cite

show abstract

“…After optimizing the conditions with response service methodology, the maximal α-KIC production reached 1.2 g/L [17,39]. A similar approach was used by Song et al (2015) to produce α-KIC, but in this case, an E. coli whole-cell biocatalyst, expressing an L-AAD, EC 1.4.3.2 from Proteus vulgaris, was used. The membrane-bound L-AAD from P. vulgaris is an oxidoreductase with flavin adenine dinucleotide as a coenzyme, which catalyze the deamination of the deamination of L-leucine to KIC.…”

Section: Phenylpyruvic Acidmentioning

confidence: 99%

“…The catalysis occurs via the binding of Proteus L-AADs to the electron transport chain to reduce O 2 to H 2 O. After optimizing the reaction conditions and the substrate feeding strategy, the α-KIC titer reached 69.1 g/L with a substrate conversion rate of 50.3% [40]. However, the α-KIC production was low, because of feedback reactions that limited its production.…”

Section: Phenylpyruvic Acidmentioning

confidence: 99%

Engineering of L-amino acid deaminases for the production of α-keto acids from L-amino acids

Nshimiyimana

Liu

2019

Bioengineered

Self Cite

View full text Add to dashboard Cite

α-keto acids are organic compounds that contain an acid group and a ketone group. L-amino acid deaminases are enzymes that catalyze the oxidative deamination of amino acids for the formation of their corresponding α-keto acids and ammonia. α-keto acids are synthesized industrially via chemical processes that are costly and use harsh chemicals. The use of the directed evolution technique, followed by the screening and selection of desirable variants, to evolve enzymes has proven to be an effective way to engineer enzymes with improved performance. This review presents recent studies in which the directed evolution technique was used to evolve enzymes, with an emphasis on L-amino acid deaminases for the whole-cell biocatalysts production of α-keto acids from their corresponding L-amino acids. We discuss and highlight recent cases where the engineered L-amino acid deaminases resulted in an improved production yield of phenylpyruvic acid, α-ketoisocaproate, α-ketoisovaleric acid, α-ketoglutaric acid, α-keto-γ-methylthiobutyric acid, and pyruvate. ARTICLE HISTORY

show abstract

“…l‐AADs from P. mirabilis catalyze stereospecific oxidative deamination of l ‐amino acids with formation of their respective keto acids and ammonia without production of hydrogen peroxide (Geueke and Hummel, ). In previous studies, we used FAD‐dependent l ‐AADs from Proteus species to produce a series of α‐keto acids, including phenylpyruvic acid (PPA) (Hou et al, , ), α‐ketoglutaric acid (α‐KG) (Hossain et al, ,), α‐ketoisocaproate (KIC) (Song et al, ), and keto‐γ‐methylthiobutyric acid (KMTB) (Hossain et al, ). We found that the supply of FAD is a limiting step in the catalysis driven by l ‐AAD.…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of cofactor flavin adenine dinucleotide (FAD) synthesis and regeneration in Escherichia coli for production of α‐keto acids

Hou

Hossain

et al. 2017

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Cofactor flavin adenine dinucleotide (FAD) plays a vital role in many FAD-dependent enzymatic reactions; therefore, how to efficiently accelerate FAD synthesis and regeneration is an important topic in biocatalysis and metabolic engineering. In this study, a system involving the synthesis pathway and regeneration of FAD was engineered in Escherichia coli to improve α-keto acid production-from the corresponding l-amino acids-catalyzed by FAD-dependent l-amino acid deaminase (l-AAD). First, key genes, ribH, ribC, and ribF, were overexpressed and fine-tuned for FAD synthesis. In the resulting E. coli strain PHCF7, strong overexpression of pma, ribC, and ribF and moderate overexpression of ribH yielded a 90% increase in phenylpyruvic acid (PPA) titer: 19.4 ± 1.1 g · L . Next, formate dehydrogenase (FDH) and NADH oxidase (NOX) were overexpressed to strengthen the regeneration rate of cofactors FADH /FAD using FDH for FADH /FAD regeneration and NOX for NAD /NADH regeneration. The resulting E. coli strain PHCF7-FDH-NOX yielded the highest PPA production: 31.4 ± 1.1 g · L . Finally, this whole-cell system was adapted to production of other α-keto acids including α-ketoglutaric acid, α-ketoisocaproate, and keto-γ-methylthiobutyric acid to demonstrate the broad utility of strengthening of FAD synthesis and FADH /FAD regeneration for production of α-keto acids. Notably, the strategy reported herein may be generally applicable to other flavin-dependent biocatalysis reactions and metabolic pathway optimizations. Biotechnol. Bioeng. 2017;114: 1928-1936. © 2017 Wiley Periodicals, Inc.

show abstract

One-step biosynthesis of α-ketoisocaproate from l-leucine by an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase from Proteus vulgaris

Cited by 33 publications

References 32 publications

Production of α‐Ketoisocaproate and α‐Keto‐β‐Methylvalerate by Engineered L‐Amino Acid Deaminase

Production of α‐Ketoisocaproate and α‐Keto‐β‐Methylvalerate by Engineered L‐Amino Acid Deaminase

Engineering of L-amino acid deaminases for the production of α-keto acids from L-amino acids

Metabolic engineering of cofactor flavin adenine dinucleotide (FAD) synthesis and regeneration in Escherichia coli for production of α‐keto acids

Contact Info

Product

Resources

About