Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis

Zhou, Shenghu; Liu, Peiran; Chen, Jian; Du, Guocheng; Li, Huazhong; Zhou, Jingwen

doi:10.1007/s00253-016-7672-8

Cited by 33 publications

(31 citation statements)

References 46 publications

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“…Two general protein engineering approaches were broadly used to improve the activities of rate-limiting enzymes in biocatalytic processes (Bommareddy et al, 2014;Chen et al, 2015a;Harrington et al, 2017;Wu et al, 2016;Zhou et al, 2016). One approach is directed evolution, combining random mutagenesis and highthroughput screening.…”

Section: Discussionmentioning

confidence: 99%

Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

Chen

Shen

Wang

et al. 2017

Biotech & Bioengineering

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Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

Chen

Shen

Wang

et al. 2017

Biotech & Bioengineering

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“…Three important benefits of the In the IHTB strategy, the promoters that drive the expression of the (2S)-naringenin biosynthetic genes were sequenced and their transcriptional strengths were identified based on results presented in our previous study (S. Zhou, Ding et al, 2017). Finally, we speculated that the rate-limiting step of (2S)-naringenin synthesis involved CHS, instead of TAL as previously reported (Santos et al, 2011;S. Zhou et al, 2016).…”

Section: Mschimentioning

confidence: 91%

“…To improve the titer of target chemicals, researchers first focused on screening for high‐activity enzymes in plants, animals, fungi, and bacteria (Jendresen et al, ; Santos et al, ). If the screened enzymes cannot satisfy present requirements of metabolic engineering, then protein engineering strategies, like directed evolution (S. Zhou & Alper, ; S. Zhou et al, ) and computer‐aided methods (Ebert & Pelletier, ; Fang, Zhang, Du, & Chen, ; Verma, Schwaneberg, & Roccatano, ), are typically performed to further improve the properties of the target enzymes. However, high‐efficiency biosynthetic pathways are through the use and regulated expression of high‐activity enzymes.…”

Section: Introductionmentioning

confidence: 99%

Fine‐tuning the (2S)‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy

Zhou

Lyu

et al. 2019

Biotech & Bioengineering

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Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed.Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry-fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl 3 and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains. K E Y W O R D S flavonoids, metabolic engineering, modular optimization, promoter Biotechnology and Bioengineering. 2019;116:1392-1404. wileyonlinelibrary.com/journal/bit 1392 |

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“…Nonlinear regression was used for fitting the data to the Michaelis-Menten equation for determining the kinetic constants [1, 15]. …”

Section: Methodsmentioning

confidence: 99%

Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis

Zhang

et al. 2017

PLoS ONE

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Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km, kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also compared the effects of the same set of single point mutations on the wild type and on the final mutant. The results indicate strong effects of epistasis, which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures.

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Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis

Cited by 33 publications

References 46 publications

Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

Fine‐tuning the (2S)‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy

Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis

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