One‐pot synthesis of amino‐alcohols using a de‐novo transketolase and β‐alanine: Pyruvate transaminase pathway in <i>Escherichia coli</i>

Ingram, Christine U.; Bommer, Martin; Smith, Mark E.; Dalby, Paul A.; Ward, John M.; Hailes, Helen C.; Lye, Gary J.

doi:10.1002/bit.21125

Cited by 130 publications

(88 citation statements)

References 53 publications

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“…1), and the proposed enzymatic activity of BauA has been reported (13). Along this line, we hypothesized that induction of the bauABCD operon by Spd and DAP was due to the common catabolic intermediate ␤-alanine and that GABA, the intermediate compound of Put catabolism, should have no effect on this operon.…”

Section: Vol 193 2011mentioning

confidence: 82%

Functional Characterization of Seven γ-Glutamylpolyamine Synthetase Genes and the bauRABCD Locus for Polyamine and β-Alanine Utilization in Pseudomonas aeruginosa PAO1

Yao

2011

J Bacteriol

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Pseudomonas aeruginosa and many other bacteria can utilize biogenic polyamines, including diaminopropane (DAP), putrescine (Put), cadaverine (Cad), and spermidine (Spd), as carbon and/or nitrogen sources. Transcriptome analysis in response to exogenous Put and Spd led to the identification of a list of genes encoding putative enzymes for the catabolism of polyamines. Among them, pauA1 to pauA6, pauB1 to pauB4, pauC, and pauD1 and pauD2 (polyamine utilization) encode enzymes homologous to Escherichia coli PuuABCD of the ␥-glutamylation pathway in converting Put into GABA. A series of unmarked pauA mutants was constructed for growth phenotype analysis. The results revealed that it requires specific combinations of pauA knockouts to abolish utilization of different polyamines and support the importance of ␥-glutamylation for polyamine catabolism in P. aeruginosa. Another finding was that the list of Spd-inducible genes overlaps almost completely with that of Put-inducible ones except the pauA3B2 operon and the bauABCD operon (␤-alanine utilization). Mutation analysis led to the conclusion that pauA3B2 participate in catabolism of DAP, which is related to the aminopropyl moiety of Spd, and that bauABCD are essential for growth on ␤-alanine derived from DAP (or Spd) catabolism via the ␥-glutamylation pathway. Measurements of the pauA3-lacZ and bauA-lacZ expression indicated that these two promoters were differentially induced by Spd, DAP, and ␤-alanine but showed no apparent response to Put, Cad, and GABA. Induction of the pauA3 and bauA promoters was abolished in the bauR mutant. The recombinant BauR protein was purified to demonstrate its interactions with the pauA3 and bauA regulatory regions in vitro. In summary, the present study support that the ␥-glutamylation pathway for polyamine utilization is evolutionarily conserved in E. coli and Pseudomonas spp. and is further expanded in Pseudomonas to accommodate a more diverse metabolic capacity in this group of microorganisms.Biogenic polyamines are a group of ubiquitous polycations found in all living organisms. They are essential for cell growth and participate in a variety of physiological functions (2,30,31). Depending on the specific biosynthetic pathways (12,22,26,29), different bacteria possess a preferential set of polyamines, which include the diamines diaminopropane (DAP), putrescine (Put), and cadaverine (Cad); the triamines spermidine (Spd) and norspermidine; and the tetramine spermine. It is generally believed that polyamines form complexes with nucleic acid-containing macromolecules through charge interactions in vivo (8,11,16). In vitro, excess binding of polyamines to DNA was reported to form very condensed complexes (3), which might cause difficulties in DNA unwinding during replication or transcription. Therefore, the intracellular concentrations of polyamines need to be tightly monitored to prevent adverse effects on cell growth.When released from the cells into environments, polyamines can be recycled by many bacteria or serve as sources of carbo...

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Section: Vol 193 2011mentioning

confidence: 82%

Functional Characterization of Seven γ-Glutamylpolyamine Synthetase Genes and the bauRABCD Locus for Polyamine and β-Alanine Utilization in Pseudomonas aeruginosa PAO1

Yao

2011

J Bacteriol

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“…Our results have shown the use of cascading free enzymes in microfluidic devices for the first time, with significant improvement in conversion yields over free enzymes in one‐pot systems (Ingram et al, 2007) and over immobilized enzymes in microfluidic systems (Abdul Halim et al, 2013; Matosevic et al, 2011) The transketolase (TK)‐transaminase (TAm) reaction cascade was optimized according to Gruber, Marques, O'Sullivan et al (2017) to overcome key issues, such as substrate and cofactor inhibition, and we have obtained for the first time full conversion in both continuous‐flow microreactors and batch systems.…”

Section: Discussionmentioning

confidence: 65%

“…Ingram et al (2007) obtained a final concentration of 5 mM ABT (corresponding to a 21% mol/mol yield conversion of L‐erythrulose to ABT) over 100 hr of reaction time. However, the corresponding conversion of MBA to AP and the depletion of ERY were each clearly below 100%.…”

Section: Resultsmentioning

confidence: 99%

“…ABT was quantified using an ACE 5 C18 RP column (150 mm × 4.6 mm, 5 μm, Advanced Chromatography Technologies) with a mobile phase comprised of 140 mM sodium acetate buffer (adjusted to pH 5 using acetic acid) at 0.5 ml min −1 with a gradient of acetonitrile from 85% to 100% over 10 min, followed by a column wash phase and re‐equilibrium step at 1 ml min −1 and detection at 254 nm. ABT for calibration purposes was synthesized according to Ingram et al (2007). …”

Section: Methodsmentioning

confidence: 99%

“…ABT is used as a building block for statins employed in the synthesis of the HIV‐protease inhibitor Nelfinavir (Kaldor et al, 1997; Kwon & Ko, 2002) and as an intermediate in the synthesis of detoxinine, a detoxifying agent for reducing the toxicity of the antibiotic treatment for rice blast disease (Delle Monache et al, 1999; Ingram et al, 2007). In this work, ABT is obtained by coupling a transketolase (TK)‐catalyzed asymmetric carbon‐carbon bond formation with a transaminase (TAm)‐catalyzed conversion of the keto‐group into a chiral amino group (Scheme 1) (Ingram et al, 2007; Rios‐Solis et al, 2011; Villegas‐Torres et al, 2015). We show, for the first time, a microreactor‐based reaction cascade with free enzymes and we obtained for the first time full conversion of this reaction sequence in both continuous‐flow microreactors and in batch.…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Gruber

Carvalho

Marques

et al. 2017

Biotech & Bioengineering

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Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml−1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml−1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

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Transketolases

Wohlgemuth¹,

Smith²,

Dalby³

et al. 2009

Encyclopedia of Industrial Biotechnology

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The enzyme transketolase is a potentially useful catalyst for asymmetric carbon–carbon bond formation. Given the importance of this reaction in organic synthesis, it is not surprising that in recent years there has been particular interest in transketolase. In this review, we describe the background to the reaction, mechanistic and structural information, application in synthesis, and the requirements for larger scale application and implementation.

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One‐pot synthesis of amino‐alcohols using a de‐novo transketolase and β‐alanine: Pyruvate transaminase pathway in Escherichia coli

Cited by 130 publications

References 53 publications

Functional Characterization of Seven γ-Glutamylpolyamine Synthetase Genes and the bauRABCD Locus for Polyamine and β-Alanine Utilization in Pseudomonas aeruginosa PAO1

Functional Characterization of Seven γ-Glutamylpolyamine Synthetase Genes and the bauRABCD Locus for Polyamine and β-Alanine Utilization in Pseudomonas aeruginosa PAO1

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Transketolases

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