Saturation-mutagenesis in two positions distant from active site of a Klebsiella pneumoniae glycerol dehydratase identifies some highly active mutants

Qi, Xianghui; Chen, Yunlai; Jiang, Ke; Zuo, Wenpu; Luo, Zhaofei; Wei, Yutuo; Du, Liqin; Wei, Hang; Huang, Ribo; Du, Qiang

doi:10.1016/j.jbiotec.2009.06.015

Cited by 21 publications

(22 citation statements)

References 35 publications

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“…The implementation of high‐throughput screening is essential for isolating clones or mutants of a desirable property from large libraries generated by random or rational mutagenesis (Lee et al, ; Lee et al, ). Among colorimetric high‐throughput screening methods, a Schiff's reagent‐based method (Qi et al, ), which is based on the aldehyde reaction principle of Schiff's reagent, has been used for aldose (sugar containing an aldehyde group) selection (Hasehira et al, ); to screen for natural rubber degradation products, aldehyde derivatives (Imai et al, ), aldehydes‐metabolites (Charneira et al, ), yeast alcohol metabolites (Shuster et al, ), glycoprotein (Hart et al, ), formaldehyde (Gibson et al, ), and as a biosensor for aliphatic aldehyde (Yang et al, ).…”

Section: Introductionmentioning

confidence: 99%

Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4‐butanediol pathway in Escherichia coli

Hwang

Park

Kim

et al. 2014

Biotech & Bioengineering

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1,4-Butanediol (1,4-BDO) is currently produced from succinate via six enzymatic reactions in an engineered Escherichia coli strain. Butyraldehyde dehydrogenase (Bld) and butanol dehydrogenase of Clostridium saccharoperbutylacetonicum were selected based on their activities of catalyzing the final two reactions in the 1,4-BDO pathway. To fit Bld into the non-natural 1,4-BDO pathway, we engineered it through random mutagenesis. Five Bld mutants were then isolated using a colorimetric Schiff's reagent-based method. Subsequent site-directed mutagenesis of Bld generated the two best Bld mutants, L273I and L273T, which produced 1,4-BDO titers fourfold greater than those of wild-type Bld. The enhanced 1,4-BDO titers obtained using L273I and L273T clearly correlated with their enhanced activities, which were caused by amino acid mutations at position 273 of Bld. The highest titer of 1,4-BDO (660 ± 40 mg/L) was obtained in a knock-out E. coli strain [ΔldhA ΔpflB ΔadhE ΔlpdA::K. lpd(E354K) Δmdh ΔarcA gltA(R164L)] coexpressing Bld273T+Bdh.

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Section: Introductionmentioning

confidence: 99%

Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4‐butanediol pathway in Escherichia coli

Hwang

Park

Kim

et al. 2014

Biotech & Bioengineering

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“…As it was explained by the authors, the branch point at glycerol partitioning is rigid, mainly due to the inbuilt properties of the enzymes involved. Thus, protein engineering, which has already entered the field of PDOR and GDHt improvement [Li et al, 2008;Ma et al, 2010;Qi et al, 2009], may constitute an interesting approach to overcome this limitation. Since the inbuilt properties of the WT enzymes involved in 1,3-PD formation disallow improvement of 1,3-PD synthesis by their over-expression, application of modified activities could potentially debottleneck 1,3-PD synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…In a study by Qi et al [2009], both first random and then directed mutagenesis were applied to modify the GDHt sequence. In the first stage, random mutagenesis was carried out by error-prone PCR.…”

Section: Protein Engineeringmentioning

confidence: 99%

Klebsiella sppas a 1, 3-propanediol producer – the metabolic engineering approach

Celińska

2011

Critical Reviews in Biotechnology

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Klebsiella spp are one of the best natural producers of 1,3-propanediol (1,3-PD). However, their usage in the biotechnological production of the diol is limited, since the species belong to the second hazard group. Nevertheless, multiple advantageous traits of Klebsiella spp justify the international effort devoted to develop a biotechnological process of 1,3-PD production with these microorganisms. Apart from the process engineering approach aiming at improvement of 1,3-PD production by Klebsiella spp, plethora of metabolic engineering approaches have been reported. Different strategies have been undertaken to genetically improve Klebsiella strains and provide them with the ability to synthesize 1,3-PD more efficiently. These include over-expression of both homologous and heterologous genes of the 1,3-PD synthesis pathway, protein and cofactor engineering, deletion of the genes involved in by-products formation. This review provides an overview of the initial and most recent reports on the metabolic engineering of Klebsiella spp with the aim of improvement of 1,3-PD biosynthesis.

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“…The genomic DNA was isolated from K. pneumoniae according to Qi et al (2009). Primers for gldABC and mutated gene were designed by Vector NTI according to gldABC sequence of K. pneumoniae deposited in GenBank under accession No.…”

Section: Cloning Expression and Site-mutagenesismentioning

confidence: 99%

“…Gene expression, cell extract preparation and enzymatic activity assay Expression of genes, extraction of recombinant enzymes, enzymatic activity assay and SDS-PAGE electrophoresis were according to those previously reported (Qi et al 2009). …”

Section: Sequence Analysis Of Gdhtmentioning

confidence: 99%

Enhancement of pH stability and activity of glycerol dehydratase from Klebsiella pneumoniae by rational design

Guo

Wei

et al. 2011

Biotechnol Lett

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Glycerol dehydratase (GDHt) is a key and rate-limiting enzyme in the pathway of 1,3-propanediol (1,3-PD) synthesis. The improvement of GDHt's stability and enzymatic activity is desirable for the biosynthesis of 1,3-PD. The gldABC gene encoding GDHt of Klebsiella pneumoniae was cloned and expressed in Escherichia coli XL10-Gold, and the mutation sites of GDHt were obtained through prediction by PoPMuSiC program. Consequently, two mutants (KpG60 and KpG525) were developed by rational design through site-mutagenesis based on 3D structure which was constructed from homology modeling. Analyses of enzymatic properties showed that pH stability of the mutants was about 1.25-2 times higher than that of the wild type, and specific activity, V(max) and K(cat)/K(m) of KpG525 were about 1.5-2 times higher than those of the wild type. This work presented a simple and useful measure to improve the performance of industrial enzyme.

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Saturation-mutagenesis in two positions distant from active site of a Klebsiella pneumoniae glycerol dehydratase identifies some highly active mutants

Cited by 21 publications

References 35 publications

Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4‐butanediol pathway in Escherichia coli

Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4‐butanediol pathway in Escherichia coli

Klebsiella sppas a 1, 3-propanediol producer – the metabolic engineering approach

Enhancement of pH stability and activity of glycerol dehydratase from Klebsiella pneumoniae by rational design

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