NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase

Prachayasittikul, Virapong; Ljung, Sarah; Isarankura‐Na‐Ayudhya, Chartchalerm; Bülow, Leif

doi:10.7150/ijbs.2.10

Cited by 16 publications

(18 citation statements)

References 32 publications

(36 reference statements)

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“…It had been reported that the fusion of two multimeric enzyme could lead to the formation of multimeric protein network which may cause steric hindrance of active domain. Soluble bi‐functional enzyme compose of two multimeric enzyme have reported to form oligomers . LeuDH and FDH in our study usually formed octamer and dimer, respectively.…”

Section: Resultssupporting

confidence: 46%

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Zhang

Wang

et al. 2017

Engineering in Life Sciences

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This study reports the application of peptide linker in the construction of bi‐functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L‐tert leucine (L‐tle) biotransformation. Seven FDH‐LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three‐dimensional structures of LeuDH‐FDH and FDH‐LeuDH models using the I‐TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N‐, C‐terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.

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Section: Resultssupporting

confidence: 46%

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Zhang

Wang

et al. 2017

Engineering in Life Sciences

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“…As an alternative to using two separate enzymes, bifunctional fusion proteins can be constructed in which the production enzyme and the cofactor‐regenerating enzyme are connected through a linker sequence to yield a single polypeptide exhibiting both catalytic activities (). Although the advantages of fusion proteins composed of enzymes that catalyse sequential reactions have been studied since the late 1980s [8,9], this technique has recently been ‘rediscovered’ as a beneficial tool for cofactor recycling [10,11].…”

Section: Introductionmentioning

confidence: 99%

Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins

Hölsch

Weuster‐Botz

2010

Biotech and App Biochem

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NADPH-dependent oxidoreductases are useful catalysts for the production of chiral synthons. However, preparative applications of oxidoreductases require efficient methods for in situ regeneration of the expensive nicotinamide cofactors. An advantageous method for cofactor regeneration is the construction of bifunctional fusion proteins composed of two enzymes, one catalysing the reduction reaction and the other one mediating the recycling of cofactors. Herein, we describe the in-frame fusion between an NADP+-accepting mutant of FDH (formate dehydrogenase) from Mycobacterium vaccae N10 and KR [3-ketoacyl-(acyl-carrier-protein) reductase] from Synechococcus sp. strain PCC 7942. The generation of linker insertion mutants led to a fusion protein exhibiting 100 and 80% of the enzymatic activities of native KR and FDH respectively. Escherichia coli cells expressing the fusion protein showed an approx. 2-fold higher initial reaction rate in the production of chiral alcohols than cells expressing the enzymes separately. The application of the engineered fusion protein in whole-cell bioreduction of pentafluoroacetophenone resulted in a substrate conversion of 99.97% with an excellent enantiomeric excess of 99.9% (S)-1-(pentafluorophenyl)ethanol.

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“…First, several protein fusions consisting of sequential enzymes have been characterized in vitro and shown to possess kinetic properties superior to those of the individual enzymes (6,7,22,29). Second, experiments carried out with purified fusion proteins in polyethylene glycol solutions to mimic the crowded environment inside the cell indicate that the positive effects of enzyme fusion may be even greater in vivo (38,25,5). Last, the production of geranylgeraniol in S. cerevisiae was significantly increased by overexpressing a fusion of the yeast proteins Bts1 and Dpp1 (33).…”

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confidence: 99%

Diversion of Flux toward Sesquiterpene Production in Saccharomyces cerevisiae by Fusion of Host and Heterologous Enzymes

Albertsen

Chen

Bach

et al. 2011

Appl Environ Microbiol

196

162

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The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.

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NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase

Cited by 16 publications

References 32 publications

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins

Diversion of Flux toward Sesquiterpene Production in Saccharomyces cerevisiae by Fusion of Host and Heterologous Enzymes

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