Physical and kinetic effects on introduction of various linker regions in β-galactosidase/galactose dehydrogenase fusion enzymes

Carlsson, Helén; Ljung, Sarah; Bülow, Leif

doi:10.1016/0167-4838(95)00240-5

Cited by 24 publications

(24 citation statements)

References 24 publications

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“…The effects of linker peptides in fusion proteins are variable (1,10,11). In a ␤-galactosidase/galactose dehydrogenase fusion enzyme, the specific activity of the galactose dehydrogenase moiety increased with a longer linker (3 versus 9 or 13 amino acids) and the sequential reaction was carried out more efficiently (7). Although a longer linker might release steric perturbation, the longer linker would be more accessible to degrading enzymes, resulting in lower stability (5).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

Seo

Koo

Lim

et al. 2000

Appl Environ Microbiol

100

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To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The K m values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the k cat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5-to 4.0-fold increase in the catalytic efficiency (k cat /K m ). The K m and k cat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-␤-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.The nonreducing disaccharide trehalose [␣-D-glucopyranosyl-(131)-␣-D-glucopyranose] has high water-holding activities, which maintain the fluidity of membranes under dry conditions (25). It also stabilizes enzymes, foods, cosmetics, and pharmaceuticals at high temperatures (8,9,38). Due to its desirable physical and chemical characteristics, commercial production of trehalose is anticipated. Escherichia coli synthesizes trehalose when exposed to high osmolarity (12,20,39,41). In E. coli, trehalose is synthesized by two separate enzymes, trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP), encoded by the genes otsA and otsB, respectively (15, 21). This is different from Saccharomyces cerevisiae, in which trehalose is synthesized by a large multisubunit complex with the catalytic activities of both TPS and TPP (6, 31, 43).Overexpression of TPS and TPP might be one way to produce trehalose. We assumed that the physical proximity of two enzymes catalyzing sequential reactions might increase the reaction rate by facilitating transfer of the reaction intermediate when they are present in a complex. A variety of techniques have been applied to better understand the proximity effect of enzymes catalyzing sequential reactions, including cross-linking and coimmobilization (23,32,34). In many of these cases,...

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Section: Discussionmentioning

confidence: 99%

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

Seo

Koo

Lim

et al. 2000

Appl Environ Microbiol

100

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“…The process likely benefits from a proximity effect of the two active sites to reduce loss of intermediates to other competing pathways. Previous attempts to use this concept in vivo have generally been unsuccessful (1,18,36) despite the fact that fusion enzymes have proven superior to free enzymes in several in vitro studies (6,7,22,29). At least two parameters should be considered in order to obtain successful exploitation of fusion proteins in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Since specific linkers can presumably favor beneficial conformations of fusion proteins (7,19,27,34), we proceeded to investigate whether the nature of the linker combining FPPS and PTS can influence patchoulol production. To this end, we focused on the configuration FPPS-PTS and examined whether patchoulol production could be further improved by altering the linker composition and length.…”

Section: Vol 77 2011 Fusion Enzyme Increases Sesquiterpene Productimentioning

confidence: 99%

“…A number of observations suggest that this strategy can be used to improve the flux through a metabolic pathway. 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).…”

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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

198

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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“…We should eliminate the sequence at the C-terminal region by PCR amplification to transfer hydrophobic properties of SQS into the soluble protein. Various sizes of linkers have been used in other studies to fuse two proteins, and long linkers were found unsuitable because they tended to be sensitive to proteolytic attack and also because the distance between the two active sites may have impeded substrate channelling between protein domains 21,22 . In case of no linker between two genes, production of the sesquiterpenoid was less effective in E. coli cells 15 .…”

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confidence: 99%

Fusion of Ginseng Farnesyl Diphosphate Synthase and <i>Centella asciatica</i> Squalene Synthase Involved in Triterpenoid Biosynthesis

Jung¹,

Kim²,

Jin³

et al. 2017

Current Science

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Physical and kinetic effects on introduction of various linker regions in β-galactosidase/galactose dehydrogenase fusion enzymes

Cited by 24 publications

References 24 publications

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

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

Fusion of Ginseng Farnesyl Diphosphate Synthase and <i>Centella asciatica</i> Squalene Synthase Involved in Triterpenoid Biosynthesis

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