Construction and characterization of a recombinant tripartite enzyme, galactose dehydrogenase/β‐galactosidase/galactokinase

Ljungcrantz, Peter; Bülow, Leif; Mosbach, Klaus

doi:10.1016/0014-5793(90)81446-u

Cited by 12 publications

(3 citation statements)

References 8 publications

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“…The next logical step for the simplification of PHB pathway expression in transgenic plants is the creation of a three‐gene fusion in which all of the enzyme activities for PHB production are contained on one polypeptide. Ljungcrantz et al . (1990) created a three‐gene fusion of galactose dehydrogenase, β‐galactosidase and galactokinase that displayed reduced activity of each of the component enzymes.…”

Section: Discussionmentioning

confidence: 99%

A novel thiolase–reductase gene fusion promotes the production of polyhydroxybutyrate in Arabidopsis

Kourtz

Dillon

Daughtry

et al. 2005

Plant Biotechnology Journal

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SummaryThe production of polyhydroxybutyrate (PHB) involves a multigene pathway consisting of thiolase, reductase and synthase genes. In order to simplify this pathway for plant-based expression, a library of thiolase and reductase gene fusions was generated by randomly ligating a short core linker DNA sequence to create in-frame fusions between the thiolase and reductase genes. The resulting fusion constructs were screened for PHB formation in Escherichia coli . This screen identified a polymer-producing candidate in which the thiolase and reductase genes were fused via a 26-amino-acid linker. This gene fusion, designated phaA-phaB , represents an active gene fusion of two homotetrameric enzymes. Expression of phaA-phaB in E. coli and Arabidopsis yielded a fusion protein observed to be the expected size by Western blotting techniques. The fusion protein exhibited thiolase and reductase enzyme activities in crude extracts of recombinant E. coli that were three-fold and nine-fold less than those of the individually expressed thiolase and reductase enzymes, respectively. When targeted to the plastid, and coexpressed with a plastid-targeted polyhydroxyalkanoate (PHA) synthase, the fusion protein enabled PHB formation in Arabidopsis , yielding roughly half the PHB formed in plants expressing individual thiolase, reductase and synthase enzymes. This work represents a first step towards simplifying the expression of the PHB biosynthetic pathway in plants.

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Section: Discussionmentioning

confidence: 99%

A novel thiolase–reductase gene fusion promotes the production of polyhydroxybutyrate in Arabidopsis

Kourtz

Dillon

Daughtry

et al. 2005

Plant Biotechnology Journal

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“…The production increase of the other two strains was not obvious, and it might be caused by the changes in enzyme structure or the inappropriate linker. The main reasons for the increase of strain YLAAF-3 might be the following: Proximity effects were exhibited by the fusion of enzymes in adjacent catalytic reactions, and transient time of reactions was decreased (Ljungcrantz et al, 1989(Ljungcrantz et al, , 1990). In addition, fusion-protein expression could reduce the degradation of intermediate metabolites by the intracellular proteases (Albertsen et al, 2011;Dai et al, 2012;Ignea et al, 2014).…”

Section: Protein Fusion Engineeringmentioning

confidence: 99%

One-Step Biosynthesis of Vitamin C in Saccharomyces cerevisiae

Zhou

Bi²,

Ding³

et al. 2021

Front. Microbiol.

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Vitamin C (VC) is comprehensively applied in foods, cosmetics, pharmaceuticals, and especially clinical medicine. Nowadays, the industrial production of VC mainly relies on the classic two-step fermentation route, and researchers have explored the way for one-step fermentation of VC in recent years. In this study, a VC biosynthesis pathway that directly produced VC from glucose was reconstructed in Saccharomyces cerevisiae, and the protein engineering and metabolic engineering strategies were adopted to improve it. First, five exogenous modules from Arabidopsis were introduced into the chassis cells by synthetic biology approaches to obtain the strain YLAA harboring VC biosynthesis. In addition, L-galactose dehydrogenase (L-GalDH) and L-galactono-1,4-lactone dehydrogenase (L-GLDH) were fused and expressed in S. cerevisiae cells for the first time, which increased the intracellular VC accumulation by 2.78-fold, reaching 9.97 ± 0.09 mg/L. Through copy number engineering, it was further confirmed that the last step catalyzed by L-GLDH is the rate-limiting step. GDP-L-galactose phosphorylase (GPP) encoded by vtc2 is another rate-limiting enzyme confirmed by GAL1p overexpression results. Finally, by balancing gene expression and cell growth, the highest production strain with overexpressing vtc2 by multicopy plasmids was constructed. The VC accumulation reached 24.94 ± 1.16 mg/L, which was currently the highest production from glucose in S. cerevisiae. The production of the recombinant strain reached nearly 44 mg/L with the exogenous addition of L-galactose or glutathione. The results further emphasized the importance of the step catalyzed by GPP. The investigation provided experience for the efficient biosynthesis of VC and the determination of rate-limiting steps.

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“…The same group reported construction of a gene fusion containing all three enzymes. 94 Other biocatalysts have been linked by gene fusion as well. Mitochondrial citrate synthase (CS) and malate dehydrogenase (MDH), two enzymes taking part in the citric acid cycle, were expressed as a fusion protein.…”

Section: Multi-enzyme Systems Obtained By Gene Fusionmentioning

confidence: 99%