Engineering of lactose metabolism inE. coli by introducing ?-galactosidase/galactokinase fusion enzymes

Carlsson, Helén; Ljungcrantz, Peter; Bülow, Leif; Mosbach, Klaus

doi:10.1007/bf01023163

Cited by 7 publications

(7 citation statements)

References 12 publications

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“…This linker separates the two proteins in space by a small distance allowing each of them to fold properly without constrains from the other protein molecule. Linkers of different length have been used but it has been shown that if a too long linker is used the proximity effect is abolished [6]. Direct fusion of enzymes without a linker may also result in an active bifunctional enzyme [7].…”

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confidence: 99%

“…However, so far this approach has been used to a relatively limited extent. The lactose utilization [6] and the osmotolerance of E. coli [14] have been influenced by introduction of the bifunctional enzymes β‐galactosidase/galactokinase and γ‐glutamyl kinase/γ‐glutamyl phosphate reductase, respectively. The starch degrading bifunctional enzyme α‐amylase/glucose isomerase was expressed in potato tubers and upon heating (65 °C for 45 min) of the crushed fresh tubers, glucose and fructose was produced from the starch present in the tubers [15].…”

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confidence: 99%

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Fusion of farnesyldiphosphate synthase and epi‐aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum

Brodelius¹,

Lundgren²,

Mercke

et al. 2002

European Journal of Biochemistry

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A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/ eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-SerGly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-b-D-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co 2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The K m values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.

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confidence: 99%

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confidence: 99%

Fusion of farnesyldiphosphate synthase and epi‐aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum

Brodelius¹,

Lundgren²,

Mercke

et al. 2002

European Journal of Biochemistry

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“…Proximity effects due to high local concentrations of intermediates have earlier been demonstrated in E. coli cells containing a scavenger enzyme, galactose dehydrogenase, competing with the galactokinase part of a ;-galactosidase galactokinase fusion protein for the galactose formed by ;-galactosidase (Carlsson et al, 1992). A similar effect was obtained when fused citrate synthase and malate dehydrogenase were expressed in mutants of S. cerevisiae deleted in citrate synthase and malate dehydrogenase genes (Elcock and McCammon, 1996;Lindbladh et al, 1994).…”

Section: Discussionmentioning

confidence: 52%

Expression of Bifunctional Enzymes with Xylose Reductase and Xylitol Dehydrogenase Activity in Saccharomyces cerevisiae Alters Product Formation during Xylose Fermentation

Anderlund

Rådström

Hahn‐Hägerdal

2001

Metabolic Engineering

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“…Subsequently it reducing the transit time required for the intermediates to spread the enzyme that catalyzes the next step in the reaction. Several elucidations in vitro and in vivo recommended that this strategy can be used to improve the flux through a metabolic pathway [ 21 – 25 ].…”

Section: Introductionmentioning

confidence: 99%

Combination ofERG9Repression and Enzyme Fusion Technology for Improved Production of Amorphadiene inSaccharomyces cerevisiae

Baadhe

Mekala

Parcha

et al. 2013

Journal of Analytical Methods in Chemistry

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The yeast strain (Saccharomyces cerevisiae) MTCC 3157 was selected for combinatorial biosynthesis of plant sesquiterpene amorpha-4,11-diene. Our main objective was to overproduce amorpha 4-11-diene, which is a key precursor molecule of artemisinin (antimalarial drug) produced naturally in plant Artemisia annua through mevalonate pathway. Farnesyl diphosphate (FPP) is a common intermediate metabolite of a variety of compounds in the mevalonate pathway of yeast and leads to the production of ergosterols, dolichol and ubiquinone, and so forth. In our studies, FPP converted to amorphadiene (AD) by expressing heterologous amorphadiene synthase (ADS) in yeast. First, ERG9 (squalane synthase) promoter of yeast was replaced with repressible methionine (MET3) promoter by using bipartite gene fusion method. Further to overcome the loss of the intermediate FPP through competitive pathways in yeast, fusion protein technology was adopted and farnesyldiphosphate synthase (FPPS) of yeast has been coupled with amorphadiene synthase (ADS) of plant origin (Artemisia annua L.) where amorphadiene production was improved by 2-fold (11.2 mg/L) and 4-fold (25.02 mg/L) in yeast strains YCF-002 and YCF-005 compared with control strain YCF-AD (5.5 mg/L), respectively.

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Engineering of lactose metabolism inE. coli by introducing ?-galactosidase/galactokinase fusion enzymes

Cited by 7 publications

References 12 publications

Fusion of farnesyldiphosphate synthase and epi‐aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum

Fusion of farnesyldiphosphate synthase and epi‐aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum

Expression of Bifunctional Enzymes with Xylose Reductase and Xylitol Dehydrogenase Activity in Saccharomyces cerevisiae Alters Product Formation during Xylose Fermentation

Combination ofERG9Repression and Enzyme Fusion Technology for Improved Production of Amorphadiene inSaccharomyces cerevisiae

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