Synthesis of GDP-mannose using coupling fermentation of recombinant Escherichia coli

Jia, Hong-hong; Lu, Fuping; Yu, Li; Liu, Xiaoguang; Liu, Yihan; Wang, Hongbin; Li, Jing; Cao, Yiyi

doi:10.1007/s10529-011-0547-2

Cited by 8 publications

(1 citation statement)

References 14 publications

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“…The GDP‐Man synthesized within 72 h (581 mg) was isolated in 34.3% yield. Using a similar one‐pot approach, however, applying instead of purified HK, PMM and ManC whole cells of E. coli , each expressing one of the three enzymes recombinantly, Honghong et al demonstrated formation of GDP‐Man (21 mM; 680 mg) from Man (65 mM) and an energy source ( d ‐fructose, 130 mM) within 22 h. The product was not isolated though.…”

Section: Introductionmentioning

confidence: 99%

A Kinase‐Independent One‐Pot Multienzyme Cascade for an Expedient Synthesis of Guanosine 5′‐Diphospho‐d‐mannose

et al. 2016

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Biomimetic synthesis routes towards the important natural d-mannosyl donor guanosine 5'-diphospho-d-mannose (GDP-Man) rely on kinase-catalyzed nucleotide triphosphate (NTP)-dependent phosphorylationso fd-mannose (Man), to give d-mannose6 -phosphate or a-d-mannose 1-phosphate (aMan 1-P) as an intermediate product.AGDP-Man synthesisn ot requiring the kinase/NTP system wouldb ep ractical and cost-effective.H ere,w eh ave developed am ultienzyme cascade towards GDP-Man, characterized in that aMan 1-P was obtained by ad iastereoselectivep hosphatase-catalyzed phosphorylation of Man. a-d-Glucose 1-phosphate (aGlc 1-P), prepared in situ through phosphorylase-catalyzed conversion of sucrosei nt he presenceo fi norganic phosphate, was used as an expedient phosphoryl donor. Thei ncipient aMan 1-P and guanosine triphosphate (GTP) were converted into GDP-Manb y ahighly manno compared to gluco selectivenucleotidyltransferase.P yrophosphatase was additionally required to hydrolyze the pyrophosphate released from the GTP, thus driving the reactiont owards GDP-Man. Thee nzymatic cascade was operated with the aMan1 -P andt he GDP-Manf ormation decoupled from one another (sequentialm ode)o r having all steps run concurrently (simultaneous mode). Detailed time course analysis revealed that kinetic pull due to the constant removal of the intermediate aMan1 -P in simultaneous-moder eactions was importantt op romote phosphorylation of Man from aGlc 1-P in high efficiency,a voiding loss of sugar 1-phosphates by hydrolysis.U nder optimized conditions for the one-pot transformation involving four enzymes,1 00 mM (67 gL À1 )G DP-Man was prepared from 140 mM sucrosea nd phosphate, using 400 mM Mana st he phosphoryla cceptor. Thep roduct was recovered by anion-exchange and size-exclusion chromatographyi n! 95% purityi na bout 50% yield (100 mg). These results demonstrate for the first time the practical use of ap hosphorylase-phosphatase combi-catalyst as an alternative to the canonicalk inase for the anomericp hosphorylation of the sugar substrate in nucleoside diphospho-sugar synthesis. Phosphorylation from inorganic phosphate via the intermediate aGlc 1-P rather than from NTP, particularly GTP,a ppears advantageous specifically in cases where the sugar acceptor is abulk commodity that can be applied in suitable excesst ot he phosphatase reaction.

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Section: Introductionmentioning

confidence: 99%

A Kinase‐Independent One‐Pot Multienzyme Cascade for an Expedient Synthesis of Guanosine 5′‐Diphospho‐d‐mannose

et al. 2016

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One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Rexer

Schildbach

Klapproth

et al. 2017

Biotech & Bioengineering

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Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl2. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)2‐Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)2‐Man1 can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.

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Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in Escherichia coli

Qin

Zhang

et al. 2016

Biotechnol Lett

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Synthesis of GDP-mannose using coupling fermentation of recombinant Escherichia coli

Cited by 8 publications

References 14 publications

A Kinase‐Independent One‐Pot Multienzyme Cascade for an Expedient Synthesis of Guanosine 5′‐Diphospho‐d‐mannose

A Kinase‐Independent One‐Pot Multienzyme Cascade for an Expedient Synthesis of Guanosine 5′‐Diphospho‐d‐mannose

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in Escherichia coli

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