Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Zhai, Yijie; Han, Donglei; Pan, Ying; Wang, Shuaishuai; Fang, Junqiang; Wang, Peng; Liu, Xianwei

doi:10.1016/j.enzmictec.2014.12.001

Cited by 29 publications

(26 citation statements)

References 38 publications

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“…Meanwhile, the chromosomal integration of Fkp along with six enzymes involved in the de novo pathway (ManB, ManC, Gmd, and WcaG) and fucosyltransferase (FutC) allowed E. coli JM109 to produce 388 mg of 2‐FL per g cell in flask culture (Baumgärtner et al, ). In another report, 122 mg/L of GDP‐ l ‐fucose was produced by fed‐batch fermentation of the engineered E. coli expressing Fkp with the series of genes involved in the guanosine nucleotides biosynthesis ( gpt , gmk , and ndk ) (Zhai et al, ).…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli to produce 2′‐fucosyllactose via salvage pathway of guanosine 5′‐diphosphate (GDP)‐l‐fucose

Chin

Seo

Kim

et al. 2016

Biotech & Bioengineering

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2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli to produce 2′‐fucosyllactose via salvage pathway of guanosine 5′‐diphosphate (GDP)‐l‐fucose

Chin

Seo

Kim

et al. 2016

Biotech & Bioengineering

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“…Via optimizing carbon source, overexpressing crucial enzymes, reinforcing the regeneration of NADPH or GTP as cofactors, and modulating the fed-batch fermentation mode, the titer of GDP-L-fucose was significantly improved from 55.2 mg/L to 305.5 mg/L and the specific GDP-L-fucose content accumulated to 3.5 mg/ g cell. In addition, the salvage pathway of GDP-L-fucose production has also been introduced into E. coli, and GTP regeneration was enhanced via overexpressing enzymes involved in the GTP biosynthesis pathway [7]. Baumgartner et al integrated recombinant genes of the de novo and salvage pathway into the chromosome of E. coli JM109 for GDP-L-fucose production and enhanced the specific intracellular GDP-L-fucose concentrations up to 5 mg/g dry cell wight (DCW) [18].…”

Section: Gdp-l-fucose Ismentioning

confidence: 99%

“…After that, the supernatant was collected via centrifugation at 10,000 × g for 10 min. Samples of the supernatant of different host strains were subjected to SDS-PAGE analysis [7].…”

Section: Sds-page Analysis For Gene Expression Levels Assaymentioning

confidence: 99%

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Combinatorial Modular Pathway Engineering for Guanosine 5′-Diphosphate-l-fucose Production in Recombinant Escherichia coli

Wan

Zhu

et al. 2020

J. Agric. Food Chem.

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Background: Guanosine 5′-diphosphate (GDP)-L-fucose is a vital nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes. Results: In this study, a combinatorial modular pathway engineering strategy was implemented to efficiently increase the intracellular titers of GDP-L-fucose in engineered Escherichia coli. The de novo GDP-L-fucose synthesis pathway was partitioned into two modules and fine-tuned in both transcriptional and translational levels, which remarkably improved the GDP-L-fucose production. In addition, the gene encoding the UDP-glucose lipid carrier transferase (WcaJ) was inactivated to eliminate the competing metabolite pathway from GDP-L-fucose to colanic acid. Furthermore, cofactors regeneration was underpinned to promote biocatalysis. Taken together, the final engineered strain EWL37, which could achieve the titers of 18.33 mg/L in shake-flask cultivation, was able to produce 106.21 mg/L (4.28 mg/g DCW) GDP-L-fucose through fed-batch cultivation. Conclusion: To date, this is the first utilization of a modular pathway optimization approach to increase the production potential of the de novo synthesized GDP-L-fucose. In general, this study manifests that via combinatorial modular pathway engineering, the GDP-L-fucose biosynthesis can be significantly improved.

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“…This may also include expression of heterologous enzymes and install a completely new pathway. For example, for GDP-fucose synthesis, besides using the de novo synthesis, the discovery of a unique bifunctional enzyme FKP from Bacteroids fragilis [ 39 ] that synthesize GDP-Fuc from fucose, ATP, GTP, provided researchers the salvage pathway for fucosylated oligosaccharides [ 33 , 40 ]. Another method is to use hosts that have an outstanding capability for sugar nucleotide synthesis.…”

Section: Introductionmentioning

confidence: 99%

The sweet branch of metabolic engineering: cherry-picking the low-hanging sugary fruits

Chen

2015

Microb Cell Fact

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In the first science review on the then nascent Metabolic Engineering field in 1991, Dr. James E. Bailey described how improving erythropoietin (EPO) glycosylation can be achieved via metabolic engineering of Chinese hamster ovary (CHO) cells. In the intervening decades, metabolic engineering has brought sweet successes in glycoprotein engineering, including antibodies, vaccines, and other human therapeutics. Today, not only eukaryotes (CHO, plant, insect, yeast) are being used for manufacturing protein therapeutics with human-like glycosylation, newly elucidated bacterial glycosylation systems are enthusiastically embraced as potential breakthrough to revolutionize the biopharmaceutical industry. Notwithstanding these excitement in glycoprotein, the sweet metabolic engineering reaches far beyond glycoproteins. Many different types of oligo- and poly-saccharides are synthesized with metabolically engineered cells. For example, several recombinant hyaluronan bioprocesses are now in commercial production, and the titer of 2′-fucosyllactose, the most abundant fucosylated trisaccharide in human milk, reaches over 20 g/L with engineered E. coli cells. These successes represent only the first low hanging fruits, which have been appreciated scientifically, medically and fortunately, commercially as well. As one of the four building blocks of life, sugar molecules permeate almost all aspects of life. They are also unique in being intimately associated with all major types of biopolymers (including DNA/RNA, proteins, lipids) meanwhile they stand alone as bioactive polysaccharides, or free soluble oligosaccharides. As such, all sugar moieties in biological components, small or big and free or bound, are important targets for metabolic engineering. Opportunities abound at the interface of glycosciences and metabolic engineering. Continued investment and successes in this branch of metabolic engineering will make vastly diverse sugar-containing molecules (a.k.a. glycoconjugates) available for biomedical applications, sustainable technology development, and as invaluable tools for basic scientific research. This short review focuses on the most recent development in the field, with emphasis on the synthesis technology for glycoprotein, polysaccharide, and oligosaccharide.

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Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Cited by 29 publications

References 38 publications

Metabolic engineering of Escherichia coli to produce 2′‐fucosyllactose via salvage pathway of guanosine 5′‐diphosphate (GDP)‐l‐fucose

Metabolic engineering of Escherichia coli to produce 2′‐fucosyllactose via salvage pathway of guanosine 5′‐diphosphate (GDP)‐l‐fucose

Combinatorial Modular Pathway Engineering for Guanosine 5′-Diphosphate-l-fucose Production in Recombinant Escherichia coli

The sweet branch of metabolic engineering: cherry-picking the low-hanging sugary fruits

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