Enhanced production of 2′-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of lactose metabolism and fucosyltransferase

Chin, Young‐Wook; Kim, Ji Yeong; Lee, Won-Heong; Seo, Jin‐Ho

doi:10.1016/j.jbiotec.2015.06.431

Cited by 89 publications

(99 citation statements)

References 31 publications

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“…Although all of lactose added at 19 h was completely consumed within 41 h, only 0.17 g/L of 2‐FL was produced. Considering the fact that the 2‐FL yield from lactose (0.01 mole/mole) was much lower than 2‐FL yield from fucose (0.14 mole/mole) (Table ), most lactose might have been used for cell growth instead of 2‐FL production, which is consistent with our previous study (Chin et al, ).…”

Section: Resultssupporting

confidence: 90%

“… c The results of fed‐batch fermentation of △L M15 BCGW‐F strain expressing the de novo GDP‐ l ‐fucose biosynthetic enzymes using glycerol and lactose was cited in Chin et al ().…”

Section: Resultsmentioning

confidence: 99%

“…coli was grown in Luria Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with appropriate antibiotics for genetic manipulation and seed culture. Batch fermentations were performed in a 500 mL baffled flask (Nalgene) containing 100 mL of a defined medium (Chin et al, 2015) containing 20 g/L glycerol and appropriate antibiotics at 25 C. The agitation speed of 250 rpm was provided for aerobic condition. When optical density (OD 600 ) reached 0.8, isopropyl-b-D-thiogalactopyranoside (IPTG), lactose, and fucose were added to a final concentration 0.1 mM, 10 g/L, and 10 g/L, respectively.…”

Section: Culture Conditionsmentioning

confidence: 99%

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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: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Culture Conditionsmentioning

confidence: 99%

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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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“…In contrast, the alternative inducer lactose has been shown to favour the production of soluble product and trigger enhanced productivity13141516. Even though lactose metabolism is topic of some recent studies17, lactose is scarcely used in biochemical engineering since induction entails the challenge of continuous supply of the disaccharide as it gets metabolized by E. coli . Furthermore, cultivations have to be conducted at limiting amounts of glucose as otherwise lactose uptake is inhibited due to the well-known phenomenon of carbon catabolite repression (e.g.…”

mentioning

confidence: 99%

Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains

Wurm

Hausjell

Ulonska

et al. 2017

Sci Rep

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When producing recombinant proteins, the use of Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. In a recent study we introduced a mechanistic model describing the correlation of the specific glucose uptake rate (qs,glu) and the corresponding maximum specific lactose uptake rate (qs,lac,max) for a pET-based E. coli BL21(DE3) strain producing a single chain variable fragment (scFv). We showed the effect of qs,lac,max on productivity and product location underlining its importance for recombinant protein production. In the present study we investigated the mechanistic qs,glu/qs,lac,max correlation for four pET-based E. coli BL21(DE3) strains producing different recombinant products and thereby proved the mechanistic model to be platform knowledge for E. coli BL21(DE3). However, we found that the model parameters strongly depended on the recombinant product. Driven by this observation we tested different dynamic bioprocess strategies to allow a faster investigation of this mechanistic correlation. In fact, we succeeded and propose an experimental strategy comprising only one batch cultivation, one fed-batch cultivation as well as one dynamic experiment, to reliably determine the mechanistic model for qs,glu/qs,lac,max and get trustworthy model parameters for pET-based E. coli BL21(DE3) strains which are the basis for bioprocess development.

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“…22,23 With additional deletion of endogenous lactose operon and adding three aspartate molecules at the Nterminal of FucT2, 6.4 g/L of 2′FL was produced in engineered E. coli BL21 star (DE3). 24 Hyaluronic acid (HA) is a glucuronic acidderived polysaccharide discovered in many animal tissues, which has remarkable applications in pharmaceutical, biomedical, and cosmetic products with high commercial value. 25 Largescale production of HA has focused on direct extraction from animal tissues and the use of bacterial expression systems in Streptococci.…”

Section: Saccharidesmentioning

confidence: 99%

Glycolysis and Its Metabolic Engineering Applications

Wang¹,

Yan²

2017

Engineering Microbial Metabolism for Chemical Synthesis

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IntroductionMicroorganisms play a pivotal role in the degradation of complex carbon sources in nature and could survive in different conditions due to the genetically coined cellular metabolism. Cellular metabolism of microorg anisms is defined as the sum of biochemical processes that involves the conversion of environmental nutrients into simple biosynthetic building blocks and energy in the cells. Within these metabolic processes, catabo lism is responsible for breakdown of complex molecules into simple molecules, producing energy, reducing power, and precursor intermedi ates for other metabolic processes like anabolism. The central catabolic pathways include Embden-Meyerhof-Parnas (EMP) pathway, pentose phosphate (PP) pathway, Entner-Doudoroff (ED) pathway, and the tricar boxylic acid (TCA) cycle. The EMP pathway, also known as glycolysis

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Enhanced production of 2′-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of lactose metabolism and fucosyltransferase

Cited by 89 publications

References 31 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

Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains

Glycolysis and Its Metabolic Engineering Applications

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