KfoA, the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates

Zhu, Hao-Miao; Sun, Bin; Li, Yijun; Meng, Dan-Hua; Zheng, Shuang; Wang, Tingting; Wang, Fengshan; Sheng, Juzheng

doi:10.1007/s00253-017-8639-0

Cited by 16 publications

(9 citation statements)

References 51 publications

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“…E. coli K4 was selected to assemble the required elements for CS biosynthesis. This strain has been extensively studied for chondroitin production 24,[29][30][31] . However, K4 produces a fructosylated chondroitin as part of its capsular polysaccharide.…”

Section: Resultsmentioning

confidence: 99%

“…1a). Enzymes for the two remaining steps (UDP-N-acetylglucosamine-/UDP-glucosamine-4-epimerase and chondroitin synthase) have been identified in E. coli K4 (Serovar O5:K4:H4) 24 . Incidentally, other bacteria like Pseudomonas aeruginosa serotype O6 25 , Yersinia enterocolitica serotype O8 26 , Pasteurella multocida Type F 27 , and E. coli O86:B7 28 possessing either or both of these chondroitin-specific enzymes have also been characterized.…”

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confidence: 99%

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Complete biosynthesis of a sulfated chondroitin in Escherichia coli

et al. 2021

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Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Complete biosynthesis of a sulfated chondroitin in Escherichia coli

et al. 2021

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“…The genes responsible for capsular polysaccharide (CPS) biosynthesis and transport in E. coli K4 are clustered and organized in three regions; regions 1 and 3 are common to all group II E. coli strains, and encode genes involved in the export and assembly of CPS on the cell surface (Rigg, Barrett, & Roberts, ). The central part, region 2, is serotype specific, and contains an insertion sequence (IS2) as well as seven genes that are directly involved in activated precursors biosynthesis ( kfoA , kfo F), polymer assembly ( kfo C), and fructosylation ( kfo E) (Liu et al, ; Ninomiya et al, ; Zhu et al, ). The function of the remaining genes ( kfo B, kfo D, and kfo G) is still unknown.…”

Section: Introductionmentioning

confidence: 99%

Physiological characterization and quantitative proteomic analyses of metabolically engineered E. coli K4 strains with improved pathways for capsular polysaccharide biosynthesis

Cimini

Russo

D’ambrosio

et al. 2018

Biotech & Bioengineering

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Among capsulated bacteria, some produce polysaccharides with unique properties that have been shown to possess relevant industrial applications and commercial value. The capsular polysaccharide (CPS) produced by Escherichia coli K4 is similar to chondroitin sulphate, and recent efforts focused on the development of genetic and fermentation strategies to increase its production titers up to technologically attractive levels. However, the control of the metabolic pathways leading to CPS synthesis together with the effect of varying the concentration of pathway intermediates on CPS final titers, is still quite unexplored, and not fully understood. In the present study four genes involved in the biosynthesis of UDP-sugar CPS precursors, namely kfoA, kfoF, pgm, and galU, were overexpressed in different combinations, and diversely affected the biosynthetic machinery. At the physiological level, results revealed a central role for kfoF, coding for UDP-glucose dehydrogenase, that increased CPS production mostly. In the attempt to unravel the molecular mechanisms regulating CPS biosynthesis, an in depth analysis of the proteome of the recombinant strains overexpressing respectively pgm and galU, and pgm, galU, and kfoF was performed and compared to the wild-type. Although, interestingly, in both strains the impact of the genetic manipulation seemed rather limited at the proteome level, results obtained from the triple mutant indicated a crosstalk between the two pathways leading to UDP-sugar precursors biosynthesis, and also an unexpected link with the purine biosynthetic pathway. Overall our results present new insights into the role of metabolic intermediates for the formation of capsular polysaccharides, utilizing a systematic approach of metabolic engineering, combined with state-of-the-art quantitative proteomic approaches, as well as genetic and physiological information.

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“…GlcNGc, taken up into the cell through the phosphotransferase system (PTS), was phosphorylated by N -acetyl- d -glucosamine kinase ( NagK ) (Uehara and Park 2004 ) to afford GlcNGc-6-P. Next, phosphoglucosamine mutase ( GlmM ), responsible for the conversion of glucosamine-6‐phosphate to glucosamine‐1‐phosphate (Jolly et al 1999 ) catalyzed the conversion of GlcNGc-6-P to GlcNGc-1-P. Next, GlcNGc was converted by the bifunctional enzyme ( GlmU ) (Mengin-Lecreulx and van Heijenoort 1994 ), to UDP-GlcNGc. UDP-glucose 4-epimerase ( KfoA ) (Zhu et al 2018 ) catalyzed the conversion of UDP-GlcNGc to UDP-GalNGc. UDP-GalNGc and UDP-GlcA were then converted to Gc-CN through the action of a chondroitin polymerase, ( KfoC ) (Zanfardino et al 2010 ).…”

Section: Discussionmentioning

confidence: 99%

N-glycolyl chondroitin synthesis using metabolically engineered E. coli

et al. 2020

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N-glycolyl chondroitin (Gc-CN) is a metabolite of N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is commonly found in mammals, but not humans. Humans can incorporate exogenous Neu5Gc into their tissues from eating red meat. Neu5Gc cannot be biosynthesized by humans due to an evolutionary mutation and has been implicated in causing inflammation causing human diseases, such as cancer. The study Neu5Gc is important in evolutionary biology and the development of potential cancer biomarkers. Unfortunately, there are several limitations to detecting Neu5Gc. The elimination of Neu5Gc involves a degradative pathway leading to the incorporation of N-glycolyl groups into glycosaminoglycans (GAGs), such as Gc-CN. Gc-CN has been found in humans and in animals including mice, lamb and chimpanzees. Here, we present the biosynthesis of Gc-CN in bacteria by feeding chemically synthesized N-glycolylglucosamine to Escherichia coli. A metabolically engineered strain of E. coli K4, fed with glucose supplemented with GlcNGc, converted it to N-glycolylgalactosamine (GalNGc) that could then be utilized as a substrate in the chondroitin biosynthetic pathway. The final product, Gc-CN was converted to disaccharides using chondroitin lyase ABC and analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring detection. This analysis showed the incorporation of GalNGc into the backbone of the chondroitin oligosaccharide.

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KfoA, the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates

Cited by 16 publications

References 51 publications

Complete biosynthesis of a sulfated chondroitin in Escherichia coli

Complete biosynthesis of a sulfated chondroitin in Escherichia coli

Physiological characterization and quantitative proteomic analyses of metabolically engineered E. coli K4 strains with improved pathways for capsular polysaccharide biosynthesis

N-glycolyl chondroitin synthesis using metabolically engineered E. coli

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