Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

Baumgärtner, Florian; Sprenger, Georg A.; Albermann, Christoph

doi:10.1016/j.enzmictec.2015.04.009

Cited by 47 publications

(58 citation statements)

References 41 publications

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“…For fairly simple HMO structures, fermentation of engineered Escherichia coli appears to be a viable strategy. For instance, HMOs including 2′-fucosyllactose (2′FL), 3FL, LNT, and LNnT have been produced by engineered E. coli with yields generally around 10 g/L [7,[45][46][47]. However, for more complex structures such as LNFPs yields are generally much lower and only LNFP I and LNFP III have been reported as fermentation products with yields below 1 g/L [48,49].…”

Section: Discussionmentioning

confidence: 99%

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides

Zeuner

Vuillemin

Holck

et al. 2018

Enzyme and Microbial Technology

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The α-1,3/4-l-fucosidases (EC 3.2.1.111; GH29) BbAfcB from Bifidobacterium bifidum and CpAfc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto-N-fucopentaose II (LNFP II) through regioselective transfucosylation of lacto-N-tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering BbAfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of BbAfcB with the corresponding 17-aminoacid α-helical loop of CpAfc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT BbAfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4-l-fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ± 2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT BbAfcB (14 ± 0.3%) and comparable to that obtained with CpAfc2 (50 ± 8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto-N-neotetraose (LNnT) could be enriched with LNFP III.

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Section: Discussionmentioning

confidence: 99%

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides

Zeuner

Vuillemin

Holck

et al. 2018

Enzyme and Microbial Technology

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“…The resulting strain produced lacto-N-tetraose at a concentration of 0.219 g/L from a mixture of glucose and lactose, and the concentration further increased up to 0.810 g/L when a mixture of lactose and galactose was used. 50,51 Using fed-batch fermentation with galactose as the sole carbon source, lacto-N-tetraose at a concentration of 12.7 g/L was produced by the engineered E. coli. 51 These results suggest that metabolic engineering can also be used to make longer oligosaccharide backbones.…”

Section: Temporary Alternative: Use Of Nonhuman Oligosaccharidesmentioning

confidence: 99%

“…50,51 Using fed-batch fermentation with galactose as the sole carbon source, lacto-N-tetraose at a concentration of 12.7 g/L was produced by the engineered E. coli. 51 These results suggest that metabolic engineering can also be used to make longer oligosaccharide backbones. Identification of precise transferases capable of adding fucose or sialic acid into the desired locations of the backbones will enable the production of complex HMOs by engineered strains.…”

Section: Temporary Alternative: Use Of Nonhuman Oligosaccharidesmentioning

confidence: 99%

Overcoming the limited availability of human milk oligosaccharides: challenges and opportunities for research and application

et al. 2016

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Human milk oligosaccharides (HMOs) are complex sugars highly abundant in human milk but currently not present in infant formula. Rapidly accumulating evidence from in vitro and in vivo studies, combined with epidemiological associations and correlations, suggests that HMOs benefit infants through multiple mechanisms and in a variety of clinical contexts. Until recently, however, research on HMOs has been limited by an insufficient availability of HMOs. Most HMOs are found uniquely in human milk, and thus far it has been prohibitively tedious and expensive to isolate and synthesize them. This article reviews new strategies to overcome this lack of availability by generating HMOs through chemoenzymatic synthesis, microbial metabolic engineering, and isolation from human donor milk or dairy streams. Each approach has its advantages and comes with its own challenges, but combining the different methods and acknowledging their limitations creates new opportunities for research and application with the goal of improving maternal and infant health.

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“…Their metabolism process highly depends on the supply of the source of carbon and nitrogen. So, nutrition starvation or fed‐batch technique may force them to produce their favorite products, including amino acids , carbohydrate , and so on. In this paper, the E. coli were supplied with peptides and cystine (Cys) to investigate their metabolites of amino acid.…”

Section: Introductionmentioning

confidence: 99%

Separation and detection of amino acid metabolites of Escherichia coli in microbial fuel cell with CE

Wang

Lin

et al. 2016

Electrophoresis

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In this work, CE-LIF was employed to investigate the amino acid metabolites produced by Escherichia coli (E. coli) in microbial fuel cell (MFC). Two peptides, l-carnosine and l-alanyl-glycine, together with six amino acids, cystine, alanine, lysine, methionine, tyrosine, arginine were separated and detected in advance by a CE-LIF system coupled with a homemade spontaneous injection device. The injection device was devised to alleviate the effect of electrical discrimination for analytes during sample injection. All analytes could be completely separated within 8 min with detection limits of 20-300 nmol/L. Then this method was applied to analyze the substrate solution containing amino acid metabolites produced by E. coli. l-carnosine, l-alanyl-glycine, and cystine were used as the carbon, nitrogen, and sulfur source for the E. coli culture in the MFC to investigate the amino acid metabolites during metabolism. Two MFCs were used to compare the activity of metabolism of the bacteria. In the sample collected at the running time 200 h of MFC, the amino acid methionine was discovered as the metabolite with the concentrations 23.3 μg/L.

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Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

Cited by 47 publications

References 41 publications

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides

Overcoming the limited availability of human milk oligosaccharides: challenges and opportunities for research and application

Separation and detection of amino acid metabolites of Escherichia coli in microbial fuel cell with CE

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