Metabolic engineering of Escherichia coli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement

Huang, Di; Yang, Kang; Liu, Jia; Xu, Yingying; Wang, Yuanyuan; Wang, Ru; Liu, Bin; Feng, Lu

doi:10.1016/j.ymben.2017.03.001

Cited by 120 publications

(175 citation statements)

References 71 publications

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“…Throughout all the optimization steps, we have accomplished 3‐FL production and specific yield with 4.6 g/L (extracellular) and 0.51 g/g DCW. Specific yield of 3‐FL production in this study (0.51 g/g DCW) is higher than that of other studies using the de novo pathway (Huang et al, ; Jung et al, ; Table ). However, when we consider the low conversion yield and the productivity of our study, further studies are still needed to improve dry cell mass (9.0 g/L) and 3‐FL titer (4.6 g/L).…”

Section: Resultscontrasting

confidence: 67%

“…Further fed‐batch fermentation studies should be done to confirm the conclusion in the future. On the other hand, despite the highest conversion yield of Huang et al (), direct comparison is difficult because enough information of the run was not provided in the paper.…”

Section: Resultsmentioning

confidence: 98%

“…The chemical synthesis of HMOs may have disadvantages due to complex pathways and possible toxicity. Several studies in fucosylated HMO synthesis showed that bio‐production using a microorganism is a better alternative synthesis process than the direct purification from human milk or chemical synthesis of HMOs (Baumgartner, Seitz, Sprenger, & Albermann, ; Chin, Seo, Kim, & Seo, ; Dumon, Samain, & Priem, ; Huang et al, ; Lin, Yuan, Li, & Lin, ). 20.28 g/L of 2′‐FL was produced in E. coli JM109 by chromosomally overexpressing de novo pathway genes (ManC, ManB, Gmd, WcaG) and FutC.…”

Section: Introductionmentioning

confidence: 99%

“…For instance, Dumon et al () was able to produce 500 mg/L of 3‐FL by overexpressing the α‐1,3‐fucosyltransferase (FutA) gene in E. coli JM107. Since then, there had not been any progress in increasing the production titer of 3‐FL to gram scale until the recent report by Huang et al Huang et al () recently reported producing 12.43 g/L of 3‐FL under batch culture via the de novo pathway through metabolic engineering of E. coli BL21(DE3).…”

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of the human milk oligosaccharide 3‐fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β‐galactosidase modification

Choi

Park

Seo

et al. 2019

Biotech & Bioengineering

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3-Fucosyllactose (3-FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2′-fucosyllactose (2′-FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function.Whereas the biological production of 2′-FL has been widely studied and made significant progress over the years, the biological production of 3-FL has been hampered by the low activity and insoluble expression of α-1,3-fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2′-FL, and lower digestibility of 3-FL than 2′-FL by bifidobacteria.In this study, we report the gram-scale production of 3-FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild-type FutA owing to the increase in k cat and the decrease in K m . We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L-fucokinase/GDP-L-fucose pyrophosphorylase to increase the supply of GDP-fucose in the cytoplasm. To increase the mass of recombinant whole-cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZΔm15) for better utilization of D-lactose. Production using the lacZΔm15 mutant yielded 3-FL concentration of 4.6 g/L with the productivity of 0.076 g·L −1 ·hr −1 and the specific 3-FL yield of 0.5 g/g dry cell weight. K E Y W O R D S fucosyllactose, fucosyltransferase, human milk, oligosaccharide, metabolic engineering *Yun Hee Choi and Bum Seok Park contributed equally to this study.

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Section: Resultscontrasting

confidence: 67%

Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of the human milk oligosaccharide 3‐fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β‐galactosidase modification

Choi

Park

Seo

et al. 2019

Biotech & Bioengineering

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“…При этом в зависимости от применяемого фермента получали фукозилирован-ные или сиалированные олигосахара [32]. Также было описано производство олигосахаридов грудного молока с использованием рекомбинантных бактерий Escherichia coli, полученных методами биологической инженерии [33]. При этом был выявлен дозозависимый эффект на рост бифидобактерий, аналогичный таковому в иссле-дованиях с применением комплекса галакто-и фрукто-олигосахаридов [34].…”

Section: олигосахариды в детских молочных смесяхunclassified

Reconsidered Concept of «Prebiotics» and Their Impact on the Child’s Organism

Лукоянова¹,

Боровик²,

Суржик³

2017

Vopr. sovr. pediatr.

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Simultaneous production of 2′‐fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency

Lee

Shin

Han

et al. 2022

Biotechnology Journal

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Background and aim Difucosyllactose (Di‐FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus, identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di‐FL as well as 2′‐fucosyllactose (2′‐FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5′‐diphosphate (GDP)‐l‐fucose biosynthetic pathway. Main methods and major results The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l‐fucokinase/GDP‐l‐fucose phosphorylase (FKP) from Bacteroides fragilis and α‐1,2‐fucosyltransferase (FucT2) from Helicobacter pylori. To decrease the intracellular accumulation of 2′‐FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the ΔLFAR‐YA/FF+P‐PLA2 strain expressing phospholipase A2 (PLA2) from Streptomyces violaceoruber, whose native signal peptide was replaced with the PelB signal peptide (P‐PLA2), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di‐FL from lactose and fucose. As a result, a batch fermentation with the ΔLFAR‐YA/FF+P‐PLA2 strain resulted in the production of 1.22 ± 0.01 g L−1 Di‐FL and 0.47 ± 0.01 g L−1 2′‐FL, whereas the control strain could only produce 0.65 ± 0.01 g L−1 2′‐FL. Conclusions and implications This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.

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Metabolic engineering of Escherichia coli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement

Cited by 120 publications

References 71 publications

Biosynthesis of the human milk oligosaccharide 3‐fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β‐galactosidase modification

Biosynthesis of the human milk oligosaccharide 3‐fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β‐galactosidase modification

Reconsidered Concept of «Prebiotics» and Their Impact on the Child’s Organism

Simultaneous production of 2′‐fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency

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