Enhancing biosynthesis of 2'‐Fucosyllactose in <i>Escherichia coli</i> through engineering lactose operon for lactose transport and α ‐1,2‐Fucosyltransferase for solubility

Park, Bum Seok; Choi, Yun Hee; Kim, Min Woo; Park, Beom Gi; Kim, Eun-Jung; Kim, Jin Young; Kim, Jung Hwa; Kim, Byung‐Gee

doi:10.1002/bit.28048

Cited by 17 publications

(21 citation statements)

References 41 publications

(60 reference statements)

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“…Park et al improved the α1,2-fucosyltransferase solubility by protein engineering on the basis of Consensus Finder and introduced a beneficial quadruple-site variant to significantly enhance the 2′-FL biosynthesis, with the titer of 40 g/L and productivity of 0.55 g/L/h by fed-batch cultivation. 25 Parschat et al developed a synthetic route for utilization of sucrose to produce 2′-FL in E. coli and integrated all the synthetic genes into the genome. The generated engineered strain produced 64 g/L 2′-FL by fed-batch cultivation, with a yield of 0.18 mol/mol sucrose.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

Liu

Zhu

Wan

et al. 2022

J. Agric. Food Chem.

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2′-Fucosyllactose (2′-FL) is the most abundant oligosaccharide in human milk. In this study, a highly efficient biosynthetic route for 2′-FL production was designed via the de novo pathway of GDP-L-fucose using engineered Escherichia coli BL21(DE3). Specifically, plasmid-based strains with previously deleted lacZ and wcaJ were further reconstructed by introducing de novo pathway genes and α1,2-fucosyltransferase-encoding wbgL to realize 2′-FL synthesis. The 2′-FL titer was enhanced to 3.92 g/L by further introducing rcsA and rcsB. Subsequently, the additional wbgL expression cassette was chromosomally integrated into recA locus to strengthen fucosylation reaction and a strong constitutive promoter (P J23119 ) was used to replace the original promoters of manC-manB and gmd-wcaG to improve 2′-FL synthesis. The maximal 2′-FL titer reached 9.06 and 79.23 g/L in shake-flask and fedbatch cultivation, respectively. The 2′-FL productivity reached 1.45 g/L/h, showing remarkable production potential in large-scale industrial application.

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Section: ■ Results and Discussionmentioning

confidence: 99%

High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

Liu

Zhu

Wan

et al. 2022

J. Agric. Food Chem.

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“…When the lac operon was replaced with one bearing lacZ Δ M15 , approximately 3% β-galactosidase activity was still retained in the engineered BL21star(DE3) and lactose could be effectively consumed and converted to 2′-FL by the engineered strain . The replacement of lac operon containing lacZ Δ M15 was also successfully performed to enhance 2′-FL biosynthesis via the salvage GDP- l -fucose pathway in BL21star(DE3) and the de novo pathway in BL21(DE3) …”

Section: ′-Fl Biosynthesis By Metabolically Engineered Strainsmentioning

confidence: 99%

“…Park et al confirmed the toxicity of LacY overexpression with T7 promoter in a multicopy plasmid. When the native promoter P lac of the lac operon was switched to P tac , the catabolite repression by high glucose concentration could be effectively restrained and thus the engineered BK21 displayed enhanced 2′-FL production using glucose as a carbon source …”

Section: ′-Fl Biosynthesis By Metabolically Engineered Strainsmentioning

confidence: 99%

“…In addition, H. pylori FutC was engineered to improve solubility and activity based on the family multiple sequence analysis for conserved residue sequences and molecular docking for substrate binding analysis. A quadruple-site variant of FutC was determined to enhance 2′-FL titer by 2.4-fold …”

Section: ′-Fl Biosynthesis By Metabolically Engineered Strainsmentioning

confidence: 99%

“…92 The replacement of lac operon containing lacZΔM15 was also successfully performed to enhance 2′-FL biosynthesis via the salvage GDP-L-fucose pathway in BL21star(DE3) 95 and the de novo pathway in BL21(DE3). 96 4.1.1.2. Modulating lacY and lacI Expression.…”

Section: ′-Fl Biosynthesis By Metabolically Engineered Strainsmentioning

confidence: 99%

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Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide

Liu

Zhu

Wang³

et al. 2022

J. Agric. Food Chem.

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Human milk oligosaccharides (HMOs), a group of structurally diverse unconjugated glycans in breast milk, act as important prebiotics and have plenty of unique health effects for growing infants. 2′-Fucosyllactose (2′-FL) is the most abundant HMO, accounting for approximately 30%, among approximately 200 identified HMOs with different structures. 2′-FL can be enzymatically produced by α1,2-fucosyltransferase, using GDP-l-fucose as donor and lactose as acceptor. Metabolic engineering strategies have been widely used for enhancement of GDP-l-fucose supply and microbial production of 2′-FL with high productivity. GDP-l-fucose supply can be enhanced by two main pathways, including de novo and salvage pathways. 2′-FL-producing α1,2-fucosyltransferases have widely been identified from various microorganisms. Metabolic pathways for 2′-FL synthesis can be basically constructed by enhancing GDP-l-fucose supply and introducing α1,2-fucosyltransferase. Various strategies have been attempted to enhance 2′-FL production, such as acceptor enhancement, donor enhancement, and improvement of the functional expression of α1,2-fucosyltransferase. In this review, current progress in GDP-l-fucose synthesis and bacterial α1,2-fucosyltransferases is described in detail, various metabolic engineering strategies for enhancing 2′-FL production are comprehensively reviewed, and future research focuses in biotechnological production of 2′-FL are suggested.

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De novo 2′‐fucosyllactose bioproduction through modular engineering of a novel Escherichia coli K12‐derived strain

Zhang

Fan

et al. 2023

Food Bioengineering

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The most abundant human milk oligosaccharide 2′-fucosyllactose (2′-FL) is a valuable component that has gained significant attention from the food industry. To biosynthesize 2′-FL, various Escherichia coli K12 derivatives have been genetically modified. To further enhance the application performance of E. coli K12, a novel E. coli K12 derivative BL27 was used as a chassis cell in this study, and modular pathway enhancement was performed to achieve de novo synthesis of 2′-FL. The futC gene encoding α-1,2-fucosyltransferase was introduced, and the wcaJ gene was knocked out to prevent the conversion of GDP-L-fucose to colanic acid. Next, the effects of overexpressing transcriptional regulators rcsA and rcsB and knocking out transcriptional regulators mcbR and waaF were evaluated to optimize the colanic acid pathway. The expression level, solubility, and activity of FutC were improved through genomic integration, TrxA-tag fusion, and double mutation in F40S/Q237S. Fermentation conditions were optimized to achieve maximum 2′-FL titers of 3.86 and 23.56 g/L in shakeflask and fed-batch cultivation, respectively. Over 85% of the products were successfully excreted into extracellular and almost no byproduct 2′,3difucosyllactose was generated. This study has explored a new microbial platform and modification strategies for the synthesis of 2′-FL and provides opportunities for its commercial production.

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Enhancing biosynthesis of 2'‐Fucosyllactose in Escherichia coli through engineering lactose operon for lactose transport and α ‐1,2‐Fucosyltransferase for solubility

Cited by 17 publications

References 41 publications

High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide

De novo 2′‐fucosyllactose bioproduction through modular engineering of a novel Escherichia coli K12‐derived strain

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