High-Titer <i>De Novo</i> Biosynthesis of the Predominant Human Milk Oligosaccharide 2′-Fucosyllactose from Sucrose in <i>Escherichia coli</i>

Parschat, Katja; Schreiber, Sandra; Wartenberg, Dirk; Engels, Benedikt; Jennewein, Stefan

doi:10.1021/acssynbio.0c00304

Cited by 38 publications

(57 citation statements)

References 52 publications

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“…The de novo pathway utilizes a low-cost carbon source, such as glucose and sucrose, instead of fucose; it is more economical and industrially applicable. Escherichia coli , Saccharomyces cerevisiae , and Corynebacterium glutamicum have been engineered to produce 2′-FL using the de novo pathway [ 16 – 18 ]. Besides the abovementioned microorganisms, Bacillus subtilis has also been selected as a host for producing functional nutraceuticals due to its generally recognized as safe (GRAS) status [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

Engineered Bacillus subtilis for the de novo production of 2′-fucosyllactose

Zhang

Liu²,

Xia³

et al. 2022

Microb Cell Fact

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Background The most abundant human milk oligosaccharide in breast milk, 2′-fucosyllactose (2′-FL), has been approved as an additive to infant formula due to its multifarious nutraceutical and pharmaceutical functions in promoting neonate health. However, the low efficiency of de novo synthesis limits the cost-efficient bioproduction of 2′-FL. Results This study achieved 2′-FL de novo synthesis in a generally recognized as safe (GRAS) strain Bacillus subtilis. First, a de novo biosynthetic pathway for 2′-FL was introduced by expressing the manB, manC, gmd, wcaG, and futC genes from Escherichia coli and Helicobacter pylori in B. subtilis, resulting in 2′-FL production of 1.12 g/L. Subsequently, a 2′-FL titer of 2.57 g/L was obtained by reducing the competitive lactose consumption, increasing the regeneration of the cofactor guanosine-5′-triphosphate (GTP), and enhancing the supply of the precursor mannose-6-phosphate (M6P). By replacing the native promoter of endogenous manA gene (encoding M6P isomerase) with a constitutive promoter P7, the 2′-FL titer in shake flask reached 18.27 g/L. The finally engineered strain BS21 could produce 88.3 g/L 2′-FL with a yield of 0.61 g/g lactose in a 3-L bioreactor, without the addition of antibiotics and chemical inducers. Conclusions The efficient de novo synthesis of 2′-FL can be achieved by the engineered B. subtilis, paving the way for the large-scale bioproduction of 2′-FL titer in the future.

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Section: Introductionmentioning

confidence: 99%

Engineered Bacillus subtilis for the de novo production of 2′-fucosyllactose

Zhang

Liu²,

Xia³

et al. 2022

Microb Cell Fact

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“…Currently, the biosynthetic pathway constructing for 2′‐FL in host bacteria such as E. coli (Parschat et al ., 2020 ), Bacillus subtilis (Deng et al ., 2019 ), Saccharomyces cerevisiae and Yarrowia lipolytica (Hollands et al ., 2019 ) has become a research hotspot. Most researches were conducted on E. coli due to the simplicity of the culture conditions and the availability of genetic tools.…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering strategies of de novo pathway for enhancing 2′‐fucosyllactose synthesis in Escherichia coli

et al. 2021

Microbial Biotechnology

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Summary 2′‐Fucosyllactose (2′‐FL), one of the most abundant human milk oligosaccharides (HMOs), is used as a promising infant formula ingredient owing to its multiple health benefits for newborns. However, limited availability and high‐cost preparation have restricted its extensive use and intensive research on its potential functions. In this work, a powerful Escherichia coli cell factory was developed to ulteriorly increase 2′‐FL production. Initially, a modular pathway engineering was strengthened to balance the synthesis pathway through different plasmid combinations with a resulting maximum 2′‐FL titre of 1.45 g l −1 . To further facilitate the metabolic flux from GDP‐ l ‐fucose towards 2′‐FL, the CRISPR‐Cas9 system was utilized to inactivate the genes including lacZ and wcaJ , increasing the titre by 6.59‐fold. Notably, the co‐introduction of NADPH and GTP regeneration pathways was confirmed to be more conducive to 2′‐FL formation, achieving a 2′‐FL titre of 2.24 g l −1 . Moreover, comparisons of various exogenous α 1,2‐fucosyltransferase candidates revealed that futC from Helicobacter pylori generated the highest titre of 2′‐FL. Finally, the viability of scaled‐up production of 2′‐FL was evidenced in a 3 l bioreactor with a maximum titre of 22.3 g l −1 2′‐FL and a yield of 0.53 mole 2′‐FL mole −1 lactose.

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“…Since polyhydroxybutyrate (PHB) can increase cell density, introducing the heterologous synthesis pathway of PHB into the E. coli S17−3 can further increase 2′-FL production to 1.03 g/L. Parschat et al 72 used sucrose as the sole carbon source to reduce production costs. This method deleted the lacZ, gcd, glK, ptsG, and pf KA genes to overexpress the de novo UDP-Dgalactose and GDP-L-fucose pathways, and used sugar efflux transporters to promote the export of 2′-FL.…”

Section: Hmos Production Using Microbial Systemsmentioning

confidence: 99%

Engineered Microbial Routes for Human Milk Oligosaccharides Synthesis

Mosleh

Abbaspourrad

2021

ACS Synth. Biol.

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Human milk oligosaccharides (HMOs) are one of the important ingredients in human milk, which have attracted great interest due to their beneficial effect on the health of newborns. The large-scale production of HMOs has been researched using engineered microbial routes due to the availability, safety, and low cost of host strains. In addition, the development of molecular biology technology and metabolic engineering has promoted the effectiveness of HMOs production. According to current reports, 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), and some fucosylated HMOs with complex structures have been produced via the engineered microbial route, with 2′-FL having been produced the most. However, due to the uncertainty of metabolic patterns, the selection of host strains has certain limitations. Aside from that, the expression of appropriate glycosyltransferase in microbes is key to the synthesis of different HMOs. Therefore, finding a safe and efficient glycosyltransferase has to be addressed when using engineered microbial pathways. In this review, the latest research on the production of HMOs using engineered microbial routes is reported. The selection of host strains and adapting different metabolic pathways helped researchers designing engineered microbial routes that are more conducive to HMOs production.

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High-Titer De Novo Biosynthesis of the Predominant Human Milk Oligosaccharide 2′-Fucosyllactose from Sucrose in Escherichia coli

Cited by 38 publications

References 52 publications

Engineered Bacillus subtilis for the de novo production of 2′-fucosyllactose

Engineered Bacillus subtilis for the de novo production of 2′-fucosyllactose

Metabolic engineering strategies of de novo pathway for enhancing 2′‐fucosyllactose synthesis in Escherichia coli

Engineered Microbial Routes for Human Milk Oligosaccharides Synthesis

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