Engineering <i>Escherichia coli</i> for <scp>d</scp>-Allulose Production from <scp>d</scp>-Fructose by Fermentation

Guo, Qiang; Zheng, Lingjie; Luo, Xuan; Gao, Xin-Quan; Liu, Chenyang; Deng, Li; Fan, Li; Zheng, Huidong

doi:10.1021/acs.jafc.1c05200

Cited by 20 publications

(29 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our achievements in this study perfectly overcome the D-fructose conversion bottleneck existing in fermentative production of D-allulose, and suggest a breakthrough in application of phosphorylation-dephosphorylation strategy in cell factories. Currently, the conversion ratio of D-fructose using Izumoring-based cell factories was only 19.6% ( Guo et al, 2021 ). In contrast, the new synthetic route can completely consume D-fructose, which may facilitate the subsequent separation of D-allulose from culture broth.…”

Section: Resultsmentioning

confidence: 99%

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation

Guo¹,

Liu²,

Zheng³

et al. 2022

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

show abstract

Section: Resultsmentioning

confidence: 99%

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation

Guo¹,

Liu²,

Zheng³

et al. 2022

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…26 The conversion ratio was the D-glucose consumed/the total D-glucose added, and the yield (g/g) was the allitol produced/the D-glucose consumed. 27 2.9. Molasses Pretreatment.…”

Section: Methodsmentioning

confidence: 99%

“…The mobile phase was fresh ultrapure water at a flow rate of 0.5 mL/min . The conversion ratio was the d -glucose consumed/the total d -glucose added, and the yield (g/g) was the allitol produced/the d -glucose consumed …”

Section: Methodsmentioning

confidence: 99%

Reconstruction of a Cofactor Self-Sufficient Whole-Cell Biocatalyst System for Efficient Biosynthesis of Allitol from d-Glucose

Zhao

Guo

et al. 2022

J. Agric. Food Chem.

View full text Add to dashboard Cite

The combined catalysis of glucose isomerase (GI), d-psicose 3-epimerase (DPEase), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) provides a convenient route for the biosynthesis of allitol from d-glucose; however, the low catalytic efficiency restricts its industrial applications. Here, the supplementation of 0.32 g/L NAD+ significantly promoted the cell catalytic activity by 1.18-fold, suggesting that the insufficient intracellular NAD(H) content was a limiting factor in allitol production. Glucose dehydrogenase (GDH) with 18.13-fold higher activity than FDH was used for reconstructing a cofactor self-sufficient system, which was combined with the overexpression of the rate-limiting genes involved in NAD+ salvage metabolic flow to expand the available intracellular NAD(H) pool. Then, the multienzyme self-assembly system with SpyTag and SpyCatcher effectively channeled intermediates, leading to an 81.1% increase in allitol titer to 15.03 g/L from 25 g/L d-glucose. This study provided a facilitated strategy for large-scale and efficient biosynthesis of allitol from a low-cost substrate.

show abstract

“…Consequently, biological production has progressively replaced other methods as the primary way to produce D-allulose [ 16 ]. It is worth our attention that in recent years, many studies have been focused on enzyme-catalyzed and microbiological methods for synthesis of D-allulose based on the Izumoring strategy [ 17 ]. The Izumoring strategy is an effective method proposed in 2006 for the bioproduction of various kinds of rare hexoses [ 18 ] and also involves D-allulose 3-epimerase (DAE), D-tagatose 3-epimerase (DTE), polyol dehydrogenases, and aldose isomerases [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

The Characterization of a Novel D-allulose 3-Epimerase from Blautia produca and Its Application in D-allulose Production

Tang

Iqbal

et al. 2022

Foods

View full text Add to dashboard Cite

D-allulose is a natural rare sugar with important physiological properties that is used in food, health care items, and even the pharmaceutical industry. In the current study, a novel D-allulose 3-epimerase gene (Bp-DAE) from the probiotic strain Blautia produca was discovered for the production and characterization of an enzyme known as Bp-DAE that can epimerize D-fructose into D-allulose. Bp-DAE was strictly dependent on metals (Mn2+ and Co2+), and the addition of 1 mM of Mn2+ could enhance the half-life of Bp-DAE at 55 °C from 60 to 180 min. It exhibited optimal activity in a pH of 8 and 55 °C, and the Km values of Bp-DAE for the different substrates D-fructose and D-allulose were 235.7 and 150.7 mM, respectively. Bp-DAE was used for the transformation from 500 g/L D-fructose to 150 g/L D-allulose and exhibited a 30% of conversion yield during biotransformation. Furthermore, it was possible to employ the food-grade microbial species Bacillus subtilis for the production of D-allulose using a technique of whole-cell catalysis to circumvent the laborious process of enzyme purification and to obtain a more stable biocatalyst. This method also yields a 30% conversion yield.

show abstract

Engineering Escherichia coli for d-Allulose Production from d-Fructose by Fermentation

Cited by 20 publications

References 46 publications

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation

Reconstruction of a Cofactor Self-Sufficient Whole-Cell Biocatalyst System for Efficient Biosynthesis of Allitol from d-Glucose

The Characterization of a Novel D-allulose 3-Epimerase from Blautia produca and Its Application in D-allulose Production

Contact Info

Product

Resources

About