The Construction of an In Vitro Synthetic Enzymatic Biosystem that Facilitates Laminaribiose Biosynthesis from Maltodextrin and Glucose

Sun, Shangshang; Wei, Xinlei; You, Chun

doi:10.1002/biot.201800493

Cited by 29 publications

(21 citation statements)

References 39 publications

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“…Orthogonal two‐enzyme phosphorylase cascades have been reported for the synthesis of disaccharides (e.g., cellobiose, α,α‐trehalose, laminaribiose, kojibiose, nigerose), oligo‐, and polysaccharides (e.g., β‐glucans, amylose). Sucrose or starch was the donor substrate to fuel the phosphate/αGlc 1‐ P shuttle .…”

Section: Introductionmentioning

confidence: 99%

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Zhong

Nidetzky

2019

Biotechnology Journal

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Cellodextrins are linear β‐1,4‐gluco‐oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP‐controlled, bottom‐up synthesis from expedient substrates is desired for their bulk production. Here, a three‐enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short‐chain (soluble) cellodextrins (DP range 3–6). The cascade reaction involves iterative β‐1,4‐glucosylation of glucose from α‐glucose 1‐phosphate (αGlc1‐P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1‐P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L−1 is obtained under near‐complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)−1. Through a simple two‐step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.

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Section: Introductionmentioning

confidence: 99%

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Zhong

Nidetzky

2019

Biotechnology Journal

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“…These data indicated that Mdc had the ability to catalyze OAA to MSA and the specific activity achieved 9.37 μmol/min/g ( Figure 2D ). In vitro multi-enzyme system has emerged as a promising bio-manufacturing platform for producing desired products ( Sun et al, 2019 ). The artificial biosynthetic pathway, including only two enzymes, may be feasible to produce malonic acid in vitro in the future.…”

Section: Resultsmentioning

confidence: 99%

Designing and Constructing a Novel Artificial Pathway for Malonic Acid Production Biologically

Zhi-qiang

Yao

et al. 2022

Front. Bioeng. Biotechnol.

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Malonic acid is used as a common component of many products and processes in the pharmaceutical and cosmetic industries. Here, we designed a novel artificial synthetic pathway of malonic acid, in which oxaloacetate, an intermediate of cytoplasmic reductive tricarboxylic acid (rTCA) pathway, is converted to malonic semialdehyde and then to malonic acid, sequentially catalyzed by a-keto decarboxylase and malonic semialdehyde dehydrogenase. After the systematic screening, we discovered the enzyme oxaloacetate decarboxylase Mdc, catalyzing the first step of the artificially designed pathway in vitro. Then, this synthetic pathway was functionally constructed in cellulolytic thermophilic fungus Myceliophthora thermophila. After enhancement of glucose uptake, the titer of malonic acid achieved 42.5 mg/L. This study presents a novel biological pathway for producing malonic acid from renewable resources in the future.

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“…Due to the rational and semiquantitative principle of promoter and RBS strength (strong/medium/weak) used in plasmid construction, we believe that the results of this study have excellent replication potential when applied to other enzyme cascades in different fields of bio-catalysis. Besides the well-known oxidoreductase systems that require regeneration of nicotinamide coenzymes and/or flavin cofactors, [1][2][3][4][5]9,10,20] two-enzyme cascades of glycoside phosphorylases similar to the one used here, [67][68][69] or cascades of two nucleoside phosphorylases, [21,22,70] are gaining increased importance. Two-enzyme cascades involving sugar nucleotide-dependent glycosyltransferases are emerging systems for synthesis.…”

Section: Resultsmentioning

confidence: 99%

Plasmid Design for Tunable Two‐Enzyme Co‐Expression Promotes Whole‐Cell Production of Cellobiose

Schwaiger

Voit

Dobiášová

et al. 2020

Biotechnology Journal

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Catalyst development for biochemical cascade reactions often follows a “whole‐cell‐approach” in which a single microbial cell is made to express all required enzyme activities. Although attractive in principle, the approach can encounter limitations when efficient overall flux necessitates precise balancing between activities. This study shows an effective integration of major design strategies from synthetic biology to a coherent development of plasmid vectors, enabling tunable two‐enzyme co‐expression in E. coli, for whole‐cell‐production of cellobiose. An efficient transformation of sucrose and glucose into cellobiose by a parallel (countercurrent) cascade of disaccharide phosphorylases requires the enzyme co‐expression to cope with large differences in specific activity of cellobiose phosphorylase (14 U mg−1) and sucrose phosphorylase (122 U mg−1). Mono‐ and bicistronic co‐expression strategies controlling transcription, transcription‐translation coupling or plasmid replication are analyzed for effect on activity and stable producibility of the whole‐cell‐catalyst. A key role of bom (basis of mobility) for plasmid stability dependent on the ori is reported and the importance of RBS (ribosome binding site) strength is demonstrated. Whole cell catalysts show high specific rates (460 µmol cellobiose min−1 g−1 dry cells) and performance metrics (30 g L−1; ∼82% yield; 3.8 g L−1 h−1 overall productivity) promising for cellobiose production.

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The Construction of an In Vitro Synthetic Enzymatic Biosystem that Facilitates Laminaribiose Biosynthesis from Maltodextrin and Glucose

Cited by 29 publications

References 39 publications

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Designing and Constructing a Novel Artificial Pathway for Malonic Acid Production Biologically

Plasmid Design for Tunable Two‐Enzyme Co‐Expression Promotes Whole‐Cell Production of Cellobiose

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