Deletion of α-amylase genes via CRISPR/Cas9 decreases the side effects of hydrolysis towards nonreducing maltoheptaose preparation

Zheng, Luhua; Li, Mengli; Chen, Jingjing; Zhang, Tao

doi:10.1016/j.fbio.2022.101801

Cited by 4 publications

(3 citation statements)

References 32 publications

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“…Deletion of ycjM and malS decreased the hydrolysis of N‐G7 by host‐generated hydrolases and exhibited positive effects 25 . Gene ycjM codes for a sucrose phosphorylase that degrades carbohydrates 26 .…”

Section: Resultsmentioning

confidence: 99%

“…Conversely, when grown in LB medium, no significant difference in growth was observed between ΔycjM-ΔmalS and BL21 (DE3). 25 These results suggest that YcjM and MalS hydrolysis may play a crucial role in facilitating the provision of monosaccharides for cellular metabolism under more abundant carbon and nitrogen sources. However, no substantial effect on cell membrane permeability was observed.…”

Section: Effects Of Lpxm Deletion On Permeabilization Of Cellsmentioning

confidence: 92%

“…Deletion of ycjM and malS decreased the hydrolysis of N-G7 by host-generated hydrolases and exhibited positive effects. 25 Gene ycjM codes for a sucrose phosphorylase that degrades carbohydrates. 26 MalS is a periplasmic ⊍-amylase, which hydrolyzes ⊍-1,4-glucosidic bonds of amylose and long maltodextrins to release hexaose.…”

Section: Effects Of Lpxm Deletion On Permeabilization Of Cellsmentioning

confidence: 99%

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Permeabilized whole cells containing co‐expressed cyclomaltodextrinase and maltooligosyltrehalose synthase facilitate the synthesis of nonreducing maltoheptaose (N‐G7) from β‐cyclodextrin

et al. 2023

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BACKGROUNDMaltodextrin is an important bulk ingredient in food and other industries; however, drawbacks such as uneven polymerization and high reducibility limit its utilization. Nonreducing maltoheptaose (N‐G7) is a good substitute for maltodextrin owing to its single degree of polymerization and its nonreducing properties. In this study, in vitro cell factory biotransformation of β‐cyclodextrin (β‐CD) to N‐G7 is demonstrated using coexpressed cyclomaltodextrinase (CDase, EC 3.2.1.54) and maltooligosyltrehalose synthase (MTSase, EC 5.4.99.15). However, the cell membrane prevents β‐CD from entering the cell owing to its large diameter.RESULTSThe amylase‐deficient permeabilized host ΔycjM‐ΔmalS‐ΔlpxM is utilized for the coexpression of recombinant CDase and MTSase. Deletion of lpxM effectively allows the entry of β‐cyclodextrin into the cell, despite its large diameter, without requiring any relevant cell membrane permeability‐promoting reagent. This results in a 28.44% increase in the efficiency of β‐CD entry into the cell, thus enabling intracellular N‐G7 synthesis without the extracellular secretion of recombinant CDase and MTSase. After reacting for 5.5 h, the highest purity of N‐G7 (65.50%) is obtained. However, hydrolysis decreases the purity of N‐G7 to 49.30%, thus resulting in a conversion rate of 40.16% for N‐G7 when the reaction lasts 6 h. Precise control of reaction time is crucial for obtaining high‐purity N‐G7.CONCLUSIONWhole‐cell catalysis avoids cell fragmentation and facilitates the creation of an eco‐friendly, energy‐efficient biotransformation system; thus, it is a promising approach for N‐G7 synthesis. © 2023 Society of Chemical Industry.

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Section: Resultsmentioning

confidence: 99%

Section: Effects Of Lpxm Deletion On Permeabilization Of Cellsmentioning

confidence: 92%