Strategies for efficient extracellular secretion of recombinant cyclomaltodextrinase by Escherichia coli

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

“…The capacity of ΔycjM-ΔmalS-ΔlpxM and ΔycjM-ΔmalS to generate the enzyme was identical. 19 Recombinant MTSase expression levels in E1-E4 were lower than that in E5, whereas recombinant CDase expression levels in E1-E4 and E6 were almost the same. Under the same fermentation conditions, the expression level of MTSase at the second multiple cloning site in the coexpression vector was lower compared with the single multiple cloning site plasmid pETmtsase, possibly because the recombinant MTSase expression was affected by recombinant CDase overexpression.…”

“…Previously, plasmid pET28a was used to construct the vectors pET‐ cdase and pET‐ mtsase and transferred into the host E. coli BL21(DE3)/ ∆ycjM ‐ ∆malS to generate the recombinant strains E5 and E6, respectively. The capacity of ∆ycjM ‐ ∆malS ‐ ∆lpxM and ∆ycjM ‐ ∆malS to generate the enzyme was identical 19 . Recombinant MTSase expression levels in E1–E4 were lower than that in E5, whereas recombinant CDase expression levels in E1–E4 and E6 were almost the same.…”

“…Genes cdase (GenBank ID: X62576) and mtsase (GenBank ID: D63343) were synthesized by GENEWIZ Biotechnology Co. Ltd (Suzhou, China), and ligated into plasmids pRSFDuet‐1, pCOLADuet‐1, pACYCDuet‐1, and pCDFDuet‐1 through homologous recombination to generate pRSF‐ cdase ‐ mtsase , pCOLA‐ cdase ‐ mtsase , pACYC‐ cdase ‐ mtsase , and pCDF‐ cdase ‐ mtsase . A triple mutant of E. coli BL21(DE3)/ ∆ycjM ‐ ∆malS ‐ ∆lpxM was used as host that was completed in a previous study using CRISPR/Cas9 19 . All successfully constructed plasmids were transformed into ∆ycjM ‐ ∆malS ‐ ∆lpxM to generate the recombinant strains E1–E4.…”

“…Antibiotics were added at certain final concentrations (kanamycin: 50 μg mL −1 , chloramphenicol: 25 μg mL −1 , and streptomycin: 50 μg mL −1 ). The culture was inoculated with modified TB medium 19 (peptone: 6 g L −1 , yeast extract: 12 g L −1 , K 2 HPO 4 .3H 2 O: 1.64 g L −1 , KH 2 PO 4 : 0.23 g L −1 and glycerol: 0.2 g L −1 ) and cultured at 37 °C. When cell growth reached the late logarithmic period, 0.5 mM isopropyl β ‐ d ‐1‐thiogalactopyranoside (IPTG) was added and further cultured at 25 °C for 20 h. The cells were collected after 20 min of centrifugation at 8000 × g and suspended in 20 mM phosphate buffer (pH 7.0).…”

“…Although cell membrane permeabilization through chemical or physical treatment is efficient on a small scale, its large‐scale implementation is challenging; instead, molecular engineering is recently being favored as an alternative 18 . This study proposes that deletion of genes associated with cell membrane lipopolysaccharide (LPS) biosynthesis ( lpxM ) and glycine‐Ca 2+ supplementation increases the extracellular release of recombinant CDase 19 . Deletion of lpxM is beneficial for increasing the permeability of cell membrane.…”