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Enzymology and Protein Engineering

Boosting Secretion of Extracellular Protein by Escherichia coli via Cell Wall Perturbation

Haiquan Yang, Xiao Lu, Jinyuan Hu, Yuan Chen, Wei Shen, Long Liu
Isaac Cann, Editor
Haiquan Yang
aThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China
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Xiao Lu
aThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China
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Jinyuan Hu
aThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China
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Yuan Chen
aThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China
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Wei Shen
aThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China
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Long Liu
aThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China
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Isaac Cann
University of Illinois at Urbana-Champaign
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DOI: 10.1128/AEM.01382-18
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  • FIG 1
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    FIG 1

    E. coli peptidoglycan synthesis and d,d-carboxypeptidase gene deletion. (A) The peptidoglycan biosynthesis pathway of E. coli. UDP-GlcNAc, UDP-N-acetylglucosamine; UDP-MurNAc, UDP-N-acetylmuramate; MurA, UDP-N-acetylglucosamine 1-carboxyvinyltransferase; MurB, UDP-N-acetylmuramate dehydrogenase; MurC, UDP-N-acetylmuramate-alanine ligase; MurD, UDP-N-acetylmuramoylalanine–d-glutamate ligase; MurE, UDP-N-acetylmuramoyl-l-alanyl–d-glutamate-2,6-diaminopimelate ligase; MurF, UDP-N-acetylmuramoyl-tripeptide-d-alanyl–d-alanine ligase; MraY, phospho-N-acetylmuramoyl-pentapeptide-transferase; MurG, UDP-N-acetylglucosamine–N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase; MtgA, monofunctional glycosyltransferase; ClassA PBP, penicillin-binding protein 1A; ClassA/B PBP, penicillin-binding protein 1A and penicillin-binding protein 2; DD-CPase, d,d-carboxypeptidases. (B) Nascent peptidoglycan chain synthesis with DD-CPase in the periplasm. During peptidoglycan synthesis, d,d-carboxypeptidases delete the C-terminal d-Ala from the majority of peptidoglycan precursor pentapeptide stem molecules. (C) Deletion of DD-CPase genes dacA and dacB (detailed gene deletion methods and data are included in the supplemental material). 1, BL21 ΔdacA ΔdacB; 2, BL21 ΔdacA ΔdacB::kan; 3, BL21 ΔdacB; and M, standard molecular weight markers.

  • FIG 2
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    FIG 2

    Effects of dacA and dacB deletion on cell growth. (A) Dry cell weight (DCW). Asterisks indicate a significant difference compared with control cells (none, P > 0.05; *, P < 0.05; and **, P < 0.01). (B) Specific growth rate, also known as relative growth rate (RGR), exponential growth rate, and continuous growth rate. RGR was calculated using the following equation from Hoffmann and Poorter (43): RGR = (lnW2 − lnW1)/(t2 − t1) where ln = natural logarithm, t1 = time 1 (e.g., in days), t2 = time 2 (e.g., in days), W1 = size at time 1, and W2 = size at time 2. Control, wild-type E. coli; ΔdacA, BL21 ΔdacA; ΔdacB, BL21 ΔdacB; and ΔdacA ΔdacB, BL21 ΔdacA ΔdacB.

  • FIG 3
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    FIG 3

    Analysis of glucosamine concentration. The y axis represents the glucosamine concentration of soluble peptidoglycan to indicate changes in intracellular soluble peptidoglycan concentration in different stains. Control, BL21-pETDuet; ΔdacA, BL21 ΔdacA pETDuet; ΔdacB, BL21 ΔdacB pETDuet; ΔdacA ΔdacB, BL21 ΔdacA ΔdacB pETDuet. Asterisks indicate a significant difference compared with control cells (none, P > 0.05; *, P < 0.05; and **, P < 0.01).

  • FIG 4
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    FIG 4

    Effects of dacA and dacB deletion on cell morphology. (A through D) Dot plot of cells using forward-scattered light (x axis) and side-scattered light (y axis). (E through H) Numbers of cells (y axis) according to the amount of forward-scattered light (x axis). (I through L) Effect of dacA and dacB deletion on cell morphology assessed by transmission electron microscopy (TEM). (A, E, and I) BL21-pETDuet (control cells). (B, F, and J) BL21 ΔdacA pETDuet. (C, G, and K) BL21 ΔdacB pETDuet. (D, H, and L) BL21 ΔdacA ΔdacB pETDuet.

  • FIG 5
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    FIG 5

    Effects of dacA and dacB deletion on extracellular recombinant green fluorescent protein (GFP) secretion. (A) Extracellular specific fluorescence intensity. AU, arbitrary units. Asterisks indicate a significant difference compared with control cells (none, P > 0.05; *, P < 0.05; and **, P < 0.01). (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. M, standard molecular weight markers; arrow, GFP; control, E. coli BL21-pETDUET-gfp (extracellular); ΔdacA, BL21 ΔdacA pETDuet-gfp; ΔdacB, BL21 ΔdacB pETDuet-gfp; ΔdacA ΔdacB, BL21 ΔdacA ΔdacB pETDuet-gfp; P-control, E. coli BL21-pETDuet-gfp (positive control, intracellular); N-control, E. coli BL21-pETDuet (negative control, intracellular).

  • FIG 6
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    FIG 6

    Effects of dacA and dacB deletion on extracellular recombinant amylase secretion. (A) Specific activity of extracellular amylase. Asterisks indicate a significant difference compared with control cells (none, P > 0.05; *, P < 0.05; and **, P < 0.01). (B) SDS-PAGE analysis of extracellular amylase. M, standard molecular weight markers; arrow, amylase; control, E. coli BL21-pETDuet-amy (extracellular); P-control, E. coli BL21-pETDuet-amy (positive control, intracellular); N-control, E. coli BL21-pETDuet (negative control, intracellular). (C) Extracellular amylase production rate. (D) Percentage of extracellular activity of total amylase activity. ΔdacA, BL21 ΔdacA pETDuet-amy; ΔdacB, BL21 ΔdacB pETDuet-amy; ΔdacA ΔdacB, BL21 ΔdacA ΔdacB pETDuet-amy.

  • FIG 7
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    FIG 7

    Effects of dacA and dacB deletion on extracellular α-galactosidase activity and outer membrane permeability. (A) Effects of dacA and dacB deletion on extracellular α-galactosidase activity. (B) Effects of dacA and dacB deletion on outer membrane permeability. Control, BL21-pETDuet; ΔdacA, BL21 ΔdacA pETDuet; ΔdacB, BL21 ΔdacB pETDuet; ΔdacA ΔdacB, BL21 ΔdacA ΔdacB pETDuet. Asterisks indicate a significant difference compared with control cells (none, P > 0.05; *, P < 0.05; and **, P < 0.01).

Tables

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  • Additional Files
  • TABLE 1

    Plasmids and strains used in this work

    Plasmid or strainRelevant genotype and/or characteristic(s)Source or reference
    Plasmids
        pMD19-T vectorTA cloningTaKaRa
        pKD13R6KY ori, Kanr, Ampr, CmrCGSC
        pKD46Ampr, helper plasmidCGSC
        pCP20Ampr, Cmr, helper plasmidCGSC
        pETDuetT7 promoters, pBR322 ori, AmprNovagen
        pET28aT7 promoters, pBR322 ori, KanrNovagen
        pETDuet-gfppETDuet derivate with gfp clonedThis work
        pET28a-amypET28a derivate with amy clonedThis work
    Gene knockout cassettes
        ΔdacA::kanKanr, knockout of gene dacAThis work
        ΔdacA::kan′Kanr, knockout of gene dacA of BL21 ΔdacB to obtain double deletion mutantThis work
        ΔdacB::kanKanr, knockout of gene dacBThis work
    Strains
        E. coli JM109Cloning hostNovagen
        E. coli BL21Wild-type E. coli BL21(DE3)Novagen
        BL21-pETDuetE. coli BL21 with plasmid pETDuetThis work
        BL21-pETDuet-gfpE. coli BL21 with plasmid pETDuet-gfpThis work
        BL21-pETDuet-amyE. coli BL21 with plasmid pETDuet-amyThis work
        BL21-pKD46E. coli BL21(DE3) derivate, including plasmid pKD46, AmprThis work
        BL21 ΔdacA::kan pKD46BL21-pKD46 derivate, deleting dacAThis work
        BL21 ΔdacB::kan pKD46BL21-pKD46 derivate, deleting dacBThis work
        BL21 ΔdacA ΔdacB::kan pKD46BL21 ΔdacB pKD46 derivate, deleting ΔdacA ΔdacBThis work
        BL21 ΔdacA::kanBL21 ΔdacA::kan pKD46 derivate, deleting plasmid pKD46This work
        BL21 ΔdacB::kanBL21 ΔdacB::kan pKD46 derivate, deleting plasmid pKD46This work
        BL21 ΔdacA ΔdacB::kanBL21 ΔdacA ΔdacB::kan pKD46 derivate, deleting plasmid pKD46This work
        BL21 ΔdacABL21 ΔdacA::kan derivate, deleting plasmid pKD46 and KanrThis work
        BL21 ΔdacBBL21 ΔdacB::kan derivate, deleting plasmid pKD46 and KanrThis work
        BL21 ΔdacA ΔdacBBL21 ΔdacA ΔdacB::kan derivate, deleting plasmid pKD46 and KanrThis work
        BL21 ΔdacA pETDuetBL21 ΔdacA derivate with plasmid pETDuetThis work
        BL21 ΔdacB pETDuetBL21 ΔdacB derivate with plasmid pETDuetThis work
        BL21 ΔdacA ΔdacB pETDuetBL21 ΔdacA ΔdacB derivate with plasmid pETDuetThis work
        BL21 ΔdacA pETDuet-gfpBL21 ΔdacA derivate with plasmid pETDuet-gfpThis work
        BL21 ΔdacB pETDuet-gfpBL21 ΔdacB derivate with plasmid pETDuet-gfpThis work
        BL21 ΔdacA pETDuet-amyBL21 ΔdacA derivate with plasmid pETDuet-amyThis work
        BL21 ΔdacB pETDuet-amyBL21 ΔdacB derivate with plasmid pETDuet-amyThis work
        BL21 ΔdacA ΔdacB pETDuet-gfpBL21 ΔdacA ΔdacB derivate with plasmid pETDuet-gfpThis work
        BL21 ΔdacA ΔdacB pETDuet-amyBL21 ΔdacA ΔdacB derivate with plasmid pETDuet-amyThis work
  • TABLE 2

    Nucleotide sequences of primers

    Oligonucleotide primer or purposeaSequence (5′ to 3′)b
    Plasmid construction
        GFP-FWCGGAATTCATGAGTAAAGGAGAAGAACTTTTC
        GFP-RVGAAGATCTTTATTTGTATAGTTCATCCATGC
        Amy-FWCCGGAATTCATGAGCGAGCTGCCGCAAATC
        Amy-RVCCGCTCGAGTTAAAAACCGCCATTGAAGGACG
    Gene knockout
        ΔdacA-FWGGCTCTTTGCACAGCCTTTATCTCTGCTGCACATGCCGATGACCTGAATAGTGTAGGCTGGAGCTGCTTC
        ΔdacA-RVCGAAGAAGTTACCTTCCGGGATTTCTTGCAGTACAACCAACGGGCGTTGTATTCCGGGGATCCGTCGACC
        ΔdacB-FWGATTACCACAGTCAGCAGATGGCGCAGCCCGCCAGTACGCAGAAAGTGATGTGTAGGCTGGAGCTGCTTC
        ΔdacB-RVCATCCACGCCCGCCTGATGCAGACCTGCACGGTACTGCAAAGAGCCGTCAATTCCGGGGATCCGTCGACC
        ΔdacA′-1-FWcGCTCTTTGCACAGCCTTTATCT
        ΔdacA′-1-RVGAAGCAGCTCCAGCCTACACGAGGAACATCAGCGAAGAACC
        ΔdacA′-2-FWGGTTCTTCGCTGATGTTCCTCGTGTAGGCTGGAGCTGCTTC
        ΔdacA′-2-RVCAGTCGCAGAAGCAACAAGGATTCCGGGGATCCGTCGACC
        ΔdacA′-3-FWGGTCGACGGATCCCCGGAATCCTTGTTGCTTCTGCGACTG
        ΔdacA′-3-RVGTTACCTTCCGGGATTTCTTG
    • ↵a FW, forward primer; RV, reverse primer.

    • ↵b Italicized letters represent the restriction enzyme sites. Underlined letters represent homologous sequences used for gene knockout.

    • ↵c Primers ΔdacA′-1 through ΔdacA′-3 were used to delete dacA of BL21 ΔdacB, and were redesigned in order to improve the efficiency of deletion. ΔdacA′-1, ΔdacA′-3, and ΔdacA′-2 were used to amplify forward and reverse homologous sequences and the Kanr gene.

Additional Files

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  • Supplemental material

    • Supplemental file 1 -

      Supplemental text (CaCl2 method for preparation of competent E. coli cells, deletion of d,d-carboxypeptidase genes, electroporation transformation, determination of extracellular protein concentration, SDS-PAGE to determine effects of dacA and dacB deletion on extracellular recombinant FGFR2 and collagen E4 secretion, amino acid sequence of E4); DNA electrophoresis (Fig. S1); extracellular protein concentrations of recombinant strains (Table S1); extracellular recombinant amylase specific activities (Table S2); effects of dacA and dacB deletion on extracellular recombinant FGFR2 and E4 secretion (Fig. S2).

      PDF, 949K

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Boosting Secretion of Extracellular Protein by Escherichia coli via Cell Wall Perturbation
Haiquan Yang, Xiao Lu, Jinyuan Hu, Yuan Chen, Wei Shen, Long Liu
Applied and Environmental Microbiology Oct 2018, 84 (20) e01382-18; DOI: 10.1128/AEM.01382-18

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Boosting Secretion of Extracellular Protein by Escherichia coli via Cell Wall Perturbation
Haiquan Yang, Xiao Lu, Jinyuan Hu, Yuan Chen, Wei Shen, Long Liu
Applied and Environmental Microbiology Oct 2018, 84 (20) e01382-18; DOI: 10.1128/AEM.01382-18
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KEYWORDS

d,d-carboxypeptidase
gene deletion
extracellular secretion
cell wall
Escherichia coli

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