Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis

2021 ◽  
Author(s):  
Panting Liu ◽  
Bo Wu ◽  
Mao Chen ◽  
Yonghua Dai ◽  
Chao Song ◽  
...  
Keyword(s):  
Cell Wall ◽  
Zymomonas Mobilis ◽  
Consolidated Bioprocessing ◽  
Single Gene ◽  
Antibiotic Sensitivity ◽  
Potential Candidate ◽  
Inhibitor Tolerance ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan ◽  
Extracellular Secretion

Zymomonas mobilis ( Z. mobilis ) is a potential candidate for consolidated bioprocessing (CBP) strain in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion affected by cell wall peptidoglycan is also not well understood. Here we constructed several Penicillin Binding Proteins (PBPs)-deficient strains derivated from Z. mobilis S192 to perturb the cell wall peptidoglycan network and investigated the effects of peptidoglycan on the endoglucanase secretion. Results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBPs mutant strains, notably, △1089/0959 (4.09-fold) and △0959 (5.76-fold) in comparison to parent strains. Besides, for PBPs-deficient strains, the growth performance was not significantly inhibited but with enhanced antibiotic sensitivity and reduced inhibitor tolerance, otherwise, cell morphology was altered obviously. The concentration of intracellular soluble peptidoglycan was increased, especially for single gene deletion. Outer membrane permeability of PBPs-deficient strains was also improved, notably, △1089/0959 (1.14-fold) and △0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our finding indicated that PBPs-deficient Z. mobilis is capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness, and also acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacterias. Herein, we perturb the peptidoglycan synthesis network via knocking out PBPs ( ZMO0197 , ZMO0959 , ZMO1089 ) in order to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study can not only lay the foundation for understanding the regulatory network of cell wall synthesis but also provide guidance for the construction of CBP strains in Z. mobilis .

Targeting Bacterial Cell Wall Peptidoglycan Synthesis by Inhibition of Glycosyltransferase Activity

2015 ◽  
Vol 87 (2) ◽  
pp. 190-199 ◽  
Author(s):  
Michael F. Mesleh ◽  
Premraj Rajaratnam ◽  
Mary Conrad ◽  
Vasu Chandrasekaran ◽  
Christopher M. Liu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan

The Site of Inhibition of Bacterial Cell Wall Peptidoglycan Synthesis by Azureomycin B, a New Antibiotic1

1980 ◽  
Vol 88 (2) ◽  
pp. 565-570 ◽  
Author(s):  
Priska SPIRI-NAKAGAWA ◽  
Ruiko ÖIWA ◽  
Yoshitake TANAKA ◽  
Haruo TANAKA ◽  
Satoshi ÖMURA
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan

scholarly journals Boosting Secretion of Extracellular Protein byEscherichia colivia Cell Wall Perturbation

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Haiquan Yang ◽  
Xiao Lu ◽  
Jinyuan Hu ◽  
Yuan Chen ◽  
Wei Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Protein Secretion ◽  
Extracellular Protein ◽  
Deletion Mutants ◽  
Cell Factory ◽  
Content Type ◽  
E Coli ◽  
Cell Wall Peptidoglycan ◽  
Extracellular Secretion

ABSTRACTEscherichia coliis one of the most widely used host microorganisms for recombinant protein expression and metabolic engineering, but it cannot efficiently secrete recombinant proteins to extracellular space. Here, extracellular protein secretion was enhanced inE. coliby deleting twod,d-carboxypeptidase genes (dacAanddacB, single and double deletions) to perturb the cell wall peptidoglycan network. Deletion ofdacAanddacBenhanced the accumulation of intracellular soluble peptidoglycan inE. coliand affected cell morphology, resulting in a more irregular cell shape and the appearance of transparent bulges. Deletion ofdacAanddacBappears to disrupt the normal rigid structure, presumably due to perturbation and destruction of the cell wall peptidoglycan network. The extracellular green fluorescent protein (GFP) fluorescence intensity of deletion mutants was increased by >2.0-fold compared with that of control cells, and that of the double deletion mutant was increased by 2.7-fold. Extracellular recombinant fibroblast growth factor receptor 2 (FGFR2) and collagen E4 secretion in deletion mutants was also enhanced compared with that in the control cells. Additionally, the extracellular recombinant amylase activity of single-deletion mutants BL21 ΔdacApETDuet-amykand BL21 ΔdacBpETDuet-amykwas increased 2.5- and 3.1-fold, respectively. The extracellular distribution of α-galactosidase by deletion mutants was also increased by >2.0-fold. Deletion ofdacAanddacBincreased outer membrane permeability, which could explain the enhanced extracellular protein secretion.IMPORTANCECell surface structure stabilization is important for extracellular secretion of proteins inEscherichia coli. As the main constituent of the cell wall, peptidoglycan contributes to cell structure robustness and stability. Here, we perturbed the peptidoglycan network by deletingdacAanddacBgenes encodingd,d-carboxypeptidase enzymes to improve extracellular protein secretion. This new strategy could enhance the capacity ofE. colias a microbial cell factory for extracellular secretion of proteins and chemicals.

scholarly journals Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis

2010 ◽  
Vol 76 (19) ◽  
pp. 6360-6369 ◽  
Author(s):  
Jeffrey G. Linger ◽  
William S. Adney ◽  
Al Darzins
Keyword(s):  
Heterologous Expression ◽  
Zymomonas Mobilis ◽  
Consolidated Bioprocessing ◽  
Biofuel Production ◽  
Cell Protein ◽  
Cellulolytic Enzymes ◽  
Simultaneous Production ◽  
Extracellular Secretion ◽  
Different Levels ◽  
Biomass To Ethanol

ABSTRACT Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.

Inhibition of peptidoglycan synthesis of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT by the antibacterial agent dodecyl glycerol

1984 ◽  
Vol 4 (8) ◽  
pp. 659-664 ◽  
Author(s):  
H. S. Ved ◽  
Evan Gustow ◽  
Ronald A. Pieringer
Keyword(s):  
Cell Wall ◽  
Antibacterial Activity ◽  
Streptococcus Mutans ◽  
Mode Of Action ◽  
Antibacterial Agent ◽  
Metabolic Regulation ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan ◽  
Streptococcus Faecium

Dodecyl glycerol inhibits the synthesis of the peptidoglycans of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT. This metabolic regulation represents the second known mode by which dodecyl glycerol expresses antibacterial activity. The first mode of action of dodecyl glycerol was shown to stimulate autolysin activity which degrades cell-wall peptidoglycan (Ved HS, Gustow E, Mahadevan V and Pieringer RA, 1984, J. Biol. Chem.259, 8115–8121).

Properties of cell wall peptidoglycan synthesized by amino acid deprived relA mutants of Escherichia coli

1984 ◽  
Vol 30 (10) ◽  
pp. 1239-1246 ◽  
Author(s):  
Désirée Vanderwel ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Amino Acid ◽  
Free Form ◽  
Amino Acid Deprivation ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan ◽  
Covalently Linked

Cell wall peptidoglycan synthesis in Escherichia coli is under stringent control. During amino acid deprivation, peptidoglycan synthesis is inhibited in re1A+ bacteria but not in re1A mutants. The relaxed synthesis of peptidoglycan by amino acid deprived re1A bacteria was inhibited by Several β-lactam antibiotics at concentrations which inhibited cell elongation in growing cultures suggesting that the transpeptidase activity of penicillin-binding protein (PBP-1B) was involved in this process. Structural studies on the peptidoglycan also indicated the involvement of transpeptidation in relaxed peptidoglycan synthesis. The peptidoglycan synthesized during amino acid deprivation was cross-linked to the existing cell wall peptidoglycan, and the degree of cross-linkage was the same as that of peptidoglycan synthesized by growing control cells. The relaxed synthesis of peptidoglycan was also inhibited by moenomycin, an inhibitor of the in vitro transglycosylase activities of PBPs, but the interpretation of this result depends on whether the transglycosylases are the sole targets of moenomycin in vivo. Most of the peptidoglycan lipoprotein synthesized by histidine-deprived re1A+ bacteria was in the free form as previously reported, possibly because of the restriction in peptidoglycan synthesis. In support of this proposal, most of the lipoprotein synthesized during histidine deprivation of re1A mutants was found to be covalently linked to peptidoglycan. Nevertheless, the peptidoglycan synthesized by amino acid deprived re1A bacteria was apparently deficient in bound lipoprotein as compared with peptidoglycan synthesized by normal growing control bacteria suggesting that the rate of lipoprotein synthesis during amino acid deprivation may be limiting.

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