Mutations in Peptidoglycan Synthesis Gene ponA Improve Electrotransformation Efficiency of Corynebacterium glutamicum ATCC 13869

Abstract

Corynebacterium glutamicum is frequently engineered to serve as a versatile platform and model microorganism. However, due to its complex cell wall structure, transformation of C. glutamicum with exogenous DNA is inefficient. Although efforts have been devoted to improve the transformation efficiency by using cell wall-weakening agents, direct genetic engineering of cell wall synthesis for enhancing cell competency has not been explored thus far. Herein, we reported that engineering of peptidoglycan synthesis could significantly increase the transformation efficiency of C. glutamicum Comparative analysis of C. glutamicum wild-type strain ATCC 13869 and a mutant with high electrotransformation efficiency revealed nine mutations in eight cell wall synthesis-related genes. Among them, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA dramatically increased the electrotransformation of strain ATCC 13869 by 19.25-fold in the absence of cell wall-weakening agents, with no inhibition on growth. The Y489C mutation had no effect on the membrane localization of PonA but affected the peptidoglycan structure. Deletion of the ponA gene led to more dramatic changes to the peptidoglycan structure but only increased the electrotransformation by 4.89-fold, suggesting that appropriate inhibition of cell wall synthesis benefited electrotransformation more. Finally, we demonstrated that the PonA^Y489C mutation did not cause constitutive or enhanced glutamate excretion, making its permanent existence in C. glutamicum ATCC 13869 acceptable. This study demonstrates that genetic engineering of genes involved in cell wall synthesis, especially peptidoglycan synthesis, is a promising strategy to improve the electrotransformation efficiency of C. glutamicumIMPORTANCE Metabolic engineering and synthetic biology are now the key enabling technologies for manipulating microorganisms to suit the practical outcomes desired by humankind. The introduction of exogenous DNA into cells is an indispensable step for this purpose. However, some microorganisms, including the important industrial workhorse Corynebacterium glutamicum, possess a complex cell wall structure to shield cells against exogenous DNA. Although genes responsible for cell wall synthesis in C. glutamicum are known, engineering of related genes to improve cell competency has not been explored yet. In this study, we demonstrate that mutations in cell wall synthesis genes can significantly improve the electrotransformation efficiency of C. glutamicum Notably, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA increased electrotransformation efficiency by 19.25-fold by affecting peptidoglycan synthesis.

Keywords: Corynebacterium glutamicum; electrotransformation; peptidoglycan; ponA.

FIG 1

Electrotransformation efficiencies of the ATCC 13869 wild-type (WT) strain and mutants. Competent cells were prepared using LBG medium without cell wall-weakening agents. The number of cells used for each electrotransformation was approximately 10⁸. Error bars indicate standard deviations from the results from three parallel experiments. Asterisks indicate significant changes in electrotransformation efficiency based on a comparison between the mutants and the wild-type strain. **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s two-tailed t test).

FIG 2

Effects of Y489C point mutation and deletion of ponA on electrotransformation efficiency. (A) Function of PonA in transglycosylation and transpeptidation of cell wall synthesis. (B) Electrotransformation efficiency and susceptibility to electroporation of the wild-type (WT), ponA^Y489C, and ΔponA strains in the absence of cell wall-weakening agents. Competent cells were prepared using LBG medium without cell wall-weakening agents. The cells used for each electrotransformation were approximately 10⁸. After electroporation and recovery, cells were spread on selective LBG plates supplemented with 25 µg/ml kanamycin to determine the electrotransformation efficiency and LBG plates without kanamycin to determine the susceptibility to electroporation. (C) Growth curves of wild-type (WT), ponA^Y489C, and ΔponA strains. Cells were cultured in LBG medium with an initial OD₆₀₀ value of 0.2. The cell density was determined every 3 hours. (D) Electrotransformation efficiency of wild-type (WT), ponA^Y489C, and ΔponA strains in the presence of cell wall-weakening agents. Competent cells were prepared using NCM medium supplemented with glycine, threonine, INH and Tween 80 according to the protocol described previously (12). Error bars indicate standard deviations from three parallel experiments. Asterisks indicate significant changes in electrotransformation efficiency and susceptibility to electroporation based on a comparison between the mutants and the wild-type strain. **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s two-tailed t test). NS indicates nonsignificant change between the wild-type strain and the mutant based on Student’s two-tailed t test (P > 0.05).

FIG 3

Membrane localization of GFP-PonA and GFP-PonA^Y489C fusion proteins in the ΔponA mutant. Microscope images under visible light and fluorescence are shown. White arrows point out the typical membrane localization of GFP-PonA and GFP-PonA^Y489C fusion proteins. Bars represent 5 μm. Fluorescence was excited at 480 nm, and the emission was monitored at 527 nm.

FIG 4

Vancomycin fluorescence (Van-FL) staining and penicillin susceptibility of the WT, ponA^Y489C mutant, and ΔponA mutant strains. (A) Microscope images of Van-FL-stained cells under fluorescence. Bars represent 5 μm. (B) Fluorescence intensities of Van-FL stained cells. When the OD₆₀₀ of the culture reached 1.0, Van-FL was added to the culture with a final concentration of 100 µg/ml. The mixture was incubated for another 30 min at 30°C. After washing 3 times with PBS buffer, the Van-FL-stained cells were used for fluorescence microscope observation (λ excitation = 480 nm, λ emission = 527 nm) and fluorescence intensity determination by using a fluorimeter (λ excitation = 490 nm, λ emission = 520 nm). Error bars indicate standard deviations from the results from three parallel experiments. Asterisks indicate significant changes in fluorescence intensity based on a comparison between the mutants and the wild-type strain. ***, P ≤ 0.001 (Student’s two-tailed t test). (C) MICs of penicillin and tetracycline for different strains. Three parallel experiments were conducted, and the same MIC was obtained. (D) Determination of penicillin MIC using M.I.C.Evaluator strips (256 to 0.015 μg/ml). One of the three parallel experiments is shown here.

FIG 5

Glutamate fermentation by the WT strain and the ponA^Y489C mutant. (A) Glutamate fermentation in shake flasks. Strains were cultivated in biotin-rich or biotin-poor fermentation medium supplemented with 80 g/liter glucose at 30°C and 220 rpm. OD₆₀₀, glucose consumption, and glutamate production were detected after 24 h of fermentation. Error bars indicate standard deviations from the results from three parallel experiments. NS indicates a nonsignificant change based on Student’s two-tailed t test (P > 0.05). (B) Glutamate fermentation in 5-liter bioreactors. Strains were cultivated in biotin-poor fermentation medium supplemented with 140 g/liter glucose. Samples were picked periodically, and the OD₆₀₀, glucose consumption, and glutamate production were detected. The data shown are the average and standard deviations of the results from three parallel determinations.