Abstract
Cell lysis is crucial for the microbial production of industrial fatty acids, proteins, biofuels, and biopolymers. In this work, we developed a novel programmable lysis system based on the heterologous expression of lysozyme. The inducible lytic system was tested in two Gram-negative bacterial strains, namely Escherichia coli and Pseudomonas putida KT2440. Before induction, the lytic system did not significantly arrest essential physiological parameters in the recombinant E. coli (ECPi) and P. putida (JBOi) strain such as specific growth rate and biomass yield under standard growth conditions. A different scenario was observed in the recombinant JBOi strain when subjected to PHA-producing conditions, where biomass production was reduced by 25% but the mcl-PHA content was maintained at about 30% of the cell dry weight. Importantly, the genetic construct worked well under PHA-producing conditions (nitrogen-limiting phase), where more than 95% of the cell population presented membrane disruption 16 h post induction, with 75% of the total synthesized biopolymer recovered at the end of the fermentation period. In conclusion, this new lysis system circumvents traditional, costly mechanical and enzymatic cell-disrupting procedures.
Highlights
The microbial synthesis of valuable chemicals using renewable feedstocks has opened new avenues for creating a more sustainable society
To achieve cell lysis during cell growth, several research groups have constructed various genetic systems based on inducible promoters that result in the production of holin and endolysin proteins (HEPs)[16,17,18,19]
We constructed a genetic autolysis system in P. putida KT2440, a natural PHA-producing strain that can be activated at different phases of growth of the bacterial culture
Summary
The microbial synthesis of valuable chemicals using renewable feedstocks has opened new avenues for creating a more sustainable society. Taking advantage of this efficiency, we constructed an m-toluic acid-inducing system to trigger the expression of the gene encoding for the C-type lysozyme of Gallus gallus As this enzyme acts on the peptidoglycan layer, which is located in the periplasm of Gram-negative organisms, we sought a secretion system that would ensure the translocation of the produced recombinant lysozyme into the periplasmic space of the cell, resulting in membrane damage of the cell. A signal peptide (SP) described for a naturally secreted protein in Pseudomonas stutzeri was fused at the N-terminus of lysozyme This resulted in high-yield cell disruption and the recovery of most synthesized biopolymers at the end of the fermentation period under nitrogen-limiting conditions in batch cultures
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