Engineering osmolysis susceptibility in Cupriavidus necator and Escherichia coli for recovery of intracellular products

Abstract

Background Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. Results To overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed. Conclusions Our strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.

Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator

“…In previous studies, ALE has been applied to C. necator for utilizing formate, glycerol, and glucose, increasing tolerance to saline solutions and carbon monoxide . However, most bacteria inherently exhibit low spontaneous mutation rates, leading to long evolutionary cycles and inefficient adaptation.…”

Isolation and Characterization of aHalomonasSpecies for Non-Axenic Growth-Associated Production of Bio-Polyesters from Sustainable Feedstocks

“…Taking advantage of this phenomenon could simplify the purification of bioplastic and reduce or even entirely abolish the need for organic solvents. Mechano-sensitive genes are pivotal for imparting resistance to changes in ionic strength, as evidenced by studies demonstrating improved osmolysis when knocked out, even of non-halophilic microorganisms [67]. While such genes are also present in CUBES01 ( mscK or mscL ), they appear to be not preventing osmolysis susceptibility of the strain.…”

Fast-track adaptive laboratory evolution ofCupriavidus necatorH16 with divalent metal cations

“…Examination using STRING [33] indicated that YgcG is functionally associated with the membrane stress resistance protein YqcG in E. coli (Figure S3). Intriguingly, the same protein was mutated at the identical position in a recent ALE study, where C. necator H16 underwent evolution for enhanced halotolerance [5]. Commencing from a W253 genotype, the identified halotolerant variant carried a W253G mutation, thereby reversing W253 back to G253.…”

“…The applications of ALE on C. necator H16 are wide-ranging. Key examples include improving glycerol utilization [4], increasing halotolerance [5], improving growth on formate [6], and enhancing carbon monoxide tolerance [7].…”