Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP), which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ∼400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1∼OM3) were identified with improved tolerance via H₂O₂ enrichment selection. The best mutant OM3 could grow in 12 mM H₂O₂ with the growth rate of 0.6 h⁻¹, whereas the growth of wild type was completely inhibited at this H₂O₂ concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS), which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H₂O₂ treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold), but also RpoS- and OxyR-regulated genes (up to 7.7-fold). qRT-PCR data and enzyme activity assay suggested that catalase (katE) could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.

Proteins, particularly enzymes, are often used in bioreactors to catalyze biosynthetic reactions. However, one of the major challenges in applying proteins in bioreactors is enzyme stability and recovery. This review is an enlightening discussion on recent trends and advancements used to alleviate these limitations completely or to minimize them. Two major strategies, polymer-based enzyme immobilization and polymeric enzyme nanoreactors have been discussed systematically. Further, this review puts light on various methods such as smart polymer-based supports which have been exploited for their ability to regulate ligand-protein interactions. In addition, chemical moieties like protein-based microcrystals, cross-linked enzyme aggregates, and polymer-based nanoreactors have also been discussed in a comprehensive way focusing more on their unique applicability and target-specific actions. Polymer-based nanoreactors are described in detail in vivo together with enhancement of the enzyme stability and controlled function based on the compartmentalization of enzymes. Among the two novel nanoreactor approaches, dendrimers have been exploited as multifunctional enzymatic carriers, while capsomes are designed to regulate poly-enzymatic reactions through intravesicular compartmentalization of a multitude of enzymatic reactions.