BackgroundAcid stress is often encountered during industrial fermentation as a result of the accumulation of acidic metabolites. Acid stress increases the intracellular acidity and can cause DNA damage and denaturation of essential enzymes, thus leading to a decrease of growth and fermentation yields. Although acid stress can be relieved by addition of a base to the medium, fermentations with acid-tolerant strains are generally considered much more efficient and cost-effective.ResultsIn this study, the global regulator H-NS was found to have significant influence on the acid tolerance of E. coli. The final OD600 of strains overexpressing H-NS increased by 24% compared to control, when cultured for 24 h at pH 4.5 using HCl as an acid agent. To further improve the acid tolerance, a library of H-NS was constructed by error-prone PCR and subjected to selection. Five mutants that conferred a significant growth advantage compared to the control strain were obtained. The final OD600 of strains harboring the five H-NS mutants was enhanced by 26–53%, and their survival rate was increased by 10- to 100-fold at pH 2.5. Further investigation showed that the improved acid tolerance of H-NS mutants coincides with the activation of multiple acid resistance mechanisms, in particular the glutamate- and glutamine-dependent acid resistance system (AR2). The improved acid tolerance of H-NS mutants was also demonstrated in media acidified by acetic acid and succinic acid, which are common acidic fermentation by-products or products.ConclusionsThe results obtained in this work demonstrate that the engineering of H-NS can enhance the acid tolerance of E. coli. More in general, this study shows the potential of the engineering of global regulators acting as repressors, such as H-NS, as a promising method to obtain phenotypes of interest. This approach could expand the spectrum of application of global transcription machinery engineering.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0966-z) contains supplementary material, which is available to authorized users.

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self-assembling peptides and a C-terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self-assembling peptide. Upon simple separation, the target protein or peptide with an authentic N-terminus is then released in the solution by intein-mediated cleavage. Different combinations of four self-assembling peptides (ELK16, L KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI-CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI-CM, which has pH-shift inducible cleavage, was found to work well with three self-assembling peptides (L KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx-strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N-terminus, which is especially effective for peptides of 30-100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.

ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems.