A phenotypic screening bioassay for Escherichia coli stress and antibiotic responses based on Fourier-transform infrared (FTIR) spectroscopy and multivariate analysis.

AIMS: To develop and optimize a Fourier-transform infrared spectroscopy (FTIRS) phenotypic screening bioassay for stress responses, regarding the effect of nutrient content, bacterial growth phase and stress agent exposure time. METHODS AND RESULTS: A high-throughput FTIRS bioassay was developed to distinguish the stress responses of Escherichia coli to sodium hydroxide, hydrochloric acid, sodium chloride, sodium hypochlorite and ethanol. Principal component analysis and hierarchical clustering were used to quantify the effect of each parameter on bioassay performance, namely its reproducibility and metabolic resolution. Bioassay performance varied greatly, ranging from poor to very good. Spectra were partitioned into biologically relevant regions to evaluate their contributions to bioassay performance, but further improvements were not observed. Bioassay optimization was validated against empirical parameters, which confirmed a closer representation of known mechanisms on the antibiotic-induced stress responses. CONCLUSIONS: The optimized bioassay used standard nutrient content, cells in the late-stationary growth phase and a one-shift exposure duration. Only the optimized bioassay adequately and reproducibly distinguished the E. coli stress and antibiotic responses. The absence of performance improvements using partitioned spectra indicated that stress responses are imprinted on the whole-spectra metabolic signature. SIGNIFICANCE AND IMPACT OF THE STUDY: Highly optimized FTIRS bioassay parameters are vital in capturing whole-spectra metabolic signatures that can be used for satisfactory and reproducible phenotypic screening of stress and antibiotic responses.

Use of chemometrics in the selection of a Saccharomyces cerevisiae expression system for recombinant cyprosin B production.

Two multivariate statistical methods, factor analysis (FA) and hierarchical cluster analysis (HCA), were applied to experimental data set to evaluate their usefulness in selecting the adequate expression system and optimal growth parameters for recombinant cyprosin B production. Using FA, the large data set was reduced to two factors representing 73.4% of variability. Factor 1, with 53.5% of variability, corresponds to recombinant cyprosin B expression and efficient secretion, while factor 2, accounting for 19.9% of variability, represents cell growth and physiological characteristics. FA and HCA allowed the establishment of correlations among different variables and the clusters obtained providing clear identification of the experimental parameters related to cyprosin B production, which results on more accurate scientific output and time saving when selection of an adequate expression system is concerned.

Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography.

During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.

Prediction of retention time of cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan-proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Phitagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE<0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE<0.022) for the two tagged cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose-2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of cutinases tagged with hydrophobic peptides in HIC.

Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoring.

A bi-enzymatic micro-analytical bioreactor integrated in a FIA system for glucose measurements is described. Its robustness and small dimensions (working volume of about 70 microl containing approximately 1.2 mg GO and 0.26 mg HRP) make it easy to operate. The column is based on immobilisation of glucose oxidase (GO) and horseradish peroxidase (HRP) on alkylamine controlled pore glass (CPG) beads. The column has excellent shelf life (no significant loss of activity after 1 year if kept at 4 degrees C), and a very high operational stability that was demonstrated through extensive usage for glucose determinations over 1 year period during which the column retained almost all of its activity. More importantly, this operational stability allows glucose monitoring in the culture media without a decay of signal over the experiment time and consequently no signal correction or re-calibration is needed. This high operational stability was also confirmed by continuous glucose conversion with 30% activity loss after converting quantity of glucose equivalent to 21600 FIA injections of 20 microl with 1.7 mM glucose. Such good performance is a result of an optimised immobilisation method and moreover of the implementation of in situ enzyme stabilisation strategy which consisted on promoting the instantaneous H2O2 consumption produced by the GO. This strategy has the additional advantage of allowing concomitant assay of the H2O2 based on the HAP catalysed co-oxidation of phenol-4-sulphonic acid (PSA) in the presence of 4-aminoantipyrine (4-AAP). The glucose measurements are reproducible with high precision against the standard HPLC method. Linear range and sensitivity depend on sample injection volume; the upper limit is about 1.1 g/l. Lower detection limit is 10mg/l. The column performance has been validated for E. coli and S. cerevisiae fermentation monitoring, and glucose measurements in an animal cell culture (rat Langerhans islets).

Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects.

Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.

Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins.

Genetic engineering was integrated with the production and purification of Fusarium solani pisi cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were cutinase wild type and two fusion proteins of cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)(2) and (WP)(4). The (WP)(4) fusion protein enabled a 300-fold increase of the cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the cutinase-(WP)(2) proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)(4) fusion proteins, respectively.

Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae.

The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.