Carles de Mas

Production of arabitol from glucose by Hansenula polymorpha.

Abstract Arabitol was produced from glucose by Hansenula polymorpha DSM 70277. Some of the factors influencing production of polyols were studied: temperature, aeration, osmotic pressure and nitrogen concentration. The best results (18.8 g/l of arabitol—no other polyol was detected—after 60 h of cultivation with a yield of 0.14 g · arabitol per g · glucose utilized) were obtained for a fermentation done in fed-batch mode, with a glucose concentration always less than 30 g/l, at 45°C, pH 4.8, stirring rate 1000 rpm and using air at 1 vvm in a 2l fermentor. Neither ethanol nor acetate were detected in this experiment.

Studies on papain action in the synthesis of Gly-Phe in two-liquid-phase media

Abstract The synthesis of the protected dipeptide BocGly-PheOMe has been carried out in an aqueous-organic two-liquid-phase system. The reaction has been catalyzed by the protease papain using trichloroethylene as organic phase with an organic phase-aqueous phase volume ratio, V org / V aq , of 4. The results of the present article can be summarized as follows: a) A relationship between enzyme deactivation, interfacial area, and interfacial tension is stated. The addition of 0.4% Tween 80 to the reaction system stabilized papain activity for> 50 h, overcoming the problems related to the presence of the organic solvent and shear stress due to mixing; b) undesirable parallel reactions are identified; c) the peptide yield was increased by working with a fed-batch strategy.

Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

The over-expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition or activation of secondary metabolism pathways. Moreover, the expression of other plasmid-encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth kinetics. In this work, we have developed a second-generation system suitable for Escherichia coli expression with an antibiotic-free plasmid maintenance mechanism based on a glycine auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression was minimized by tuning the expression levels of the repressor protein (LacI) and glyA using a library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. The engineered antibiotic-free expression system was applied to the L-fuculose phosphate aldolase (FucA) over-production, showing an increase in production up to 3.8-fold in terms of FucA yield (mg g(-1)DCW) and 4.5-fold in terms of FucA activity (AU g(-1)DCW) compared to previous expression. Moreover, acetic acid production was reduced to 50%, expressed as gAc gDCW(-1). Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity.

Simulation and prediction of protein production in fed‐batch E. coli cultures: An engineering approach

An overall model describing the dynamic behavior of fed‐batch E. coli processes for protein production has been built, calibrated and validated. Using a macroscopic approach, the model consists of three interconnected blocks allowing simulation of biomass, inducer and protein concentration profiles with time. The model incorporates calculation of the extra and intracellular inducer concentration, as well as repressor–inducer dynamics leading to a successful prediction of the product concentration. The parameters of the model were estimated using experimental data of a rhamnulose‐1‐phosphate aldolase‐producer strain, grown under a wide range of experimental conditions. After validation, the model has successfully predicted the behavior of different strains producing two different proteins: fructose‐6‐phosphate aldolase and ω‐transaminase. In summary, the presented approach represents a powerful tool for E. coli production process simulation and control. Biotechnol. Bioeng. 2016;113: 772–782.