José Luis Montesinos

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

Effect of different carbon sources on lipase production by Candida rugosa.

Different carbon sources affecting growth and lipase production in Candida rugosa were studied by using batch cultures on defined medium. Carbohydrates and acids non-related to fats did not induce lipase production. The highest yields of enzyme were obtained with lipids or fatty acids as carbon sources. Tween 80 stimulated lipase biosynthesis and secretion outside the cell. Combinations of two types of substrates, carbohydrates and fatty acids, did not improve lipase production, and in some cases, their consumption was produced in a sequential pattern. Glucose presented a repressing effect on lipase production. Moreover, glucose was found to be effective in stimulating lipase secretion by cells with a high level of cell-bound lipase activity because of their previous growth in oleic acid.

MELISSA: a loop of interconnected bioreactors to develop life support in space.

The development of a loop of interconnected continuous bioreactors, aimed to provide life support in space, is reported. The complete loop concept consists of four bioreactors and one higher plant compartment. For its realization the continuous and controlled operation of the bioreactors is characterized, up to the pilot scale level, first for each individual reactor, second for the interconnected reactor operation. The results obtained with the two more advanced bioreactors in the Micro Ecological Life Support System Alternative (MELISSA) loop are described more specifically. These reactors consist of a packed-bed reactor working with an immobilized co-culture of Nitrosomonas and Nitrobacter cells, and an external loop gas-lift photobioreactor for the culture of the cyanobacteria Spirulina platensis. Their individual operation for long duration runs has been achieved and characterized, and their interconnected operation at pilot scale is reported.

Recovery and treatment of Spirulina platensis cells cultured in a continuous photobioreactor to be used as food

The development of auto-regenerative biological life support systems for men in Space will be based on photosynthetic organisms, such as higher plants and algae, providing edible material. In this work, Spirulina platensis grown in a continuous photobioreactor was used to design a process for its recovery and further treatment to be used as food. Two different possibilities are studied (liquid or dry food). In each case, different steps are considered in the design of the process and further characterised: cell harvesting, washing, pasteurisation and spry-drying. Special emphasis is made on biomass quality, both in terms of potential microbial contamination and changes in its composition during the different steps of the process. Cell harvesting was conducted with a net recovery of solids and water higher than 95% with a solids concentration factor about 20–30. Biomass quality was shown as satisfactory in all the treatments tested.

Physiological control on the expression and secretion of Candida rugosa lipase

The fungus Candida rugosa secretes an extracellular lipase whose production is induced by the addition of fatty acids to the culture broth. This lipase is indeed composed by several protein isoforms partly differing in their catalytic properties. Synthesis and secretion of lipase proteins by C. rugosa cells were studied in culture media containing either glucose or oleic acid as the carbon source. It was shown that, according to their regulation, lipase-encoding genes might be grouped in two classes, one of which is constitutively expressed and the other is induced by fatty acids. The synthesis of inducible enzymes is inhibited at the level of transcription by the addition of glucose and, conversely, oleic acid appears to hinder the synthesis of the constitutive lipase. Growth conditions supporting high level expression both in batch and in continuous culture give rise to the intracellular accumulation of enzyme, possibly due to the existence of a rate-limiting step in the transport of the newly synthesized protein. These results suggest the possibility to develop fermentation processes aimed at the control of the enzyme composition.

Effects of different fatty acids in lipase production by Candida rugosa

SummaryOleic acid has been reported as a good inducer of lipase production by Candida rugosa. In order to know if this enzyme is induced by oleic acid itself or by a metabolite, different short chain fatty acids were tested. Butyric acid was the best carbon source to growth microorganism but it did not induce lipase production. Although caprylic and capric acid were the best inducers of lipase production, at concentrations up 1 g/l they have toxic effect in Candida rugosa growth. Thus, from the point of view of industrial production oleic acid could be considered as the best substrate tested.

Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies.

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.

Production of native and recombinant lipases by Candida rugosa: a review.

The yeast Candida rugosa produces multiple lipase isoenzymes sharing high sequence homology but with some differences in their catalytic properties. The regulation of C. rugosa lipase (CRL) synthesis and secretion in C. rugosa obeys a complex pattern. Fermentation processes for both wildtype and mutant C. rugosa strains are available for lipase production. Native CRL preparations have been extensively used for biotransformations. However, their inherent mixture of isoforms with variable profiles complicates interpretation and brings into question the reproducibility achieved between preparations. Although heterologous CRLs gene expression had been hampered owing to a nonuniversal codon usage, recent advances have made heterologous CRLs available. This will expand and improve the industrial utility of CRLs even further. The purpose of this review is to provide a summary of the recent advances on the production of native and recombinant lipases by C. rugosa.

Stability studies and effect of the initial oleic acid concentration on lipase production by Candida rugosa

The production of lipase by Candida rugosa in batch cultures was studied. The initial concentration of the carbon source employed, oleic acid, had an important effect on the final lipolytic activity levels. The maximum lipase/substrate yield and specific productivity obtained correspond to an initial oleic acid concentration of 2 g/l. At higher concentrations, up to 8 g/l oleic acid, specific productivity decreased. Lipase production was not observed below 1 g/l oleic acid. Lipase inactivation in culture broth due to surface forces and shear stress at the gas/liquid interface was not observed. There was no shear stress denaturation at stirring rates of 250, 500 and 750 rpm. No temperature inactivation was detected up to 50° C. Two different lipases with a similar molecular weight of 60kDa were purified from culture broth.

IMPROVING LIPASE PRODUCTION FROM CANDIDA RUGOSA BY A BIOCHEMICAL ENGINEERING APPROACH

It has been tested that the use of oleic acid as sole carbon source and as inducer of the production has an important effect in the lipase production by Candida rugosa under aerobic conditions. A simple structured mathematical model coupled with a methodology to estimate biomass, specific growth rate and substrate was developed and applied to the production of Candida rugosa lipase in batch, fed-batch and continuous operation to obtain a reproducible product. The best operation mode tested was a controlled specific growth rate fed-batch with a 10-fold increase in productivity related to batch operation. Downstream of the culture broth has demonstrated that the ratio between the different isoenzymes presented can be modulated by the selection of the operational strategy and this ratio is quite different comparing with commercial lipases. Thus, their catalytic properties in front of chiral reactions could be different.