Stress tolerance and growth physiology of yeast strains from the Brazilian fuel ethanol industry.

Improved biofuels production requires a better understanding of industrial microorganisms. Some wild Saccharomyces cerevisiae strains, isolated from the fuel ethanol industry in Brazil, present exceptional fermentation performance, persistence and prevalence in the harsh industrial environment. Nevertheless, their physiology has not yet been systematically investigated. Here we present a first systematic evaluation of the widely used industrial strains PE-2, CAT-1, BG-1 and JP1, in terms of their tolerance towards process-related stressors. We also analyzed their growth physiology under heat stress. These strains were evaluated in parallel to laboratory and baker's strains. Whereas the industrial strains performed in general better than the laboratory strains under ethanol or acetic acid stresses and on industrial media, high sugar stress was tolerated equally by all strains. Heat and low pH stresses clearly distinguished fuel ethanol strains from the others, indicating that these conditions might be the ones that mostly exert selective pressure on cells in the industrial environment. During shake-flask cultivations using a synthetic medium at 37 °C, industrial strains presented higher ethanol yields on glucose than the laboratory strains, indicating that they could have been selected for this trait-a response to energy-demanding fermentation conditions. These results might be useful to guide future improvements of large-scale fuel ethanol production via engineering of stress tolerance traits in other strains, and eventually also for promoting the use of these fuel ethanol strains in different industrial bioprocesses.

Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the ß-lactam producer Penicillium chrysogenum.

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of ß-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA.

Simple and robust method for estimation of the split between the oxidative pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway in microorganisms.

The flux through the oxidative pentose phosphate (PP) pathway was estimated in Bacillus clausii, Saccharomyces cerevisiae, and Penicillium chrysogenum growing in chemostats with [1-(13)C]glucose as the limiting substrate. The flux calculations were based on a simple algebraic expression that is valid irrespective of isotope rearrangements arising from reversibilities of the reactions in the PP pathway and the upper part of the Embden-Meyerhof-Parnas pathway. The algebraically calculated fluxes were validated by comparing the results with estimates obtained using a numerical method that includes the entire central carbon metabolism. Setting the glucose uptake rate to 100, the algebraic expression yielded estimates of the PP pathway flux in B. clausii, S. cerevisiae, and P. chrysogenum of 20, 42, and 75, respectively. These results are in accordance with the results from the numerical method. The information on the labeling patterns of glucose and the proteinogenic amino acids were obtained using gas chromatography / mass spectrometry, which is a very sensitive technique, and therefore only a small amount of biomass is needed for the analysis. Furthermore, the method developed in this study is fast and readily accessible, as the calculations are based on a simple algebraic expression.

Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression.

The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h(-1) or in a batch culture with a specific growth rate of 0.37 h(-1). Experiments were carried out using [1-(13)C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern.

Lipase production by Penicillium restrictum using solid waste of industrial babassu oil production as substrate.

Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, olive oil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.

Mathematical modelling of metabolism.

Mathematical models of the cellular metabolism have a special interest within biotechnology. Many different kinds of commercially important products are derived from the cell factory, and metabolic engineering can be applied to improve existing production processes, as well as to make new processes available. Both stoichiometric and kinetic models have been used to investigate the metabolism, which has resulted in defining the optimal fermentation conditions, as well as in directing the genetic changes to be introduced in order to obtain a good producer strain or cell line. With the increasing availability of genomic information and powerful analytical techniques, mathematical models also serve as a tool for understanding the cellular metabolism and physiology.

Recombinant gene expression in Escherichia coli cultivation using lactose as inducer.

The use of lactose as inducer of foreign gene expression under control of the lac UV5 promoter was investigated in recombinant Escherichia coli. Chicken muscle troponin C (TnC) gene was transcripted by T7 RNA polymerase and expressed in bioreactor cultivations after a feed-forward controlled fed-batch growth phase. Cell concentrations of 22 g l-1 dry cell weight (DCW)--before induction started--were used to achieve a TnC content of 19.5% of total cell protein through an induction strategy that combined the addition of a specific lactose amount of 4.7 g g-1 DCW divided into three pulses and the addition of yeast extract (1 g l-1) together with the second and the third lactose pulses. The results presented suggest that the residual lactose concentration plays an important role on the production of the heterologous protein.