Trehalose accumulation in Saccharomyces cerevisiae cells: experimental data and structured modeling

Several studies have shown that the reserve disaccharide trehalose also protects yeast cells under environmental stress. Besides, it is acknowledged that trehalose improves yeast viability so its synthesis has become paramount in baker’s yeast industry, as well as in wine and beer production. In this work, starving carbon or nitrogen conditions were imposed to Saccharomyces cerevisiae growing in fed-batch cultivations in order to induce biosynthesis and accumulation of trehalose. Our experimental results show that yeast can accumulate trehalose up to 13% of biomass dry weight (0.13 gtrehalose g^−1biomass) under carbon or nitrogen starvation. A compartmentalized and biochemically structured model is proposed in order to mathematically describe these experimental findings. The model predictions were compared with literature results obtained under growth conditions different from those established in our experiments, resulting in a very good agreement and thus indicating that the proposed hypotheses are essentially correct

A New Tool for the Quantification of Microorganism Interaction Dynamics

A two-reservoir, membrane bioreactor for carrying out studies of mixed population dynamics in batch fermentation is presented. Mixing requirements and design aspects for the validity of the approach are presented and discussed. Equations describing mixing times between the reservoirs are given and compared to the experimental results. The validity of the approach is demonstrated by the study of several types of situations in the bioreactor. The main validation consists of the comparison between the results obtained in an actual mixed culture and the results obtained by keeping the strains separated. Finally, this new tool is used to study the interaction kinetics between two oenological bacteria. These experiments in liquid media help to determine the seeding conditions for a chosen strain to overgrow another strain through a quantification of the interaction dynamics

A simple metabolic flux balance analysis of biomass and bioethanol production in Saccharomyces cerevisiae fed-batch cultures

Production of Saccharomyces cerevisiae yeast for applications in the food industry or in bioethanol production still presents important techno-economic challenges as an industrial bioprocess. Mathematical modeling of cellular metabolism in biological production usually improves process yields, though for industrial applications, the model should be as simple as possible in order to sustain model usefulness and technical feasibility. A comparative analysis between a black box description and a simple metabolic network accounting for the main metabolic events involved in yeast growth and bioethanol production is proposed here. In both cases, a thorough analysis of reaction rates allowed for the ethanol concentrations produced in fed-batch yeast cultures, although our results showed more accurate estimations with the metabolic flux balance methodology. Moreover, an interpretation of the yeast physiological state in fed-batch cultures at different glucose feed concentrations was accomplished by means of a stoichiometric analysis linked to the simplified metabolic network. The results confirmed that increasing glucose uptake rates, controlled mainly by the glucose concentration in the input flow, produced an up-regulation in reductive catabolism, resulting in higher ethanol excretion. The biomass production relied mostly on oxidative catabolism, which is controlled by the glucose and oxygen uptake rates. Thus, ethanol or biomass production is strongly dependent on the physiological state of yeast in the culture, which can be inferred from a suitable metabolic flux balance approach

Metabolic flux distribution and thermodynamic analysis of green fluorescent protein production in recombinant Escherichia coli: the effect of carbon source and CO2 partial pressure

Increasing recombinant protein production yields from bacterial cultures remains an important challenge in biotechnology. Acetate accumulation due to high dissolved carbon dioxide (pCO2) concentrations in the medium has been identified as a factor that negatively affects such yields. Under appropriate culture conditions, acetate could be re-assimilated by bacterial cells to maintain heterologous proteins production. In this work, we developed a simplified metabolic network aiming to establish a reaction rate analysis for a recombinant Escherichia coli when producing green fluorescent protein (GFP) under controlled pCO2 concentrations. Because E. coli is able to consume both glucose and acetate, the analysis was performed in two stages. Our results indicated that GFP synthesis is an independent process of cellular growth in some culture phases. Additionally, recombinant protein production is influenced by the available carbon source and the amount of pCO2 in the culture medium. When growing on glucose, the increase in the pCO2 concentration produced a down-regulation of central carbon metabolism by directing the carbon flux toward acetate accumulation; as a result, cellular growth and the overall GFP yield decreased. However, the maximum specific rate of GFP synthesis occurred with acetate as the main available carbon source, despite the low activity in the other metabolic pathways. To maintain cellular functions, including GFP synthesis, carbon flux was re-distributed toward the tricarboxylic acid cycle and the pentose phosphate pathway to produce ATP and NADH. The thermodynamic analysis allowed demonstrating the feasibility of the simplified network for describing the metabolic state of a recombinant system

Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest