Atypical Glycolysis in Clostridium Thermocellum

Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum. The requirement for PP i in this pathway can be satisfied only to a small extent by biosynthetic reactions, in con- trast to what is generally assumed for a PPi-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PPi must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PPi generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol

Quantitative Determination of Glucose Transfer Between Cocurrent Laminar Water Streams in a H-Shaped Microchannel

To explore the applicability of a laminar fluid diffusion interface (LFDI) for the controlled feeding of microbioreactors, glucose diffusion experiments were carried out in a rounded H-shaped microstructure etched in a glass substrate. The diffusion channel of the microstructure had a length of 4 mm and a depth of 50 μm with a trapezoidal cross section with a width of 100 μm at the bottom and 200 μm at the surface of the channel. The microchannel was operated at residence times of less than 1 s ensuring high-mass-transfer rates. It was confirmed, both by microscopic observations as well as computational fluid dynamics (CFD) studies that the flow characteristics in the microchannel were fully laminar. Special attention was paid to flow splitting at the end of the channel, because the CFD simulations indicated that the performance of the device was sensitive to unequal flow splitting. The difference in outflow volume of the two streams was measured to be small (1.25% ± 0.6%). The measured glucose concentration in both exit ports at a fixed residence time was found to be stable in time and reproducible in multiple experiments. CFD simulation was shown to be a powerful tool for estimating the mass transfer in the LFDI, even at very short residence times. The results obtained in this work show the applicability of LFDI for the controlled diffusive supply of a solute to a water stream, with as possible application substrate and/or precursor feeding to microreactors

Selection and subsequent physiological characterization of industrial Saccharomyces cerevisiae strains during continuous growth at sub- and- supra optimal temperatures

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.btre.2020.e00462.A phenotypic screening of 12 industrial yeast strains and the well-studied laboratory strain CEN.PK113-7D at cultivation temperatures between 12°C and 40°C revealed significant differences in maximum growth rates and temperature tolerance. From those 12, two strains, one performing best at 12°C and the other at 40°C, plus the laboratory strain, were selected for further physiological characterization in well-controlled bioreactors. The strains were grown in anaerobic chemostats, at a fixed specific growth rate of 0.03h1 and sequential batch cultures at 12°C, 30°C, and 39°C. We observed significant differences in biomass and ethanol yields on glucose, biomass protein and storage carbohydrate contents, and biomass yields on ATP between strains and cultivation temperatures. Increased temperature tolerance coincided with higher energetic efficiency of cell growth, indicating that temperature intolerance is a result of energy wasting processes, such as increased turnover of cellular components (e.g. proteins) due to temperature induced damage.We would like to thank Judith Cohen and Kristen H. David for technical assistance with the chemostat fermentations and José Ma Heras (Lallemand Ibéria, SA) for kindly providing the industrial strains. This research was carried out within the ERA-IB project “YeastTempTation” (ERA-IB-2-6/0001/2014) and partially supported by the Portuguese Foundation for Science and Technology (FCT) through strategic funding UID/BIO/04469/2020 and BioTecNorte (NORTE-01-0145-FEDER-000004).info:eu-repo/semantics/publishedVersio

An Engineered Yeast Efficiently Secreting Penicillin

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's

Similar temperature dependencies of glycolytic enzymes: an evolutionary adaptation to temperature dynamics?

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Temperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in Saccharomyces cerevisiae during temperature changes. RESULTS: Saccharomyces cerevisiae was grown under different temperature regimes and glucose availability conditions. These included glucose-excess batch cultures at different temperatures and glucose-limited chemostat cultures, subjected to fast linear temperature shifts and circadian sinoidal temperature cycles. An observed temperature-independent relation between intracellular levels of glycolytic metabolites and residual glucose concentration for all experimental conditions revealed that it is the substrate availability rather than temperature that determines intracellular metabolite profiles. This observation corresponded with predictions generated in silico with a kinetic model of yeast glycolysis, when the catalytic capacities of all glycolytic enzymes were set to share the same normalized temperature dependency. CONCLUSIONS: From an evolutionary perspective, such similar temperature dependencies allow cells to adapt more rapidly to temperature changes, because they result in minimal perturbations of intracellular metabolite levels, thus circumventing the need for extensive modification of enzyme levels

Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum:A systems biology approach

Background: In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited chemostat cultivations. Results: During these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-alpha-(d-aminoadipyl)-L-alpha-cystenyl-D-alpha-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production. Conclusions: Our findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population