Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay

We investigate how to characterize the kinetic parameters of an aminotransaminase using a non-standard coupled (or auxiliary) enzyme assay, where the peculiarity arises for two reasons. First, one of the products of the auxiliary enzyme is a substrate for the primary enzyme and, second, we explicitly account for the reversibility of the auxiliary enzyme reaction. Using singular perturbation theory, we characterize the two distinguished asymptotic limits in terms of the strength of the reverse reaction, which allows us to determine how to deduce the kinetic parameters of the primary enzyme for a characterized auxiliary enzyme. This establishes a parameter-estimation algorithm that is applicable more generally to similar reaction networks. We demonstrate the applicability of our theory by performing enzyme assays to characterize a novel putative aminotransaminase enzyme, CnAptA (UniProtKB Q0KEZ8) from Cupriavidus necator H16, for two different omega-amino acid substrates

Preliminary Results on Light Conditions Manipulation in Octopus vulgaris (Cuvier, 1797) Paralarval Rearing

High paralarvae mortality is a major bottleneck currently hindering the control over the lifecycle of common octopus (Octopus vulgaris Cuvier, 1797). It is believed that this problem might be related to either zoo-technical and/or nutritional aspects. The present paper is focused on the study of different zoo-technical aspects related to light conditions on the rearing of paralarvae, including the effects of polarization in prey ingestion, the use of a blue filter to simulate natural conditions, and the use of focused light to avoid reflections of the rearing tank’s walls. In the first experiment, O. vulgaris paralarvae ingestion of Artemia sp. and copepods (Tisbe sp.) was assessed under either normal or polarized light. In the second experiment, the effect of a blue filter with natural light or focused artificial light on growth and mortality was assessed over 15 days of rearing. Ingestion rate was not influenced by light polarization. Nonetheless, a significantly higher ingestion of Artemia sp. with respect to copepods was observed. The blue filter promoted the use of natural light conditions in Octopus paralarval culture, while focused light reduced the collision of the paralarvae against the walls. However, no significant differences were found in paralarval growth nor survivalEn prens

Characterisation of the native β-alanine pathway in Cupriavidus necator H16: an attractive route towards 3-hydroxypropionic acid production.

The sustainable production of chemicals and fuels through microbial fermentation will play a pivotal role in reducing our dependence on fossil resources as well as decreasing global emissions of greenhouse gases, and in particular CO2. A particularly promising strategy is to produce the requisite molecules using autotrophic microbial chassis that are capable of growing on CO2 as a sole carbon source. One such chassis is Cupriavidus necator H16, formerly Rastonia eutropha. It is a Gram-negative, non-pathogenic, asporogenous bacterium found in aerobic and anaerobic, non-halophilic environments. It is a facultative, chemolithoautotroph able to grow aerobically on CO2 and H2 as sole carbon and energy source, respectively. It can also grow heterotrophically on a variety of organic substrates. 3-Hydroxypropionic acid (3-HP) is one of these potential chemicals. It is a platform chemical, which can be converted into a wide variety of acids and biodegradable polyesters, among other highly valued industrial compounds. Currently, industrial demand for 3-HP is entirely met through petrochemical routes which are associated with environmental pollution. A biological process involving genetically engineered microorganisms would potentially be a sustainable manner of production that industry will surely support and value. Metabolic modelling has previously identified a number of potential routes to 3-HP of which a process that proceeds via β-alanine (BAL) represents the most energy efficient pathway. Whilst C. necator possesses a putative native BAL pathway, it does not presently produce 3-HP. The current study sought to investigate the potential of the native BAL metabolic route in C. necator for the production of 3-HP, focusing our efforts on the phenotypical and enzymatic characterisation of the last two metabolic steps of the pathway, from the pyruvate-dependent transamination reaction between BAL and malonate semialdehyde (MSA) to the reduction of MSA to 3-HP. This analysis demonstrated that a putative transaminase (CnAptA) and two putative dehydrogenases (CnHpdH and CnHbdH) were largely responsible for the failure of C. necator to produce 3-HP. Following strain development, a heterologous pathway was implemented in the organism which led to the total conversion of BAL into 3-HP. This innovation could form the basis of the future production of sustainable and commercially viable amounts of 3-HP using CO2 as the sole carbon source in C. necator H16

Characterisation of the native β-alanine pathway in Cupriavidus necator H16: an attractive route towards 3-hydroxypropionic acid production.

The sustainable production of chemicals and fuels through microbial fermentation will play a pivotal role in reducing our dependence on fossil resources as well as decreasing global emissions of greenhouse gases, and in particular CO2. A particularly promising strategy is to produce the requisite molecules using autotrophic microbial chassis that are capable of growing on CO2 as a sole carbon source. One such chassis is Cupriavidus necator H16, formerly Rastonia eutropha. It is a Gram-negative, non-pathogenic, asporogenous bacterium found in aerobic and anaerobic, non-halophilic environments. It is a facultative, chemolithoautotroph able to grow aerobically on CO2 and H2 as sole carbon and energy source, respectively. It can also grow heterotrophically on a variety of organic substrates. 3-Hydroxypropionic acid (3-HP) is one of these potential chemicals. It is a platform chemical, which can be converted into a wide variety of acids and biodegradable polyesters, among other highly valued industrial compounds. Currently, industrial demand for 3-HP is entirely met through petrochemical routes which are associated with environmental pollution. A biological process involving genetically engineered microorganisms would potentially be a sustainable manner of production that industry will surely support and value. Metabolic modelling has previously identified a number of potential routes to 3-HP of which a process that proceeds via β-alanine (BAL) represents the most energy efficient pathway. Whilst C. necator possesses a putative native BAL pathway, it does not presently produce 3-HP. The current study sought to investigate the potential of the native BAL metabolic route in C. necator for the production of 3-HP, focusing our efforts on the phenotypical and enzymatic characterisation of the last two metabolic steps of the pathway, from the pyruvate-dependent transamination reaction between BAL and malonate semialdehyde (MSA) to the reduction of MSA to 3-HP. This analysis demonstrated that a putative transaminase (CnAptA) and two putative dehydrogenases (CnHpdH and CnHbdH) were largely responsible for the failure of C. necator to produce 3-HP. Following strain development, a heterologous pathway was implemented in the organism which led to the total conversion of BAL into 3-HP. This innovation could form the basis of the future production of sustainable and commercially viable amounts of 3-HP using CO2 as the sole carbon source in C. necator H16

Harnessing yeast peroxisomes for biosynthesis of fatty-acid-derived biofuels and chemicals with relieved side-pathway competition

Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and/or insufficient precursor supply. Compartmentalization in cellular organelles can isolate synthetic pathways from competing pathways, and provide a compact and suitable environment for biosynthesis. Peroxisomes are cellular organelles where fatty acids are degraded, a process that is inhibited under typical fermentation conditions making them an interesting workhouse for production of fatty-acid-derived molecules. Here, we show that targeting synthetic pathways to peroxisomes can increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins up to 700%. In addition, we demonstrate that biosynthesis of these chemicals in the peroxisomes results in significantly decreased accumulation of byproducts formed by competing enzymes. We further demonstrate that production can be enhanced up to 3-fold by increasing the peroxisome population. The strategies described here could be used for production of other chemicals, especially acyl-CoA-derived molecules

Preliminary Results on Light Conditions Manipulation in Octopus vulgaris (Cuvier, 1797) Paralarval Rearing.

High paralarvae mortality is a major bottleneck currently hindering the control over the lifecycle of common octopus (Octopus vulgaris Cuvier, 1797). It is believed that this problem might be related to either zoo-technical and/or nutritional aspects. The present paper is focused on the study of different zoo-technical aspects related to light conditions on the rearing of paralarvae, including the effects of polarization in prey ingestion, the use of a blue filter to simulate natural conditions, and the use of focused light to avoid reflections of the rearing tank’s walls. In the first experiment, O. vulgaris paralarvae ingestion of Artemia sp. and copepods (Tisbe sp.) was assessed under either normal or polarized light. In the second experiment, the effect of a blue filter with natural light or focused artificial light on growth and mortality was assessed over 15 days of rearing. Ingestion rate was not influenced by light polarization. Nonetheless, a significantly higher ingestion of Artemia sp. with respect to copepods was observed. The blue filter promoted the use of natural light conditions in Octopus paralarval culture, while focused light reduced the collision of the paralarvae against the walls. However, no significant differences were found in paralarval growth nor survival

The genetic basis of 3-hydroxypropanoate metabolism in Cupriavidus necator H16.

Arenas-Lopez C, Locker J, Orol D, et al. The genetic basis of 3-hydroxypropanoate metabolism in Cupriavidus necator H16. Biotechnology for biofuels. 2019;12(1): 150.Background: 3-Hydroxypropionic acid (3-HP) is a promising platform chemical with various industrial applications. Several metabolic routes to produce 3-HP from organic substrates such as sugars or glycerol have been implemented in yeast, enterobacterial species and other microorganisms. In this study, the native 3-HP metabolism of Cupriavidus necator was investigated and manipulated as it represents a promising chassis for the production of 3-HP and other fatty acid derivatives from CO2 and H2.; Results: When testing C. necator for its tolerance towards 3-HP, it was noted that it could utilise the compound as the sole source of carbon and energy, a highly undesirable trait in the context of biological 3-HP production which required elimination. Inactivation of the methylcitrate pathway needed for propionate utilisation did not affect the organism's ability to grow on 3-HP. Putative genes involved in 3-HP degradation were identified by bioinformatics means and confirmed by transcriptomic analyses, the latter revealing considerably increased expression in the presence of 3-HP. Genes identified in this manner encoded three putative (methyl)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) and two putative dehydrogenases (hpdH and hbdH). These genes, which are part of three separate mmsA operons, were inactivated through deletion of the entire coding region, either singly or in various combinations, to engineer strains unable to grow on 3-HP. Whilst inactivation of single genes or double deletions could only delay but not abolish growth, a triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain was unable utilise 3-HP as the sole source of carbon and energy. Under the used conditions this strain was also unable to co-metabolise 3-HP alongside other carbon and energy sources such as fructose and CO2/H2. Further analysis suggested primary roles for the different mmsA operons in the utilisation of beta-alanine generating substrates (mmsA1), degradation of 3-HP (mmsA2), and breakdown of valine (mmsA3).; Conclusions: Three different (methyl)malonate semialdehyde dehydrogenases contribute to 3-HP breakdown in C. necator H16. The created triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain represents an ideal chassis for autotrophic 3-HP production