Autotrophic Production of the Sesquiterpene α-Humulene with in a Controlled Bioreactor.

is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO into industrially valuable chemicals. To translate the potential of into technical applications, it is necessary to optimize and scale up production processes.

Comparison of plasmid stabilization systems during heterologous isopropanol production in fed-batch bioreactor.

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures.

Co-expression of an isopropanol synthetic operon and eGFP to monitor the robustness of Cupriavidus necator during isopropanol production.

Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C.

Study of plasmid-based expression level heterogeneity under plasmid-curing like conditions in Cupriavidus necator.

Plasmid expression level heterogeneity in Cupriavidus necator was studied in response to stringent culture conditions, supposed to enhance plasmid instability, through plasmid curing strategies. Two plasmid curing strategies were compared based on their efficiency at generating heterogeneity in batch: rifampicin addition and temperature increase. A temperature increase from 30° to 37 °C was the most efficient plasmid curing strategy.

Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures.

It is of major interest to ensure stable and performant microbial bioprocesses, therefore maintaining high strain robustness is one of the major future challenges in industrial microbiology. Strain robustness can be defined as the persistence of genotypic and/or phenotypic traits in a system. In this work, robustness of an engineered strain is defined as plasmid expression stability, cultivability, membrane integrity and macroscopic cell behavior and was assessed in response to implementations of sugar feeding strategies (pulses and continuous) and two plasmid stabilization systems (kanamycin resistance and Post-Segregational Killing hok/sok).

Accelerostat study in conventional and microfluidic bioreactors to assess the key role of residual glucose in the dimorphic transition of Yarrowia lipolytica in response to environmental stimuli.

Yarrowia lipolytica, with a diverse array of biotechnological applications, is able to grow as ovoid yeasts or filamentous hyphae depending on environmental conditions. This study has explored the relationship between residual glucose levels and dimorphism in Y. lipolytica.

Insights into Clostridium tetani: From genome to bioreactors.

Tetanus vaccination is of major importance for public health in most countries in the world. The World Health Organization indicated that 15,000 tetanus cases were reported in 2018 (Organization, World Health, 2019). Currently, vaccine manufacturers use tetanus toxin produced by Clostridium tetani fermentation in complex media.

Plasmid expression level heterogeneity monitoring via heterologous eGFP production at the single-cell level in Cupriavidus necator.

A methodology for plasmid expression level monitoring of eGFP expression suitable for dynamic processes was assessed during fermentation. This technique was based on the expression of a fluorescent biosensor (eGFP) encoded on a recombinant plasmid coupled to single-cell analysis. Fluorescence intensity at single-cell level was measured by flow cytometry.

Isopropanol production from carbon dioxide in Cupriavidus necator in a pressurized bioreactor.

A bioreactor was designed to provide high gas mass transfer to reach cell and product titres in the g L level from CO for realistic, laboratory scale, engineered autotrophic strain evaluation. The design was based on independent CO, H and air inputs and the ability to operate at high pressures. The bioreactor configuration and cultivation strategy enabled growth of Cupriavidus necator strains for long periods, to reach over 3 g L dry cell weight.

Alka(e)ne synthesis in Cupriavidus necator boosted by the expression of endogenous and heterologous ferredoxin-ferredoxin reductase systems.

To boost aldehyde deformylating oxygenase (ADO) activity in a Cupriavidus necator strain expressing a synthetic alkane pathway, the expression of two ferredoxin-ferredoxin reductase systems was tested. The genes of a native fd/FNR-like system were identified in C. necator and expressed in a previously engineered alka(e)ne producing strain.

Impacts of environmental conditions on product formation and morphology of Yarrowia lipolytica.

The yeast Yarrowia lipolytica is an industrially important microorganism with distinctive physiological and metabolic characteristics. A variety of external factors (e.g.

Modeling and optimization of lipid accumulation by Yarrowia lipolytica from glucose under nitrogen depletion conditions.

Oleaginous yeasts have been seen as a feasible alternative to produce the precursors of biodiesel due to their capacity to accumulate lipids as triacylglycerol having profiles with high content of unsaturated fatty acids. The yeast Yarrowia lipolytica is a promising microorganism that can produce lipids under nitrogen depletion conditions and excess of the carbon source. However, under these conditions, this yeast also produces citric acid (overflow metabolism) decreasing lipid productivity.

Modulation of the Glycerol Phosphate availability led to concomitant reduction in the citric acid excretion and increase in lipid content and yield in Yarrowia lipolytica.

In order to improve TriAcylGycerol (TAG) lipids accumulation in the yeast Yarrowia lipolytica on glucose, double over-expression of the major acyl-CoA:diacylglycerol acyltransferase encoding gene (ylDGA2) and of the glycerol-phosphate dehydrogenase encoding gene (ylGPD1) was carried out. The genes were over-expressed in a strain impaired for the mobilization of the accumulated lipids, through the deletion of the genes encoding acyl-coenzyme A oxidases (POX1-6 genes) and the deletion of the very efficient lipase attached to the lipid bodies, encoded by ylTGL4. This metabolic engineering strategy had the objective of pulling the C-flow into the TAG synthesis by increasing the availability of glycerol-3-phosphate and its binding to fatty acids for the TAG synthesis.

Expanding the genetic tool box for Cupriavidus necator by a stabilized L-rhamnose inducible plasmid system.

The Gram negative bacterium Cupriavidus necator is well known for the accumulation of poly(3-hydroxybutyrate) and its fast lithoautotrophic growth, leading in high cell densities. Although the host was engineered for the heterologous production of diverse chemicals and biopolymers in recent years, tool box of stabilized inducible expression systems is still limited. To avoid plasmid loss during fermentation processes and to allow expression of complex proteins, a tunable L-rhamnose inducible system was established and characterized using enhanced green fluorescent protein (eGFP).

Influence of oxygen availability on the metabolism and morphology of Yarrowia lipolytica: insights into the impact of glucose levels on dimorphism.

Dynamic behavior of Yarrowia lipolytica W29 strain under conditions of fluctuating, low, and limited oxygen supply was characterized in batch and glucose-limited chemostat cultures. In batch cultures, transient oscillations between oxygen-rich and -deprived environments induced a slight citric acid accumulation (lower than 29 mg L). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO value, 100% N), limited (zero pO value, 75% of cell needs), and low (pO close to 2%) dissolved oxygen (DO) levels.

Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production.

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4gL isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered.

Dynamic behavior of Yarrowia lipolytica in response to pH perturbations: dependence of the stress response on the culture mode.

Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and morphological changes in response to environmental conditions. The effect of pH perturbations of different types (pulses, Heaviside) on the dynamic behavior of Y. lipolytica W29 strain was characterized under two modes of culture: batch and continuous.

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production.

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO2: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content).

Lipids containing medium-chain fatty acids are specific to post-whole genome duplication Saccharomycotina yeasts.

Background: Yeasts belonging to the subphylum Saccharomycotina have been used for centuries in food processing and, more recently, biotechnology. Over the past few decades, these yeasts have also been studied in the interest of their potential to produce oil to replace fossil resources. Developing yeasts for massive oil production requires increasing yield and modifying the profiles of the fatty acids contained in the oil to satisfy specific technical requirements.

Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed-batch and continuous cultures.

Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin-Benson-Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization.

Isopropanol production with engineered Cupriavidus necator as bioproduction platform.

Alleviating our society's dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO2. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)].

D-stat culture for studying the metabolic shifts from oxidative metabolism to lipid accumulation and citric acid production in Yarrowia lipolytica.

Lipid accumulation in oleaginous yeasts is triggered by nutrient imbalance in the culture medium between the carbon source in excess and the nitrogen source in limiting concentration. However Yarrowia lipolytica when cultivated on glucose as the sole carbon source, mainly produces citric acid upon nitrogen limitation over lipid accumulation (only 5-10% triacylglycerol). Therefore for developing bioprocess for the production of triacylglycerol from renewable carbon source as glucose it is of first importance to control this imbalance in order to avoid citric acid production during TAG accumulation.

Impact of oleic acid as co-substrate of glucose on "short" and "long-term" Crabtree effect in Saccharomyces cerevisiae.

Background: Optimization of industrial biomass directed processes requires the highest biomass yield as possible. Yet, some useful yeasts like Saccharomyces cerevisiae are subject to the Crabtree effect under glucose excess. This phenomenon can occur in large scale tank where heterogeneities in glucose concentrations exist.

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae.

Background: Finely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions.

Dynamic model of temperature impact on cell viability and major product formation during fed-batch and continuous ethanolic fermentation in Saccharomyces cerevisiae.

The impact of the temperature on an industrial yeast strain was investigated in very high ethanol performance fermentation fed-batch process within the range of 30-47 °C. As previously observed with a lab strain, decoupling between growth and glycerol formation occurred at temperature of 36 °C and higher. A dynamic model was proposed to describe the impact of the temperature on the total and viable biomass, ethanol and glycerol production.