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. We demonstrated that promoter evaluation based on single-cell analysis versus classic global analysis brings valuable insights. Single-cell analysis pointed out the fact that intrinsic fluorescence increased with the strength of the promoter up to a threshold. Beyond that, cell permeability increases to excrete the fluorescent protein in the medium. The metabolic load due to the increase in the eGFP production in the case of strong constitutive promoters leads to slower growth kinetics compared with plasmid-free cells. With the strain Cupriavidus necator Re2133, growth rate losses were measured from 3% with the weak constitutive promoter Plac to 56% with the strong constitutive promoter Pj5. Through this work, it seems crucial to find a compromise between the fluorescence intensity in single cells and the metabolic load; in our conditions, the best compromise found was the weak promoter Plac. The plasmid expression level monitoring method was tested in the presence of a heterogeneous population induced by plasmid-curing methods. For all the identified subpopulations, the plasmid expression level heterogeneity was significantly detected at the level of fluorescence intensity in single cells. After cell sorting, growth rate and cultivability were assessed for each subpopulation. In conclusion, this eGFP biosensor makes it possible to follow the variations in the level of plasmid expression under conditions of population heterogeneity.Key Points•Development of a plasmid expression level monitoring method at the single-cell level by flow cytometry.•Promoter evaluation by single-cell analysis: cell heterogeneity and strain robustness.•Reporter system optimization for efficient subpopulation detection in pure cultures.

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. The improved production of alka(e)nes in this Re2061-pMAB1 strain confirmed the activity of the native Fd/FNR system in C. necator. Concomitantly, the expression of the heterologous system from Synechococcus elongatus was investigated identically, leading to a second strain, Re2061-pMAB2. In the bioreactor, the aldehyde production was strongly reduced compared with the original alka(e)ne producer, leading to alka(e)nes production up to 0.37 and 1.48 g/L (22 and 82 mg/gCDW ), respectively. The alka(e)ne production yield of Re2061-pMAB2 accounted for 15% of the theoretical yield. We report here the highest level and yield of alka(e)nes production by an engineered bacterium to date.

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. This work presents two mathematical models for lipid production by Y. lipolytica from glucose. The first model is based on Monod and inhibition kinetics, and the second one is based on the Droop quota model approach, which is extended to yeast. The two models showed good agreements with the experimental data used for calibration and validation. The quota based model presented a better description of the dynamics of nitrogen and glucose dynamics leading to a good management of N/C ratio which makes this model interesting for control purposes. Then, quota model was used to evaluate, by means of simulation, a scenario for optimizing lipid productivity and lipid content. For that, a control strategy was designed by approximating the flow rates of glucose and nitrogen with piecewise linear functions. Simulation results achieved productivity of 0.95 g L-1 hr-1 and lipid content fraction of 0.23 g g-1 , which indicates that this strategy is a promising alternative for the optimization of lipid production.

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., pH, temperature, and nutrient availability) influences the behavior of the yeast and may act as stress conditions which the cells must withstand and adapt. In this mini review, the impacts of environmental factors on the morphology and metabolite production by Y. lipolytica are summarized. In this regard, detailed insights into the effectors involved in the dimorphic transition of Y. lipolytica, the cultivation conditions employed, as well as the methods applied for the morphological characterization are highlighted. Concerning the metabolism products, a special focus is addressed on lipid and citric acid metabolites which have attracted significant attention in recent years. The dependence of lipid and citric acid productivity on key process parameters, such as media composition and physico-chemical variables, is thoroughly discussed. This review attempts to provide a recent update on the topic and will serve as a meaningful resource for researchers working in the field.

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-1 isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15gL-1. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8gL-1 was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16gL-1 after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9-18% increase in the isopropanol yield on fructose.

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-1). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO2 value, 100% N2), limited (zero pO2 value, 75% of cell needs), and low (pO2 close to 2%) dissolved oxygen (DO) levels. The macroscopic behavior (kinetic parameters, yields, viability) of Y. lipolytica was not significantly affected by the exposure to DO fluctuations under both modes of culture. Nevertheless, conditions of oxygen limitation resulted in the destabilization of the glucose-limited growth during the continuous cultivations. Morphological responses of Y. lipolytica to DO oscillations were different between batch and chemostat runs. Indeed, a yeast-to-mycelium transition was induced and progressively intensified during the batch fermentations (filamentous subpopulation reaching 74% (v/v)). While, in chemostat bioreactors, the culture consisted mainly of yeast-like cells (mean diameter not exceeding 5.7 µm) with a normal size distribution. During the continuous cultures, growth at low DO concentration did not induce any changes in Y. lipolytica morphology. Dimorphism (up to 80.5% (v/v) of filaments) was only detected under conditions of oxygen limitation in the presence of a residual glucose excess (more than 0.75 g L-1). These data suggest an impact of glucose levels on the signaling pathways regulating dimorphic responses in Y. lipolytica.

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. In batch cultures, different pH (4.5, 5.6 (optimal condition), and 7) were investigated in order to identify the pH inducing a stress response (metabolic and/or morphologic) in Y. lipolytica. Macroscopic behavior (kinetic parameters, yields, viability) of the yeast was slightly affected by pH. However, contrary to the culture at pH 5.6, a filamentous growth was induced in batch experiments at pH 4.5 and 7. Proportions of the filamentous subpopulation reached 84 and 93 % (v/v) under acidic and neutral conditions, respectively. Given the significant impact of neutral pH on morphology, pH perturbations from 5.6 to 7 were subsequently assayed in batch and continuous bioreactors. For both process modes, the growth dynamics remained fundamentally unaltered during exposure to stress. Nevertheless, morphological behavior of the yeast was dependent on the culture mode. Specifically, in batch bioreactors where cells proliferated at their maximum growth rate, mycelia were mainly formed. Whereas, in continuous cultures at controlled growth rates (from 0.03 to 0.20 h-1) even closed to the maximum growth rate of the stain (0.24 h-1), yeast-like forms predominated. This pointed out differences in the kinetic behavior of filamentous and yeast subpopulations, cell age distribution, and pH adaptive mechanisms between both modes of culture.

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). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670mg/L total hydrocarbons containing 435mg/l of alkanes consisting of 286mg/l of pentadecane, 131mg/l of heptadecene, 18mg/l of heptadecane, and 236mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO2 as the sole carbon source.

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. For example, derivatives of medium-chain fatty acids (MCFAs, containing 6-14 carbons) are used for the production of biodiesels, cleaning products, lubricants and cosmetics. Few studies are available in the literature on the production of MCFAs in yeasts. RESULTS: We analyzed the MCFA content in Saccharomyces cerevisiae grown in various conditions. The results revealed that MCFAs preferentially accumulated when cells were grown on synthetic media with a high C/N ratio at low temperature (23 °C). Upon screening deletion mutant strains for genes encoding lipid droplet-associated proteins, we found two genes, LOA1 and TGL3, involved in MCFA homeostasis. A phylogenetic analysis on 16 Saccharomycotina species showed that fatty acid profiles differed drastically among yeasts. Interestingly, MCFAs are only present in post-whole genome duplication yeast species. CONCLUSIONS: In this study, we produced original data on fatty acid diversity in yeasts. We demonstrated that yeasts are amenable to genetic and metabolic engineering to increase their MCFA production. Furthermore, we revealed that yeast lipid biodiversity has not been fully explored, but that yeasts likely harbor as-yet-undiscovered strains or enzymes that can contribute to the production of high-value fatty acids for green chemistry.

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)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a ß-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l(-1) isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l(-1) of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g(-1) h(-1), yield 0.32 Cmole Cmole(-1)) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole(-1)) and the overall specific productivity (0.044 g g(-1) h(-1)) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures.

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. Therefore yeasts encounter local environments with glucose excess leading to ethanol production to the detriment of biomass formation. We previously demonstrated that oleic acid as a co-substrate in glucose-limited chemostat allowed to delay and modulate the "short-term" Crabtree effect in Saccharomyces cerevisiae. Here we further investigated the effect of oleic acid as a modulator of the Crabtree effect. RESULTS: The impact of oleic acid as co-substrate on the Crabtree effect was investigated in terms of i) strain specificity, ii) reversibility of the potential effect with aerobic glucose-excess batches and iii) durability and maximal capacities under high ethanol stress with glucose-excess fed-batches. First, the addition of oleic acid resulted in an increase of the critical dilution rate by 8% and the specific carbon uptake rate by 18%. Furthermore, a delay was observed for the onset of ethanol production when a batch was inoculated with cells previously grown in glucose-oleate chemostat. Finally, the culture of adapted cells in a glucose-oleate fed-batch led to a redirection of the carbon flux toward biomass production, with a 73% increase in the biomass yield. CONCLUSIONS: This work demonstrated clearly that the perturbation by oleic acid as co-substrate resulted in a decrease in the "short-term" and "long-term" Crabtree effects. This impact was not strain dependent and reversible. Thus, industrial applications of this biochemical strategy could be envisaged to tackle heterogeneities issues in large scale tanks or to prepare starter yeasts for various applications.

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. RESULTS: Both strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis. CONCLUSIONS: This work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further developing robust strains with improved ethanol yield.

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. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L-1 could be achieved. When the isopropanol concentration has reached about 8 g. L-1, cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L-1 of IPA compared to the reference strain (8 g. L-1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L-1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L- 1.

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

Cupriavidus necator is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO2 into industrially valuable chemicals. To translate the potential of C. necator into technical applications, it is necessary to optimize and scale up production processes. A previous proof-of-principle study showed that C. necator can be used for the de novo production of the terpene α-humulene from CO2 up to concentrations of 11 mg L-1 in septum flasks. However, an increase in final product titer and space-time yield will be necessary to establish an economically viable industrial process. To ensure optimized growth and production conditions, the application of an improved process design in a gas bioreactor with the control of pH, dissolved oxygen and temperature including a controlled gas supply was investigated. In the controlled gas bioreactor, the concentration of α-humulene was improved by a factor of 6.6 and the space-time yield was improved by a factor of 13.2. These results represent an important step toward the autotrophic production of high-value chemicals from CO2. In addition, the in situ product removal of α-humulene was investigated and important indications of the critical logP value were obtained, which was in the range of 3.0-4.2.

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. To generate a heterogeneous population in terms of plasmid expression levels, successive batches at supra-optimal culture temperature (i.e. 37 °C) were initially conducted. Three distinct fluorescent subpopulations P0 (not fluorescent), P1 (low fluorescence intensity, median = 1 103) and P2 (high fluorescence intensity, median = 6 103) were obtained. From there, the chemostat culture was implemented to study the long-term stress response under well-controlled environment at defined dilution rates. For dilution rates comprised between 0.05 and 0.10 h-1, the subpopulation P2 (62% vs 90%) was favored compared to P1 cells (54% vs 1%), especially when growth rate increased. Our biosensor was efficient at discriminating subpopulation presenting different expression levels under stringent culture conditions. Plus, we showed that controlling growth kinetics had a stabilizing impact on plasmid expression levels, even under heterogeneous expression conditions.

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. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol/acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighted the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered.

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. The complex components, commonly derived from animal sources, introduce potential variability in cultures. To achieve replicable fermentation and to avoid toxic or allergic reactions from animal-source compounds, several studies have tried to switch from complex to chemically defined media without affecting toxin titers. The present review introduces the current knowledge on i) C. tetani strain diversity, whole-genome sequences and metabolic networks; ii) toxin regulation and synthesis; and iii) culture media, cultivation processes and growth requirements. We critically reviewed the reported data on metabolism in C. tetani and completed comparative genomic and proteomic analyses with other Clostridia species. We integrated genomic data based on whole-genome sequence annotation, supplemented with cofactor specificities determined by protein sequence identity, in a new map of C. tetani central metabolism. This is the first data review that integrates insights from omics experiments on C. tetani. The overview of C. tetani physiology described here could provide support for the design of new chemically defined media devoid of complex sources for toxin production.

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). Fed-batch bioreactor cultures, relevant mode to reach high cell densities and higher cell generation number, were implemented to investigate the robustness of C. necator engineered strains. Host cells bore a recombinant plasmid encoding for a plasmid expression level monitoring system, based on eGFP fluorescence quantified by flow cytometry. We first showed that well-controlled continuous feeding in comparison to a pulse-based feeding allowed a better carbon use for protein synthesis (avoiding organic acid excretion), a lower heterogeneity of the plasmid expression and a lower cell permeabilization. Moreover, the plasmid stabilization system Post-Segregational Killing hok/sok, an autonomous system independent on external addition of compounds, showed the best ability to maintain plasmid expression level stability insuring a greater population homogeneity in the culture. Therefore, in the case of engineered C. necator, the PSK system hok/sok appears to be a relevant and an efficient alternative to antibiotic resistance system for selection pressure, especially, in the case of bioprocess development for economic and environmental reasons.

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. Under pH stress conditions, the morphological and physiological characteristics of the yeast were examined during well-controlled accelerostat cultures using both a 1 L-laboratory scale and a 1 mL-microfluidic bioreactor. The accelerostat mode, via a smooth increase of dilution rate (D), enabled the cell growth rate to increase gradually up to the cell wash-out (D ≥µmax of the strain), which was accompanied by a progressive increase in residual glucose concentration. The results showed that Y. lipolytica maintained an ovoid morphology when residual glucose concentration was below a threshold value of around 0.35-0.37 mg L-1. Transitions towards more elongated forms were triggered at this threshold and progressively intensified with the increase in residual glucose levels. The effect of cAMP on the dimorphic transition was assessed by the exogenous addition of cAMP and the quantification of its intracellular levels during the accelerostat. cAMP has been reported to be an important mediator of environmental stimuli that inhibit filamentous growth in Y. lipolytica by activating the cAMP-PKA regulatory pathway. It was confirmed that the exogenous addition of cAMP inhibited the mycelial morphology of Y. lipolytica, even with glucose concentrations exceeding the threshold level. The results suggest that dimorphic responses in Y. lipolytica are regulated by sugar signaling pathways, most likely via the cAMP-PKA dependent pathway.

Quantitative evaluation of yeast's requirement for glycerol formation in very high ethanol performance fed-batch process.

BACKGROUND: Glycerol is the major by-product accounting for up to 5% of the carbon in Saccharomyces cerevisiae ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardizing growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process. RESULTS: The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native GPD1 promoter in the yeast genome by previously generated well-characterized TEF promoter mutant versions in a gpd2Delta background. Two TEF promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as TEFmut7 and TEFmut2. The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in TEFmut7 and 88% in TEFmut2. The overall ethanol production yield on glucose was improved from 0.43 g g(-1) in the wild type to 0.44 g g(-1) measured in TEFmut7 and 0.45 g g(-1) in TEFmut2. Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for TEFmut7 and TEFmut2 respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 +/- 4 g L(-1)), growth-inhibiting ethanol concentration (87 +/- 3 g L(-1)) and volumetric ethanol productivity (2.1 +/- 0.15 g l(-1) h(-1)) measured in wild-type remained virtually unchanged in the engineered strains. CONCLUSIONS: This work demonstrates the power of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Under the conditions used in this study (VHEP fed-batch), the two strains with "fine-tuned" GPD1 expression in a gpd2Delta background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains gpd1Delta or gpd2Delta. Although glycerol reduction is known to be even higher in a gpd1Delta gpd2Delta double deletion strain, our strains could much better cope with process stress as reflected by better growth and viability.