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.
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•Heterologous expression of a cyanobacterial alkane pathway into a PHB deleted C. necator strain was successfully achieved in Re2061-pLC10 strain producing up to 337mg/l of alkanes.•Extractive biphasic fermentation improved production of alka(e)nes up to 435mg/l together with 236mg/l of hexadecanal.•First demonstration of alka(e)nes production by a non-native alkane producer from CO2 as sole C-source.
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.
•A bioreactor was designed to cultivate C.necator under a safe environment and high pressure.•Cultivations led to growth and production of C. necator up to 3 gDCW L-1 and 3.5 g L-1 isopropanol.•First ...demonstration of g L-1-scale molecule production from autotrophic engineered bacteria.
A bioreactor was designed to provide high gas mass transfer to reach cell and product titres in the g L-1 level from CO2 for realistic, laboratory scale, engineered autotrophic strain evaluation. The design was based on independent CO2, H2 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-1 dry cell weight. No negative impact of the high pressure was observed on viability of the strains up to more than 4 bar overpressure. The cultivation was then carried out using an engineered isopropanol producing strain; in this case, 3.5 g L-1 isopropanol was obtained from CO2 as the sole carbon source. This is the first reported demonstration of a successful production from engineered bacteria of product in the g L-1 range on CO2, raising the prospect of future development of CO2-based bioprocesses.
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.
•GroESL improved cellular tolerance to isopropanol.•PSK hok/sok expression improve plasmid stability up to a threshold IPA concentration.•Metabolic load due to GroESL and PSK hok/sok expression deleterious to overall isopropanol production compared to the reference strain.
•Overexpression of ylDGA2 and ylGPD1 led to 3-fold increase in TAG content.•Overexpression of ylDGA2 and ylGPD1 led to 4-fold increase in lipid yield.•DGA2-GPD1 overexpressing strain was more robust ...towards N/C fluctuations.
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.
This strain showed a strong improvement in production performances on glucose in terms of lipid content (increase from 18 to 55%), lipid yield (increase from 0,035 to 0.14gg −1) and by-product formation (decrease in citric acid yield from 0.68 to 0.4gg −1).
For developing bioprocess for the production of triacylglycerol from renewable carbon sources as glucose it is of first importance to control the C/N ratio in order to avoid citric acid excretion during lipid accumulation. Our engineered strain showed a delay in the onset of citric acid excretion as suggested by the 15% modulation of the critical C/N ratio.
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 P
lac
to 56% with the strong constitutive promoter P
j5
. 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 P
lac
. 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
.
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 highlighed 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.
•Co-expression of heterologous isopropanol pathway and eGFP-biosensor to evaluate plasmid expression stability at single-cell level.•The co-production of eGFP and Isopropanol impacts strain robustness and performances comparing to the single overexpression (Isopropanol or eGFP).•Increased cell permeabilization and plasmid loss with increasing isopropanol and acetone concentration.
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.
•Comparison of plasmid curing strategies: rifampicin vs supra-optimal temperature.•Higher efficiency of supra-optimal temperature plasmid curing strategy.•Higher number of generations necessary to trigger subpopulations in bioreactor vs flask.•Modulation of the subpopulation distribution using chemostat culture.
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.
•Over-expression of the native groEL and groES led to a better tolerance of C. necator towards exogenous isopropanol.•Over-expression of groELS in an engineered isopropanol producing C. necator led to improve the production on fructose.•Isopropanol production from CO2 as sole C-source was demonstrated using our engineered C. necator strain in bioreactor.
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.
•Overview of Clostridium tetani strain diversity and genome sequences.•Schematic representation of a metabolic network including cofactor specificities•Current knowledge on toxin regulation and synthesis.•Report of C. tetani cultures and its requirements for growth and toxin synthesis.•First attempts to produce tetanus toxin in chemically defined medium.
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