Impact of nitrogen deficiency on succinic acid production by engineered strains of Yarrowia lipolytica.

Yarrowia lipolytica strains PGC01003 and PGC202 engineered for succinic acid production were studied and compared to the wild type strain W29. For the first time, these two strains were characterized in a chemically defined medium. Strain growth and organic acid production were investigated in fed-batch mode with glycerol as carbon and energy source. This study evaluated the impact of nitrogen deficiency strategy to redirect carbon flux toward succinic acid synthesis. Strain PGC01003 produced 19 g L-1 succinic acid with an overall yield of 0.23 g g-1 and an overall productivity of 0.23 g L-1 h-1, while strain PGC202 produced 33 g L-1 succinic acid with an overall yield of 0.12 g g-1 and a productivity of 0.57 g L-1 h-1. Nitrogen limitation effectively stopped biomass growth and increased succinic acid yield of PGC01003 and PGC202 by 18 % and 62 %, respectively. However, the specific succinic acid production rate was reduced by 77 % and 66 %, respectively.

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.

Understanding of polyhydroxybutyrate production under carbon and phosphorus-limited growth conditions in non-axenic continuous culture.

In a waste into resource strategy, a selection of polyhydroxybutyrate (PHB)-accumulating organisms from activated sludge was achieved in an open continuous culture under acetic acid and phosphorus limitation. Once the microbial population was selected at a dilution rate (D), an increase in phosphorus limitation degree was applied in order to study the intracellular phosphorus plasticity of selected bacteria and the resulting capacity to produce PHB. Whatever D, all selected populations were able to produce PHB. At a D, the phosphorus availability determined the phosphorus-cell content which in turn fixed the amount of cell. All the remaining carbon was thus directed toward PHB. By decreasing D, microorganisms adapted more easily to higher phosphorus limitation leading to higher PHB content. A one-stage continuous reactor operated at D=0.023h(-)(1) gave reliable high PHB productivity with PHB content up to 80%. A two-stage reactor could ensure better productivity while allowing tuning product quality.

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. The maximal growth yield was determined at 0.16 Cmole Cmole(-1) in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (µmax = 0.18 h(-1) ) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l(-1) .

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.

Phosphorus limitation strategy to increase propionic acid flux towards 3-hydroxyvaleric acid monomers in Cupriavidus necator.

Properties of polyhydroxybutyrate-co-hydroxyvalerate (P(3HB-co-3HV)) depend on their 3HV content. 3HV can be produced by Cupriavidus necator from propionic acid. Few studies explored carbon distribution and dynamics of 3HV and 3HB monomers production, and none of them have been done with phosphorus as limiting nutrient. In this study, fed-batch cultures of C. necator with propionic acid, as sole carbon source or mixed with butyric acid, were performed. Phosphorus deficiency allowed sustaining 3HV production rate and decreasing 3HB production rate, leading to an instant production of up to 100% of 3HV. When a residual growth is sustained by a phosphorus feeding, the maximum 3HV percentage produced from propionic acid is limited to 33% (Mole.Mole(-1)). The association of a second carbon source like butyric acid lead to higher conversion of propionic acid into 3HV. This study showed the importance of the limiting nutrient and of the culture strategy to get the appropriate product.

Polyhydroxybutyrate production by direct use of waste activated sludge in phosphorus-limited fed-batch culture.

Polyhydroxybutyrate (PHB) production directly by waste activated sludge (WAS) was investigated in aerobic fed-batch conditions using acetic acid as substrate. PHB production was induced by phosphorus limitation. WAS of different origin were tested with various degrees of phosphorus limitation and PHB contents of up to 70% (gCOD PHB/gCOD particulate) were obtained. This strategy showed the importance of maintaining cell growth for PHB production in order to increase PHB concentration and that the degree of phosphorus limitation has a direct impact on the quantity of PHB produced. Pyrosequencing of 16S rRNA transcripts showed changes in the active bacteria of the WAS microbial community as well as the acclimation of populations depending on sludge origin. The monitoring of the process appeared as the key factor for optimal PHB production by WAS. Different strategies are discussed and compared in terms of carbon yield and PHB content with the feast and famine selection process.

Impact of sustaining a controlled residual growth on polyhydroxybutyrate yield and production kinetics in Cupriavidus necator.

In this study a complementary modeling and experimental approach was used to explore how growth controls the NADPH generation and availability, and the resulting impact on PHB (polyhydroxybutyrate) yields and kinetics. The results show that the anabolic demand allowed the NADPH production through the Entner-Doudoroff (ED) pathway, leading to a high maximal theoretical PHB production yield of 0.89 C mole C mole(-1); whereas without biomass production, NADPH regeneration is only possible via the isocitrate dehydrogenase leading to a theoretical yield of 0.67 C mole C mole(-1). Furthermore, the maximum specific rate of NADPH produced at maximal growth rate (to fulfil biomass requirement) was found to be the maximum set in every conditions, which by consequence determines the maximal PHB production rate. These results imply that sustaining a controlled residual growth improves the PHB specific production rate without altering production yield.