Eric Lombard

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.

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.

In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis

This work combines physical and biochemical analyses to scrutinize liquefaction and saccharification of complex lignocellulose materials. A multilevel analysis (macroscopic: rheology, microscopic: particle size and morphology and molecular: sugar product) was conducted at the lab-scale with three matrices: microcrystalline cellulose (MCC), Whatman paper (WP) and extruded paper-pulp (PP). A methodology to determine on-line viscosity is proposed and validated using the concept of Metzner and Otto (1957) and Rieger and Novaks (1973). The substrate suspensions exhibited a shear-thinning behaviour with respect to the power law. A structured rheological model was established to account for the suspension viscosity as a function of shear rate and substrate concentration. The critical volume fractions indicate the transition between diluted, semi-diluted and concentrated regimes. The enzymatic hydrolysis was performed with various solid contents: MCC 273.6 gdm/L, WP 56.0 gdm/L, PP 35.1 gdm/L. During hydrolysis, the suspension viscosity decreased rapidly. The fibre diameter decreased two fold within 2 h of starting hydrolysis whereas limited bioconversion was obtained (10-15%).

Assessment of Candida shehatae viability by flow cytometry and fluorescent probes.

Quantification of different physiological states of Candida shehatae cells was performed by flow cytometry associated with two fluorescent probes. Propidium iodide and carboxyfluorescein diacetate acetoxymethyl ester fluorescent dyes were chosen based on data from the literature. A staining procedure, developed from the previous works was applied to the yeast. Then, the protocol was improved to fit with fermentation constraints such as no physiological interference between the staining procedure and the cells, shortest preparation time and small amounts of dyes. From this optimisation, propidium iodide was included in the sample at 8 mg/L whereas carboxyfluorescein was first diluted in Pluronic® agent and used at 3mg/L, samples were incubated for 10 min at 40°C. Repeatability and accuracy were evaluated to validate this flow cytometry procedure for viability determination.