RESUMEN
Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.
Asunto(s)
Ingeniería Metabólica/métodos , Saccharomyces cerevisiae , Ácido Sórbico/análogos & derivados , Fermentación , Glucosa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Sórbico/química , Ácido Sórbico/aislamiento & purificación , Ácido Sórbico/metabolismoRESUMEN
This integrated study shows that waste glycerol can be bio-valorized by the fabrication of electrospun scaffolds for stem cells. Human mesenchymal stem cells (hMSC) provide an interesting model of regenerating cells because of their ability to differentiate into osteo-, chrondro-, adipo- and myogenic lineages. Moreover, hMSC have modulatory properties with potential on treatment of immunologic diseases. Electrospun fiber meshes offer tunable mechanical and physical properties that can mimic the structure of the native extracellular matrix, the natural environment where cells inhabit. Following a biorefinery approach, crude glycerol directly recovered from a biodiesel post-reaction stream was fed as major C source to Cupriavidus necator DSM 545 to produce polyhydroxyalkanoates at polymer titers of 9-25g/L. Two of the P(3HB-4HB-3HV) terpolymers produced, one containing 11.4% 4HB and 3.5% 3HV and the other containing 35.6% 4HB and 3.4% 3HV, were electrospun into fibers of average diameters of 600 and 1400nm, respectively. hMSC were cultured for 7 days in both fiber meshes, showing their ability to support stem cell growth at acceptable proliferation levels. Comparative results clearly demonstrate that scaffold topology is critical, with electrospun PHA fibers succeeding on the support of significant cell adhesion and proliferation, where planar PHA films failed.
Asunto(s)
Glicerol/química , Polihidroxialcanoatos/química , Andamios del Tejido/química , Biomarcadores/metabolismo , Adhesión Celular , Técnicas de Cultivo de Célula , Diferenciación Celular , Supervivencia Celular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fenómenos Mecánicos , Nanofibras/química , Nanofibras/ultraestructura , Polihidroxialcanoatos/biosíntesis , Células Madre/citología , Células Madre/metabolismo , ResiduosRESUMEN
Poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) (P(3HB-4HB-3HV)) terpolymers of low 3-hydroxyvalerate (3HV) content (1.7-6.4%) with 4-hydroxybutyrate (4HB) molar fractions from 1.8% to 35.6% were produced by fed-batch cultivation of Cupriavidus necator DSM545. Waste glycerol, γ-butyrolactone and propionic acid were used as main carbon source, 4HB and 3HV precursors, respectively. Uniaxial tensile tests were performed on the corresponding biopolymers. The Young's modulus and tensile strength of P(3HB-4HB-3HV) decreased, whereas the elongation at break increased with the 4HB molar%, following the general trend described for poly(3-hydroxybutyrate-4-hydroxybutyrate) (P(3HB-4HB)) but with pronounced lower elasticity. Differential scanning calorimetry results indicate that the temperature of crystallization and enthalpy of melting decreased as the 4HB% increased. No crystallization was observed in terpolymers containing more than 30% of heteromonomers (4HB and 3HV) even though multiple melting events were detected. Terpolymer fractions of different composition were obtained by solvent-fractionation of the original bacterial terpolymers.
Asunto(s)
Biopolímeros/metabolismo , Cupriavidus necator/metabolismo , Polihidroxialcanoatos/metabolismo , Rastreo Diferencial de Calorimetría , Cromatografía en Gel , Cristalización , Espectroscopía de Resonancia Magnética , Resistencia a la Tracción , TermodinámicaRESUMEN
Short-chain polyhydroxyalkanoate co-polymers (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) (P(3HB-co-4HB)) and terpolymers (poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate)) (P(3HB-4HB-3HV)) were produced using high-cell density fed-batch cultures of Cupriavidus necator DSM 545. C-source for growth and 3HB synthesis was waste glycerol (GRP) from a biodiesel plant. Incorporation of 4HB monomers was promoted by γ-butyrolactone (GBL). Propionic acid (PA), a stimulator of 4HB accumulation, increased the 4HB molar ratio 2-fold, but also acted as 3HV precursor, yielding P(3HB-4HB-3HV). Dissolved oxygen (DOC) was a key parameter for % PHA accumulation and volumetric productivity (Prod(vol)). 4HB molar ratio increased in the presence of PA and with extended accumulation time. By manipulating DOC and cultivation time, P(3HB-4HB) with between 11.4 and 21.5 molar% of 4HB were attained. Similarly, P(3HB-4HB-3HV) was obtained with 4HB molar% between 24.8% and 43.6% and 3HV% from 5.6% to 9.8%. Mw varied between 5.5 × 10(5) and 1.37 × 10(6)Da. PHA production from GRP helps reducing production costs with concomitant GRP valorization.
Asunto(s)
Cupriavidus necator/metabolismo , Glicerol/química , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Liofilización , Espectroscopía de Resonancia MagnéticaRESUMEN
The fatty acid (FA) composition of the bacterial membrane of Cupriavidus necator DSM 545 was assessed during the time course of two-stage fed-batch cultivations for the production of short-chain polyhydroxyalkanoates (PHA). Changes in the relative proportion of straight, methyl and cyclopropyl saturated, unsaturated, hydroxy substituted and polyunsaturated FA were observed, depending on the C sources and cultivation conditions used to favor the synthesis of poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) or poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) (P(3HB-4HB-3HV)), under N limiting conditions. The relative percentage of each FA class was studied using glucose or waste glycerol (GRP), as main C source for P(3HB) production. The FA profile was also assessed when GRP was used together with i) γ-butyrolactone (GBL) (precursor of 4HB monomers) for P(3HB-4HB) synthesis and ii) GBL and propionic acid (PA) (3HV precursor) to yield P(3HB-4HB-3HV). The effect of GBL and PA utilization as PHA monomer precursors on the FA profile of the cell membrane was studied under two different dissolved oxygen concentrations (DOC).
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Cupriavidus necator/metabolismo , Polihidroxialcanoatos/metabolismo , Animales , Biotecnología , Membrana Celular/metabolismo , Tamaño de la Célula , Células Cultivadas , Cupriavidus necator/citología , Ácidos Grasos/análisis , Ácidos Grasos/metabolismo , Fermentación , Glicerol , Análisis de los Mínimos Cuadrados , Polihidroxialcanoatos/análisisRESUMEN
Lactococcus lactis subsp. lactis biovar diacetylactis CRL264 is a natural strain isolated from cheese (F. Sesma, D. Gardiol, A. P. de Ruiz Holgado, and D. de Mendoza, Appl. Environ. Microbiol. 56:2099-2103, 1990). The effect of citrate on the growth parameters at a very acidic pH value was studied with this strain and with derivatives whose citrate uptake capacity was genetically manipulated. The culture pH was maintained at 4.5 to prevent alkalinization of the medium, a well-known effect of citrate metabolism. In the presence of citrate, the maximum specific growth rate and the specific glucose consumption rate were stimulated. Moreover, a more efficient energy metabolism was revealed by analysis of the biomass yields relative to glucose consumption or ATP production. Thus, it was shown that the beneficial effect of citrate on growth under acid stress conditions is not primarily due to the concomitant alkalinization of the medium but stems from less expenditure of ATP, derived from glucose catabolism, to achieve pH homeostasis. After citrate depletion, a deleterious effect on the final biomass was apparent due to organic acid accumulation, particularly acetic acid. On the other hand, citrate metabolism endowed cells with extra ability to counteract lactic and acetic acid toxicity. In vivo 13C nuclear magnetic resonance provided strong evidence for the operation of a citrate/lactate exchanger. Interestingly, the greater capacity for citrate transport correlated positively with the final biomass and growth rates of the citrate-utilizing strains. We propose that increasing the citrate transport capacity of CRL264 could be a useful strategy to improve further the ability of this strain to cope with strongly acidic conditions.