Construction of isopropanol-producing bacterial strains and product recovery Simon Dusséaux1, Truus de Vrije2, Christian Croux1, Philippe Soucaille1, Ana M. López-Contreras2 and Isabelle Meynial-Salles1 1 : INRA LISBP UMR INSA-INRA 792, 135 Avenue de Rangueil, F-31077 Toulouse, France 2 : Food and Biobased Research, Wageningen UR, Bornse Weilanden 9, 6708WG Wageningen, The Netherlands INTRODUCTION RESULTS Clostridium acetobutylicum was metabolically engineered to construct a stable strain that would produce an Isopropanol Butanol Ethanol mixture (IBE) with a low amount of other by-products. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in the C. acetobutylicum ATCC 824 ∆cac15∆upp∆buk strain that can no longer produce butyrate due a stable deletion of butyrate kinase and phosphotransbutyrylase encoding genes. Acetoacetyl-CoA The evaluation of the impact of culture pH on controlled batch fermentation of the 824∆bukpCLF952 showed that best conditions were reached at pH 4.8 : Secondary alcohol dehydrogenase (sAdh) CoAT Adc (ctfA,ctfB) The initial evaluation of the recombinant strains in batch culture in CGM medium at pH5 showed that when the isopropanol operon was expressed from the thl promoter isopropanol, butanol and ethanol titers were higher Acetone Acetoacetate Isopropanol Acetyl-CoA Acetate adc ter catP adc ter adc catP adc sadh pCLF942 pCLF952 pCLF942 pCLF942 6674 bp ColE1 IBE yield : 0.34 g.g-1 glucose 6674 bp ColE1 repL ctfA Isopropanol yield : 0.083 g.g-1 glucose ctfA -35 pTHL -35 IBE concentration : 20.63 g/L ctfB ctfB repL Isopropanol Final titer: 5 g/L sadh pPtb Productivity : 0.8 g/l.h -10 -10 Pthl = constitutive promoter Pptb = constitutive promoter: 2 or 3 > Pthl (Girbal et al. 2003) pCLF952 and pCLF942 plasmids used for expression of the synthetic isopropanol pathway into C. acetobutylicum : pthl: thiolase promoter; Pptb : phosphotransbutyrylase promoter ; ctfA: gene encoding the CoA transferase subunit A; ctfB: gene encoding the CoA transferase subunit B; sadh: gene encoding the secondary alcohol dehydrogenase; adc: gene encoding the acetoacetate decarboxylase Glucose The isopropanol production in batch cultures seems to be limited by the [acetate]intracellular and the high KM of CoAT for acetate (1200 mM) H2 Nfor 2 Pyruvate Pfor CO2 Fed-Ox NAD(P)+ Fed-Red NAD(P)H Growth and metabolic profiles of 824 Δbuk pCLF952 fermentations at various fermentation pH values 2H+ 2 Acetate Acetoacetate NADP+ NADPH CoAT NAD+ Acetoacetyl-CoA NAD+ β-hydroxybutyryl-CoA Secondary alcohol dehydrogenase Crotonyl-CoA C. beijerinkii NRRLB593 Butyrate NADH NAD+ NADH sAdh BK 2 Ethanol 2 Acetaldehyde NADH Adc Acetone Isopropanol 2 Acetyl-CoA 2 Acetyl-P Butyryl-P PTB NADH NADH NAD+ NAD+ Butyryl-CoA NADH Butyraldehyde NAD+ Butanol Schematic view of the metabolism of the engineered 824 ∆buk pCLF942 or pCLF952 strains. CoAT: CoA transferase; Adc: acetoacetate decarboxylase; sAdh: secondary alcohol dehydrogenase; Buk: butyrate kinase; Ptb: phosphotransbutyrylase; Pfor: pyruvate ferredoxin oxidoreductase. Fermentation parameters have been optimized to maximize titers, yields on glucose and productivities. Furthermore, by applying a flux analysis, we identified one bottleneck of the isopropanol pathway to further improve the biofuel yield by an enzyme engineering approach. Finally, to relieve product toxicity and increase productivity of the process, the removal of the IBE from the broth during fermentation was studied using two kinds of methods i) gas stripping and ii) adsorption (High Si-zeolite or activated carbon). CONCLUSIONS A stable metabolically engineered C. acetobutylicum strain was developed for an efficient IBE production with a low amount of by-products The high performances described in this work for IBE production in batch culture have never been obtained with clostridial species Productivities could be 1.5 times increased by gas stripping recovery of the products. A new method using adsorption (using activated carbon or a zeolite) at the place of gas stripping was developed and validated in Wageningen. This new method is being evaluated by modeling to determine energy requirement compared to conventional methods. Adsorption column External pH, [acetate] intracellular , CoAT in vivo activity , isopropanol IBE can be produced from Xylose and Xylan (from oat-spelt) at similar productivities than on glucose When the IBE was removed during fermentation either by gas stripping (using N2 gas) 1or by adsorption (using activated carbon or a zeolite), the productivity of the cultures increased by 50 % compared to the simple batch cultures. Based on the kinetic parameters measured during batch the fermentations performed with C. beijerinckii, a natural isopropanol producer, or with the 824∆bukpCLF952 recombinant strain, the efficiency of the IBE extraction by gas stripping was evaluated and results showed that 1.5 times increase in IBE productivity could be expected: C. beijerinckii WT C. acetobutylicum Δbuk pCLF952 (pH4.8) Batch control Gas stripped Batch ontrol2 Gas-stripped1 Sugar cons. rate (g/Lh) 0.57 0.9 2.32 3.31 IBE productivity (g/Lh) 0.16 0.29 0.80 1.23 I productivity (g/Lh) 0.05 0.09 0.19 0.29 Yield IBE (g/ g sugar) 0.32 Nd 0.34 N2 + IBE + Water Condenser Condenser 1 estimated 2 Dusseaux et al (2013) N2 REFERENCES Wash water Contacts IBE gas stripping or adsorption recovery : Ana Lopez-Contreras, DLO, Wageningen, The Netherlands : [email protected] Strain metabolic engineering : Isabelle Meynial-Salles, INRA LISBP Toulouse France : [email protected] De Vrije T, Budde M, van der Wal H, Claassen PAM, López-Contreras AM: "In situ" removal of isopropanol, butanol and ethanol from fermentation broth by gas stripping. Bioresource Technology 2013, 137:153-159 (http://dx.doi.org/110.1016/j.biortech.2013.1003.1098). Dusséaux S, Croux C, Soucaille P, Meynial-Salles I. 2013. Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture. Metab Eng.,18,1-8. Girbal, L., Mortier-Barrière, I., Raynaud, F., Rouanet, C., Croux, C., Soucaille, P., 2003. Development of a Sensitive Gene Expression Reporter System and an Inducible PromoterRepressor System for Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 69, 4985-4988.
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