Acetone production with metabolically engineered strains of Acetobacterium woodii

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_act_thlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4 mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters P_thlA and P_pta-ack. Batch fermentations using A. woodii [pMTL84151_act_thlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high K_m value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_act_thlA] was increased from 1.2 mg L⁻¹ h⁻¹ in bottle fermentation to 26.4 mg L⁻¹ h⁻¹ in continuous gas fermentation.     

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.     

Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli

A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 g l^?1 cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5–2 g l^?1 α-ketoglutarate or 1.0 g l^?1 citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6 l fermentor study, a 23.5 g l^?1 cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.     

Targeted mutagenesis of the Clostridium acetobutylicum acetone–butanol–ethanol fermentation pathway

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone–butanol–ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100 mM ethanol, no acetone and very little (7 mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.     

Production of isopropanol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.     

Characterization of a highly thermostable ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824

Higher energy content and hydrophobicity make bio-based n-butanol a preferred building block for chemical and biofuels manufacturing. Butanol is obtained by Clostridium sp. based ABE fermentation process. While the ABE process is well understood, the enzyme systems involved have not been elucidated in detail. The important enzyme ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824 (Hbd) was purified and characterized. Surprisingly, Hbd shows extremely high temperature (T > 60 °C), pH (4–11) and solvent (1-butanol, isobutanol, ethanol) stability. Hbd catalyzes acetoacetyl CoA hydration to ß-hydroxybutyryl CoA up to pH 9.5, where the reaction is reversed. Substrate (acacCoA, ß-hbCoA) and cofactor (NADH, NAD⁺, NADPH and NADP⁺) specificities were determined. We identified NAD⁺ as an uncompetitive inhibitor. Identification of process relevant enzymes such as Hbd is key to optimize butanol production via cellular or cell-free enzymatic systems.     

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2–2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD^D485G variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad^D485G) ABE products resulted in a blend with nearly 50 wt%≤C₉ products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9 g L⁻¹ while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80 wt% of catalysis products having a carbon chain length≥C₁₁ which amounts to 9.8 g L⁻¹ of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.     

Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702

Biosynthetic thiolases (EC 2.3.1.9) are key enzymes in the branched catabolism of diverse clostridia as their activity and regulation influence the production of organic acids and solvents. In Clostridium butyricum, they are also involved in the production of hydrogen as a sustainable and environmentally benign energy source. In this study, the gene coding for thiolase from C. butyricum DSM 10702 was cloned by genome walking. It was found to consist of 1179 bp coding for a protein with 393 amino acids and a deduced molecular weight of 41.4 kDa. The enzyme was fused to an N-terminal his-tag, expressed in Escherichia coli, purified to near homogeneity and characterised for biochemical and kinetic properties. Gel filtration chromatography revealed that the catalytically active enzyme consists of a homotetramer. The enzyme showed a K_M of ～32 μM towards acetoacetyl-CoA and of ～21 μM towards CoASH at 30 °C and pH 8.0. Claisen condensation of acetyl-CoA by thiolase was analysed in a coupled enzyme assay, where β-hydroxybutyryl-CoA dehydrogenase was applied catalysing the subsequent NADH-dependant reduction of the formed condensation product acetoacetyl-CoA. For this purpose the latter enzyme was cloned from C. butyricum DSM 10702 and recombinantly expressed in E. coli. The K_M of thiolase towards acetyl-CoA was ～674 μM at 30 °C and pH 7.5. Acetyl-CoA condensation was inhibited even at micromolar concentrations of CoASH indicating that CoASH has an important regulatory function in vivo.     

Uptake and activation of acetate and butyrate in Clostridium acetobutylicum

Summary The pathway for uptake of acids during the solvent formation phase of an acetone-butanol fermentation by Clostridium acetobutylicum ATCC 824 was studied. ¹³C NMR investigations on actively metabolizing cells showed that butyrate can be taken up from the medium and quantitatively converted to butanol without accumulation of intermediates. The activities of acetate phosphotransacetylase, acetate kinase and phosphate butyryltransferase rapidly decreased to very low levels when the organism began to form solvents. This indicates that the uptake of acids does not occur via a reversal of these acid forming enzymes. No short-chain acyl-CoA synthetase activity or butyryl phosphate reducing activity could be detected. Based on our results and a critical analysis of literature data on acetone-butanol fermentations, it is suggested that an acetoacetyl-CoA: acetate (butyrate) CoA-transferase is solely responsible for uptake and activation of acetate and butyrate in C. acetobutylicum. The transferase exhibits a broad carboxylic acid specificity. The key enzyme in the uptake is acetoacetate decarboxylase, which is induced late in the fermentation and pulls the transferase reaction towards formation of acetoacetate. The major implication is that it is not feasible to obtain a batch-wise butanol fermentation without acetone formation and retention of a good yield of butanol.     

Xylose-based hydrolysate from eucalyptus extract as feedstock for poly(lactate-co-3-hydroxybutyrate) production in engineered Escherichia coli

Woody extract-derived hemicellulosic hydrolysate, which was obtained from dissolving pulp manufacturing, was utilized as feedstock for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] in engineered Escherichia coli. The hydrolysate was composed of mainly xylose and galactose, and contained impurities mainly acetate, which was found to inhibit the polymer synthesis rather than the cell growth. Thus, acetate and other impurities were removed through active charcoal and ion-exchange columns. Using the purified hydrolysate, P(LA-co-3HB) was successfully produced (cell dry weight 8.6 g/L, polymer concentration 5.4 g/L, LA fraction 5.5 mol%, polymer content 62.4%), the amount of which was comparable to that obtained using reagent grade xylose and galactose. Therefore, the hydrolysate from woody extract is considered as an abundant, inexpensive and efficient feedstock applicable to consolidated process for P(LA-co-3HB) production, when the removal of acetic acid was satisfactorily accomplished.     

Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies

We previously reported development of a metabolic pathway in Escherichia coli for overproduction of medium-chain methyl ketones (MK), which are relevant to the biofuel and flavor-and-fragrance industries. This MK pathway was a re-engineered version of β-oxidation designed to overproduce β-ketoacyl-CoAs and involved overexpression of the fadM thioesterase gene. Here, we document metabolic engineering modifications that have led to a MK titer of 3.4 g/L after ~45 h of fed-batch glucose fermentation and attainment of 40% of the maximum theoretical yield (the best values reported to date for MK). Modifications included balancing overexpression of fadR and fadD to increase fatty acid flux into the pathway, consolidation of the pathway from two plasmids into one, codon optimization, and knocking out key acetate production pathways. In vitro studies confirmed that a decarboxylase is not required to convert β-keto acids into MK and that FadM is promiscuous and can hydrolyze several CoA-thioester pathway intermediates.     

Improving the performance of solventogenic clostridia by reinforcing the biotin synthetic pathway

An efficient production process is important for industrial microorganisms. The cellular efficiency of solventogenic clostridia, a group of anaerobes capable of producing a wealth of bulk chemicals and biofuels, must be improved for competitive commercialization. Here, using Clostridium acetobutylicum, a species of solventogenic clostridia, we revealed that the insufficient biosynthesis of biotin, a pivotal coenzyme for many important biological processes, is a major limiting bottleneck in this anaerobe’s performance. To address this problem, we strengthened the biotin synthesis of C. acetobutylicum by overexpressing four relevant genes involved in biotin transport and biosynthesis. This strategy led to faster growth and improved the titer and productivity of acetone, butanol and ethanol (ABE solvents) of C. acetobutylicum in both biotin-containing and biotin-free media. Expressionally modulating these four genes by modifying the ribosome binding site further promoted cellular performance, achieving ABE solvent titer and productivity as high as 21.9 g/L and 0.30 g/L/h, respectively, in biotin-free medium; these values exceeded those of the wild-type strain by over 30%. More importantly, biotin synthesis reinforcement also conferred improved ability of C. acetobutylicum to use hexose and pentose sugars, further demonstrating the potential of this metabolic-engineering strategy in solventogenic clostridia.     

Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli

A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588) — a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240 AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.     

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol_OA/Cmol_lac) and a higher P(3HB) production (18.88 g_P(3HB)/L) were achieved, comparing to the original recombinant strain (0.11 Cmol_OA/Cmol_lac and 7.8 g_P(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol_P(3HB)/Cmol_lac vs 0.10 Cmol_P(3HB)/Cmol_lac of the original strain).     

Modifying the product pattern of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis>

Clostridial acetone–butanol–ethanol (ABE) fermentation is a natural source for microbial n-butanol production and regained much interest in academia and industry in the past years. Due to the difficult genetic accessibility of Clostridium acetobutylicum and other solventogenic clostridia, successful metabolic engineering approaches are still rare. In this study, a set of five knock-out mutants with defects in the central fermentative metabolism were generated using the ClosTron technology, including the construction of targeted double knock-out mutants of C. acetobtuylicum ATCC 824. While disruption of the acetate biosynthetic pathway had no significant impact on the metabolite distribution, mutants with defects in the acetone pathway, including both acetoacetate decarboxylase (Adc)-negative and acetoacetyl-CoA:acyl-CoA transferase (CtfAB)-negative mutants, exhibited high amounts of acetate in the fermentation broth. Distinct butyrate increase and decrease patterns during the course of fermentations provided experimental evidence that butyrate, but not acetate, is re-assimilated via an Adc/CtfAB-independent pathway in C. acetobutylicum. Interestingly, combining the adc and ctfA mutations with a knock-out of the phosphotransacetylase (Pta)-encoding gene, acetate production was drastically reduced, resulting in an increased flux towards butyrate. Except for the Pta-negative single mutant, all mutants exhibited a significantly reduced solvent production.     

Geranyl diphosphate synthase: An important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli

The expression level of geranyl diphosphate synthase (GPPS) was suspected to play a key role for geraniol production in recombinant Escherichia coli harboring an entire mevalonate pathway operon and a geraniol synthesis operon. The expression of GPPS was optimized by using ribosomal binding sites (RBSs) designed to have different translation initiation rates (TIRs). The RBS strength in TIR window of 500 arbitrary unit (au)–1400 au for GPPS appears to be suitable for balancing the geraniol biosynthesis pathway in this study. With the TIR of 500 au, the highest production titer of geraniol was obtained at a level of 1119 mg/L, which represented a 6-fold increase in comparison with the previous titer of 183 mg/L. The TIRs of GPPS locating out of range of the optimal window (500–1400 au) caused significant decreases of cell growth and geraniol production. It was suspected to result from metabolic imbalance and plasmid instability in geraniol production by inappropriate expression level of GPP synthase. Our results collectively indicated GPPS as an important regulation point in balancing a recombinant geraniol synthesis pathway. The GPPS-based regulation approach could be applicable for optimizing microbial production of other monoterpenes.     

Thermophilic α-glucan phosphorylase from Clostridium thermocellum: Cloning,characterization and enhanced thermostability

ORF Cthe0357 from the thermophilic bacterium Clostridium thermocellum ATCC 27405 that encodes a putative α-glucan phosphorylase (αGP) was cloned and expressed in Escherichia coli. The protein with a C-terminal His-tag was purified by Ni²⁺ affinity chromatography; the tag-free protein obtained from a cellulose-binding module–intein–αGP fusion protein was purified through affinity adsorption on amorphous cellulose followed by intein self-cleavage. Both purified enzymes had molecular weights of ca. 81,000 and similar specific activities. The optimal conditions were pH 6.0–6.5 and 60 °C for the synthesis direction and pH 7.0–7.5 and 80 °C for the degradation direction. This enzyme had broad substrate specificities for different chain length dextrins and soluble starch. The thermal inactivation of this enzyme strongly depended on temperature, protein concentration, and certain addictives that were shown previously to benefit the protein thermostability. The half lifetime of 0.05 mg αGP/mL at 50 °C was extended by 45-fold to 90 h through a combined addition of 0.1 mM Mg²⁺, 5 mM DTT, 1% NaCl, 0.1% Triton X-100, and 1 mg/mL BSA. The enzyme with prolonged stability would work as a building block for cell-free synthetic enzymatic pathway biotransformations, which can implement complicated biocatalysis through assembly of a number of enzymes and coenzymes.     

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2 g/L of AECC and produce 22.1 g/L of solvents (4.25 g/L acetone, 11.5 g/L butanol and 6.37 g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.     

Production of an anti-MUC1 C595 dbFv antibody fragment in recombinant Escherichia coli

The production of C595 diabody fragment (dbFv) in Escherichia coli (E. coli) HB2151 clone has been explored. The comparison of fermentation processes mode demonstrated that a higher biomass inoculum operation enhanced C595 dbFv production. It was demonstrated that a concentration of 12.1 mg l⁻¹ broth of dbFv and a cell concentration of 23.6 g l⁻¹ broth were achieved at the end of 75 l fermentation.