Engineering Acetobacterium Woodii for the Production of Isopropanol and Acetone From Carbon Dioxide and Hydrogen

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5 mM isopropanol and 7.6 mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438 mM acetic acid, 7.6 mM acetone, and 14.5 mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time.Research in Professor Peter Dürre's lab was funded by the ERA-IB7 project OBAC (FKZ 031B0274B). Research in Professor Christian Kennes' lab was also funded by the collaborative European ERA-IB7 project OBAC as well as PCIN2016-148. The authors in the latter laboratory, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55)Xunta de Galicia; ED431C 2021/55Alemania. Bundesministerium für Bildung und Forschung (BMBF); 031B0274

mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis

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Differential regulation of two thiolase genes from Clostridium acetobutylicum DSM 792.

Thiolase of Clostridium acetobutylicum is an important enzyme involved in both, acid and solvent fermentation. Two thiolase genes (thlA and thlB) have been cloned and sequenced from Clostridium acetobutylicum DSM 792, showing high homology to each other and to thiolases of PHA-synthesizing bacteria. The thlA gene is identical to the gene already cloned and sequenced from strain ATCC 824 (Stim- Herndon et al., 1995, Gene 154: 81-85). Using primer extension and S1 nuclease analysis a transcriptional start site was identified 102 bp upstream of the thlA start codon. This site was preceded by a region that exhibits high similarity to the s70 consensus promoter sequences of Gram-positive and -negative bacteria. Regulation of thlA and thlB was studied at the transcriptional level to elucidate the specific function of each gene. Non-radioactive primer extension analysis using fluorescein-labelled oligonucleotides and Northern blot analysis revealed high levels of thlA transcripts in acid- and solvent-producing cells. During an induced shift of a continuous culture from acid to solvent formation, the transcript level transiently decreased to a minimum, 3 to 7 h after induction. The thlA transcript length is about 1.4 kb, indicating a monocistronic organisation, whereas genetic organization and reverse transcription (RT)-PCR analysis indicated that thlB forms an operon with two other adjacent genes, thlR and thlC’. Transcription and regulation of the thlB operon was studied using RTPCR and showed a very low expression in acid- and solvent-producing cells. Heterologously expressed clostridial ThlB showed high thiolase activity in Escherichia coli. The N-terminal part of ThlR possesses a potential helix-turn-helix motif and shows significant homology to regulatory proteins belonging to the TetR/AcrR family of transcriptional regulators. ThlR possibly acts as a transcriptional repressor of thlB operon expression. The data provide strong evidence that ThlA is involved in the metabolism of both acid and solvent formation, whereas the physiological function of ThlB has yet to be elucidated

Butanol production from lignocellulosic biomass: revisiting fermentation performance indicators with exploratory data analysis

After just more than 100 years of history of industrial acetone–butanol–ethanol (ABE) fermentation, patented by Weizmann in the UK in 1915, butanol is again today considered a promising biofuel alternative based on several advantages compared to the more established biofuels ethanol and methanol. Large-scale fermentative production of butanol, however, still suffers from high substrate cost and low product titers and selectivity. There have been great advances the last decades to tackle these problems. However, understanding the fermentation process variables and their interconnectedness with a holistic view of the current scientific state-of-the-art is lacking to a great extent. To illustrate the benefits of such a comprehensive approach, we have developed a dataset by collecting data from 175 fermentations of lignocellulosic biomass and mixed sugars to produce butanol that reported during the past three decades of scientific literature and performed an exploratory data analysis to map current trends and bottlenecks. This review presents the results of this exploratory data analysis as well as main features of fermentative butanol production from lignocellulosic biomass with a focus on performance indicators as a useful tool to guide further research and development in the field towards more profitable butanol manufacturing for biofuel applications in the future.publishedVersio

Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum

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New Insights into the Physiology of the Propionate Producers <i>Anaerotignum propionicum</i> and <i>Anaerotignum neopropionicum</i> (Formerly <i>Clostridium propionicum</i> and <i>Clostridium neopropionicum</i>)

Propionate is an important platform chemical that is available through petrochemical synthesis. Bacterial propionate formation is considered an alternative, as bacteria can convert waste substrates into valuable products. In this regard, research primarily focused on propionibacteria due to high propionate titers achieved from different substrates. Whether other bacteria could also be attractive producers is unclear, mostly because little is known about these strains. Therefore, two thus far less researched strains, Anaerotignum propionicum and Anaerotignum neopropionicum, were investigated with regard to their morphologic and metabolic features. Microscopic analyses revealed a negative Gram reaction despite a Gram-positive cell wall as well as surface layers for both strains. Furthermore, growth, product profiles, and the potential for propionate formation from sustainable substrates, i.e., ethanol or lignocellulosic sugars, were assessed. Results showed that both strains can oxidize ethanol to different extents. While A. propionicum only partially used ethanol, A. neopropionicum converted 28.3 mM ethanol to 16.4 mM propionate. Additionally, the ability of A. neopropionicum to produce propionate from lignocellulose-derived substrates was analyzed, leading to propionate concentrations of up to 14.5 mM. Overall, this work provides new insights into the physiology of the Anaerotignum strains, which can be used to develop effective propionate producer strains