E. Terry Papoutsakis

Bio: E. Terry Papoutsakis is an academic researcher from Delaware Biotechnology Institute. The author has contributed to research in topics: Clostridium acetobutylicum & Fermentation. The author has an hindex of 5, co-authored 6 publications receiving 312 citations. Previous affiliations of E. Terry Papoutsakis include University of Delaware.

Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

Abstract: Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adhB-593) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum

Abstract: Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5′UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5′UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

Characterization and evaluation of corn steep liquid in acetone-butanol-ethanol production by Clostridium acetobutylicum

Abstract: Corn steep liquor (CSL) obtained from a commercial starch manufacturing process was analyzed and tested as a complex nutrient source for ABE (acetone, butanol, and ethanol) production by Clostridium acetobutylicum PJC4BK_AdhE1. CSL contained a wealth of trace elements and nitrogenous components, proteins and amino acids. ABE production increased the content of CSL was raised up to 6% (v/v) in medium and then decreased at higher contents. In 6% CSL-containing medium, C. acetobutylicum PJC4BK_AdhE1 produced 21.4 g/L of ABE with a yield of 0.41 g/g in 18 h of fermentation. Although the final titer of ABE was similar in CSL containing media and Clostridial Growth Medium (CGM, containing yeast extract and asparagines as complex nutrients), the yield and productivity of ABE in 6% CSL-containing medium were found to be higher than 32 and 26%, respectively.

Genome analysis of a hyper acetone-butanol-ethanol (ABE) producing Clostridium acetobutylicum BKM19.

Abstract: Previously the development of a hyper acetone-butanol-ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re-sequenced by a high-throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlAV5A ) is showed a 32% higher activity than that of the wild-type thiolase (thlAWT ). In batch fermentation, butanol production is increased by 26% and 23% when the thlAV5A gene is overexpressed in the wild-type C. acetobutylicum ATCC 824 and in its derivative, the thlA-knockdown TKW-A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5th residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain.

Microbial electron transport and energy conservation - the foundation for optimizing bioelectrochemical systems.

Abstract: Microbial electrochemical techniques describe a variety of emerging technologies that use electrode–bacteria interactions for biotechnology applications including the production of electricity, waste and wastewater treatment, bioremediation and the production of valuable products Central in each application is the ability of the microbial catalyst to interact with external electron acceptors and/or donors and its metabolic properties that enable the combination of electron transport and carbon metabolism And here also lies the key challenge A wide range of microbes has been discovered to be able to exchange electrons with solid surfaces or mediators but only a few have been studied in depth Especially electron transfer mechanisms from cathodes towards the microbial organism are poorly understood but are essential for many applications such as microbial electrosynthesis We analyze the different electron transport chains that nature offers for organisms such as metal respiring bacteria and acetogens, but also standard biotechnological organisms currently used in bio-production Special focus lies on the essential connection of redox and energy metabolism, which is often ignored when studying bioelectrochemical systems The possibility of extracellular electron exchange at different points in each organism is discussed regarding required redox potentials and effect on cellular redox and energy levels Key compounds such as electron carriers (eg, cytochromes, ferredoxin, quinones, flavins) are identified and analyzed regarding their possible role in electrode–microbe interactions This work summarizes our current knowledge on electron transport processes and uses a theoretical approach to predict the impact of different modes of transfer on the energy metabolism As such it adds an important piece of fundamental understanding of microbial electron transport possibilities to the research community and will help to optimize and advance bioelectrochemical techniques

A comprehensive metabolic map for production of bio-based chemicals

Abstract: Production of industrial chemicals using renewable biomass feedstock is becoming increasingly important to address limited fossil resources, climate change and other environmental problems. To develop high-performance microbial cell factories, equivalent to chemical plants, microorganisms undergo systematic metabolic engineering to efficiently convert biomass-derived carbon sources into target chemicals. Over the past two decades, many engineered microorganisms capable of producing natural and non-natural chemicals have been developed. This Review details the current status of representative industrial chemicals that are produced through biological and/or chemical reactions. We present a comprehensive bio-based chemicals map that highlights the strategies and pathways of single or multiple biological reactions, chemical reactions and combinations thereof towards production of particular chemicals of interest. Future challenges are also discussed to enable production of even more diverse chemicals and more efficient production of chemicals from renewable feedstocks.

Bio-based production of C2–C6 platform chemicals

Abstract: Platform chemicals composed of 2-6 carbons derived from fossil resources are used as important precursors for making a variety of chemicals and materials, including solvents, fuels, polymers, pharmaceuticals, perfumes, and foods. Due to concerns regarding our environment and the limited nature of fossil resources, however, increasing interest has focused on the development of sustainable technologies for producing these platform chemicals from renewable resources. The techniques and strategies for developing microbial strains for chemicals production have advanced rapidly, and it is becoming feasible to develop microbes for producing additional types of chemicals, including non-natural molecules. In this study, we review the current status of the bio-based production of major C2-C6 platform chemicals, focusing on the microbial production of platform chemicals that have been used for the production of chemical intermediates, building block compounds, and polymers.