scholarly journals Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Zi-Yong Liu ◽  
De-Chen Jia ◽  
Kun-Di Zhang ◽  
Hai-Feng Zhu ◽  
Quan Zhang ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Ethanol Production ◽  
Biomass Production ◽  
Ethanol Oxidation ◽  
Ethanol Yield ◽  
Ethanol Metabolism ◽  
Content Type ◽  
Atp Production ◽  
Clostridium Ljungdahlii ◽  
Gas Fermentation

ABSTRACT Bioethanol production from syngas using acetogenic bacteria has attracted considerable attention in recent years. However, low ethanol yield is the biggest challenge that prevents the commercialization of syngas fermentation into biofuels using microbial catalysts. The present study demonstrated that ethanol metabolism plays an important role in recycling NADH/NAD+ during autotrophic growth. Deletion of bifunctional aldehyde/alcohol dehydrogenase (adhE) genes leads to significant growth deficiencies in gas fermentation. Using specific fermentation technology in which the gas pressure and pH were constantly controlled at 0.1 MPa and 6.0, respectively, we revealed that ethanol was formed during the exponential phase, closely accompanied by biomass production. Then, ethanol was oxidized to acetate via the aldehyde ferredoxin oxidoreductase pathway in Clostridium ljungdahlii. A metabolic experiment using 13C-labeled ethanol and acetate, redox balance analysis, and comparative transcriptomic analysis demonstrated that ethanol production and reuse shared the metabolic pathway but occurred at different growth phases. IMPORTANCE Ethanol production from carbon monoxide (CO) as a carbon and energy source by Clostridium ljungdahlii and “Clostridium autoethanogenum” is currently being commercialized. During gas fermentation, ethanol synthesis is NADH-dependent. However, ethanol oxidation and its regulatory mechanism remain incompletely understood. Energy metabolism analysis demonstrated that reduced ferredoxin is the sole source of NADH formation by the Rnf-ATPase system, which provides ATP for cell growth during CO fermentation. Therefore, ethanol production is tightly linked to biomass production (ATP production). Clarification of the mechanism of ethanol oxidation and biosynthesis can provide an important reference for generating high-ethanol-yield strains of C. ljungdahlii in the future.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

scholarly journals A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

2012 ◽  
Vol 79 (4) ◽  
pp. 1102-1109 ◽  
Author(s):  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Genetic System ◽  
Content Type ◽  
Suicide Vector ◽  
Double Deletion ◽  
Clostridium Ljungdahlii ◽  
Genomic Studies

ABSTRACTMethods for genetic manipulation ofClostridium ljungdahliiare of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies ofC. ljungdahliihave not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation inC. ljungdahliiwere identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility inC. ljungdahlii. Deletion offliAyielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases,adhE1andadhE2, were deleted individually or together. Deletion ofadhE1, but notadhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of theadhE1-deficient strain. Expression ofadhE1intranspartially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation ofC. ljungdahliito optimize autotrophic biocommodity production.

scholarly journals Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

2012 ◽  
Vol 78 (16) ◽  
pp. 5708-5716 ◽  
Author(s):  
Sun-Mi Lee ◽  
Taylor Jellison ◽  
Hal S. Alper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Directed Evolution ◽  
Ethanol Yield ◽  
Xylose Isomerase ◽  
Mutant Enzyme ◽  
Xylose Utilization ◽  
Xylose Consumption ◽  
Content Type ◽  
Consumption Rates

ABSTRACTThe heterologous expression of a highly functional xylose isomerase pathway inSaccharomyces cerevisiaewould have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways inS. cerevisiaesuffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of thePiromycessp. xylose isomerase (encoded byxylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing agre3knockout andtal1andXKS1overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

scholarly journals Metabolic Response of Clostridium ljungdahlii to Oxygen Exposure

2015 ◽  
Vol 81 (24) ◽  
pp. 8379-8391 ◽  
Author(s):  
Jason M. Whitham ◽  
Oscar Tirado-Acevedo ◽  
Mari S. Chinn ◽  
Joel J. Pawlak ◽  
Amy M. Grunden
Keyword(s):  
Ethanol Production ◽  
Metabolic Response ◽  
Reductase Activity ◽  
Preliminary Investigation ◽  
Rna Seq ◽  
Product Analysis ◽  
Content Type ◽  
Batch Cultures ◽  
Oxygen Exposure ◽  
Clostridium Ljungdahlii

ABSTRACTClostridium ljungdahliiis an important synthesis gas-fermenting bacterium used in the biofuels industry, and a preliminary investigation showed that it has some tolerance to oxygen when cultured in rich mixotrophic medium. Batch cultures not only continue to grow and consume H2, CO, and fructose after 8% O2exposure, but fermentation product analysis revealed an increase in ethanol concentration and decreased acetate concentration compared to non-oxygen-exposed cultures. In this study, the mechanisms for higher ethanol production and oxygen/reactive oxygen species (ROS) detoxification were identified using a combination of fermentation, transcriptome sequencing (RNA-seq) differential expression, and enzyme activity analyses. The results indicate that the higher ethanol and lower acetate concentrations were due to the carboxylic acid reductase activity of a more highly expressed predicted aldehyde oxidoreductase (CLJU_c24130) and thatC. ljungdahlii's primary defense upon oxygen exposure is a predicted rubrerythrin (CLJU_c39340). The metabolic responses of higher ethanol production and oxygen/ROS detoxification were found to be linked by cofactor management and substrate and energy metabolism. This study contributes new insights into the physiology and metabolism ofC. ljungdahliiand provides new genetic targets to generateC. ljungdahliistrains that produce more ethanol and are more tolerant to syngas contaminants.

scholarly journals Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA, Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

2016 ◽  
Vol 199 (3) ◽  
Author(s):  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
Sean J. Murphy ◽  
Xiongjun Shao ◽  
Liang Tian ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Single Gene ◽  
Maximum Yield ◽  
Primary Alcohol ◽  
Content Type ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dehydrogenase Gene ◽  
High Ethanol

ABSTRACT Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at about 90% of the theoretical maximum yield (2 ethanol molecules per glucose equivalent) and a titer of 70 g/liter. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here, we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ∼50%, whereas multiple gene deletions of all annotated alcohol dehydrogenase genes except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked alcohol dehydrogenase (ADH) activity (acetaldehyde-reducing direction) by 93%. IMPORTANCE In this study, we set out to identify the alcohol dehydrogenases necessary for high ethanol production in T. saccharolyticum. Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase gene. Here, we show that both adhA and adhE are needed for high ethanol yield in the engineered strain LL1049. This is the first report showing adhA is important for ethanol production in a native adhA host, which has important implications for achieving higher ethanol yields in other microorganisms.

scholarly journals Bioethanol Production via Syngas Fermentation

2018 ◽  
Vol 156 ◽  
pp. 03025 ◽  
Author(s):  
Irika Anggraini ◽  
Made Tri Ari Penia Kresnowati ◽  
Ronny Purwadi ◽  
Tjandra Setiadi
Keyword(s):  
Ethanol Production ◽  
High Performance ◽  
Organic Nitrogen ◽  
Main Product ◽  
Ethanol Yield ◽  
Optimum Process ◽  
Clostridium Ljungdahlii ◽  
Buffer Composition ◽  
Organic Nitrogen Source ◽  
High Ethanol

Bioconversion of C-1 carbon in syngas through microbial fermentation presents a huge potential to be further explored for ethanol production. Syngas can be obtained from the gasification of lignocellulosic biomass, by which most of carbon content of the biomass was converted into CO and CO2. These gases could be further utilized by carbon-fixing microorganism such as Clostridium sp. to produce ethanol as the end product. In order to obtain an optimum process, a robust and high performance strain is required and thus high ethanol yield as the main product can be expected. In this study, series of batch fermentation was carried out to select high performance strains for ethanol production. Bottle serum fermentations were performed using CO-gas as the sole carbon source to evaluate the potential of some Clostridia species such as Clostridium ljungdahlii, C. ragsdalei, and C. carboxidovorans in producing ethanol at various concentration of yeast extract as the organic nitrogen source, salt concentration, and buffer composition. Strain with the highest ethanol production in the optimum media will be further utilized in the upscale fermentation.

scholarly journals Erratum for Liu et al., “Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide”

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Zi-Yong Liu ◽  
De-Chen Jia ◽  
Kun-Di Zhang ◽  
Hai-Feng Zhu ◽  
Quan Zhang ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Ethanol Metabolism ◽  
Clostridium Ljungdahlii

scholarly journals An open-source multiple-bioreactor system for replicable gas-fermentation experiments: Nitrate feed results in stochastic inhibition events, but improves ethanol production of Clostridium ljungdahlii with CO2 and H2

2019 ◽  
Author(s):  
Christian-Marco Klask ◽  
Nicolai Kliem-Kuster ◽  
Bastian Molitor ◽  
Largus T. Angenent
Keyword(s):  
Steady State ◽  
Ethanol Production ◽  
Open Source ◽  
Production Rates ◽  
Clostridium Ljungdahlii ◽  
Gas Fermentation ◽  
Bioreactor System ◽  
Ph Conditions ◽  
The Impact ◽  
Ph Controlled

AbstractThe pH-value in fermentation broth has a large impact on the metabolic flux and growth behavior of acetogens. A decreasing pH level throughout time due to undissociated acetic acid accumulation is anticipated under uncontrolled pH conditions such as in bottle experiment. As a result, the impact of changes in the metabolism (e.g., due to a genetic modification) might remain unclear or even unrevealed. In contrast, pH-controlled conditions can be easily achieved in commercially available bioreactors. However, their acquisition is costly and their operation is time consuming, and therefore the experiment is often limited to a single bioreactor run. Here, we present a self-built, relatively cheap, and easy to handle open-source multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. The functionality of the MBS was tested in three experiments by cultivating the acetogen Clostridium ljungdahlii with CO2 and H2 at steady-state conditions (=chemostat). The experiments were addressing the questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; (2) does feeding acetate alter the production rate of ethanol; and (3) does feeding nitrate influence the product spectrum under controlled pH conditions with CO2 and H2? We applied four different periods in each experiment ranging from pH 6.0 to pH 4.5. Our data show high reproducibility for gas-fermentation experiments with C. ljungdahlii, using the MBS. We found that feeding acetate did not improve ethanol production, but rather impaired growth and reduced acetate production. Using nitrate as sole N-source, on the other hand, enhanced biomass production even at a low pH. However, we observed differences in growth, acetate, and ethanol production rates between triplicate bioreactors (n=3). We explained the different performances because of stochastic inhibition events, which we observed through the accumulation of nitrite, and which led to complete crashes at different operating times. One of these bioreactors recovered after the crash and showed enhanced ethanol production rates while simultaneously producing less acetate. The MBS offers a great opportunity to perform bench-scale bioreactor experiments at steady-state conditions with replicates, which is especially attractive for academia.

scholarly journals Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

2015 ◽  
Vol 81 (16) ◽  
pp. 5440-5448 ◽  
Author(s):  
Andrew C. Tolonen ◽  
Trevor R. Zuroff ◽  
Mohandass Ramya ◽  
Magali Boutard ◽  
Tristan Cerisy ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Complex Cation ◽  
Pyruvate Decarboxylase ◽  
Pathway Engineering ◽  
Solvent Tolerance ◽  
Potential Cost ◽  
Content Type ◽  
Clostridium Phytofermentans

ABSTRACTNovel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacteriumClostridium phytofermentanswas adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression ofZymomonas mobilispyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.

scholarly journals High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes

2011 ◽  
Vol 77 (23) ◽  
pp. 8288-8294 ◽  
Author(s):  
D. Aaron Argyros ◽  
Shital A. Tripathi ◽  
Trisha F. Barrett ◽  
Stephen R. Rogers ◽  
Lawrence F. Feinberg ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Wild Type Strain ◽  
Ethanol Yield ◽  
Fold Increase ◽  
Substantial Improvement ◽  
Wild Type ◽  
Content Type ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Lactic Acids ◽  
High Ethanol

ABSTRACTThis work describes novel genetic tools for use inClostridium thermocellumthat allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the nativeC. thermocellum hptgene and theThermoanaerobacterium saccharolyticum tdkgene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The ΔldhΔptamutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of bothC. thermocellumandT. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.

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