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 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.

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 The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene,adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

2015 ◽  
Vol 197 (8) ◽  
pp. 1386-1393 ◽  
Author(s):  
Jonathan Lo ◽  
Tianyong Zheng ◽  
Shuen Hon ◽  
Daniel G. Olson ◽  
Lee R. Lynd
Keyword(s):  
Ethanol Production ◽  
Aldehyde Dehydrogenase ◽  
Clostridium Thermocellum ◽  
Deletion Strain ◽  
Aldh Activity ◽  
Content Type ◽  
Ethanol Formation ◽  
Cell Extracts ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dehydrogenase Gene

ABSTRACTThermoanaerobacterium saccharolyticumandClostridium thermocellumare anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene,adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of theadhEgene inC. thermocellumandT. saccharolyticum. Deletion ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In bothadhEdeletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. InT. saccharolyticum, theadhEdeletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in theT. saccharolyticumparent strain but did not increase activity in theadhEdeletion strain, suggesting that ALDH activity was inhibited. InC. thermocellum, theadhEdeletion strain lost >90% of ALDH and ADH activity in cell extracts. TheC. thermocellumadhEdeletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase.IMPORTANCEThermoanaerobacterium saccharolyticumandClostridium thermocellumare bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation ofadhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In strains withoutadhE, we note changes in biochemical activity, product formation, and growth.

scholarly journals Deletion ofnfnABin Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

2015 ◽  
Vol 197 (18) ◽  
pp. 2920-2929 ◽  
Author(s):  
Jonathan Lo ◽  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
Natalie Ruppertsberger ◽  
Shital A. Tripathi ◽  
...  
Keyword(s):  
Ethanol Yield ◽  
Oxidoreductase Activity ◽  
Fermentation Products ◽  
Content Type ◽  
Ferredoxin Oxidoreductase ◽  
Ethanol Formation ◽  
Cell Extracts ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Adh Activity ◽  
Reversible Transfer

ABSTRACTNfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP+. The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains ofThermoanaerobacterium saccharolyticumengineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts ofT. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. ThenfnABgene was deleted in different strains ofT. saccharolyticumto determine its effect on end product formation. In wild-typeT. saccharolyticum, deletion ofnfnABresulted in a 46% increase in H2formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss ofnfnABcaused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnABstrain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnABstrain with unchanged ethanol yield had NADH-linked ADH activity. Deletion ofnfnABcaused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lostnfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct fromnfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity.IMPORTANCEThermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P)+. The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP+and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion ofnfnABand demonstrate a role for NfnAB in metabolism and ethanol formation inThermoanaerobacterium saccharolyticumand show that this may be an important feature among other thermophilic ethanologenic anaerobes.

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 Cloning, sequencing and expression inEscherichia coliof the primary alcohol dehydrogenase gene fromThermoanaerobacter ethanolicusJW200

2000 ◽  
Vol 190 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Peter J Holt ◽  
Richard E Williams ◽  
Keith N Jordan ◽  
Christopher R Lowe ◽  
Neil C Bruce
Keyword(s):  
Alcohol Dehydrogenase ◽  
Primary Alcohol ◽  
Dehydrogenase Gene ◽  
Sequencing And Expression

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 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 Rewiring Lactococcus lactis for Ethanol Production

2013 ◽  
Vol 79 (8) ◽  
pp. 2512-2518 ◽  
Author(s):  
Christian Solem ◽  
Tore Dehli ◽  
Peter Ruhdal Jensen
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Lactococcus Lactis ◽  
Model Organism ◽  
Pyruvate Decarboxylase ◽  
Major Product ◽  
High Yield ◽  
Content Type ◽  
Synthetic Promoters ◽  
In The Wild

ABSTRACTLactic acid bacteria (LAB) are known for their high tolerance toward organic acids and alcohols (R. S. Gold, M. M. Meagher, R. Hutkins, and T. Conway, J. Ind. Microbiol.10:45–54, 1992) and could potentially serve as platform organisms for production of these compounds. In this study, we attempted to redirect the metabolism of LAB model organismLactococcus lactistoward ethanol production. Codon-optimizedZymomonas mobilispyruvate decarboxylase (PDC) was introduced and expressed from synthetic promoters in different strain backgrounds. In the wild-typeL. lactisstrain MG1363 growing on glucose, only small amounts of ethanol were obtained after introducing PDC, probably due to a low native alcohol dehydrogenase activity. When the same strains were grown on maltose, ethanol was the major product and lesser amounts of lactate, formate, and acetate were formed. Inactivating the lactate dehydrogenase genesldhX,ldhB, andldhand introducing codon-optimizedZ. mobilisalcohol dehydrogenase (ADHB) in addition to PDC resulted in high-yield ethanol formation when strains were grown on glucose, with only minor amounts of by-products formed. Finally, a strain with ethanol as the sole observed fermentation product was obtained by further inactivating the phosphotransacetylase (PTA) and the native alcohol dehydrogenase (ADHE).

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