Ethanol Synthesis Pathway Research Articles

Rare earth ion Nd3+ promotes production of cellulose ethanol by Clostridium thermocellum ATCC 27405

Clostridium thermocellum can directly produce ethanol from cellulose by consolidated bioprocessing. We studied how rare earth ions impact the ethanol titer and found that Nd3+ can significantly increase the production of ethanol from cellulose by C. thermocellum ATCC 27405. With the addition of 10−6 M Nd3+ in the initial incubation, the ethanol titer increased significantly, but the cellulose degradation ratio and cell growth did not change significantly. The intracellular distribution of Nd3+ was determined. Most Nd3+ was located on the surface of the cell wall. Nd3+ increased cell permeability, which was one of the reasons for the ethanol titer increased. We further explored the underlying mechanism from the perspective of key enzymes in the ethanol synthesis pathway. The enzyme activity and enzyme expression level of alcohol dehydrogenase (ADH) of strain C1 with added Nd3+ were lower than those of the original strain C0. However, both the enzyme activity and enzyme expression levels of acetaldehyde dehydrogenase (ALDH), pyruvate-ferredoxin oxidoreductase (PFO) and 6-phosphofructokinase (PFK) were increased. This rule was still valid for the sixth-generation strain C2 with Nd3+ removed. It was verified that the increase in the expression level of ALDH was not caused by the mutation of the ALDH promoter sequence. This research explored the key factors that limited the titer of cellulose ethanol in consolidated bioprocessing from an unconventional perspective and provided a new strategy for improving cellulose ethanol production.

Engineering cyanobacteria with enhanced growth in simulated flue gases for high-yield bioethanol production

Abstract Flue gases emitted from industrial plants mainly contribute to global CO2 emissions. Sequestrations of CO2 from the flue gas could reduce the impact of CO2 on global warming. In this study, recombinant cyanobacterial strains with enhanced photosynthetic activity, cell growth, and ethanol production were generated by co-overexpressing ictB, ecaA, and groESL, along with a heterologous ethanol synthesis pathway (pdc-adhII genes from Zymomonas mobilis) in S. elongatus PCC7942. The engineered S. elongatus PCC7942 exhibited a significant improvement in cell growth and ethanol production under a simulated flue gas consisted of 25% CO2, 80−90 ppm SO2, 90−100 ppm NO. The present work represents the first attempt of direct photoconversion of CO2 from flue gases to ethanol via expression of ictB, ecaA, groESL, and pdc-adhII in S. elongatus. The transgenic cyanobacterium becomes useful for sequestrations of CO2 directly from flue gases with the simultaneous production of bioethanol.

Heterologous Expression of Ethanol Synthesis Pathway in Glycogen Deficient Synechococcus elongatus PCC 7942 Resulted in Enhanced Production of Ethanol and Exopolysaccharides.

In this study, the Synechococcus elongatus PCC 7942 (hereafter S. elongatus) was engineered by the glgC knockout as well as the insertion of the pdc-adh genes from two different microorganisms. The insertion of pdc-adh genes increased the ethanol synthesis with further improvement in the productivity upon the destruction of glycogen synthesis pathway and the supplementation of cofactor. The abolition of glycogen synthesis pathway led to a considerable increase of the engineered S. elongatus metabolites involved in the ethanol synthesis pathway. Moreover, the studies on cofactor addition highlighted the importance of Mg+2, Zn+2, thiamine pyrophosphate, and NADP+ in ethanol synthesis. The yields of 3856 mg/L ethanol and 109.5 µg/108 cells exopolysaccharides were obtained in the engineered S. elongatus using a photo-bioreactor under optimized conditions. This enhanced production in ethanol and exopolysaccharides are attributed to the flux of carbon from glycogen synthesis pathway and proper availability of essential components.

Knockout of acetoacetate degradation pathway gene atoDA enhances the toxicity tolerance of Escherichia coli to isopropanol and acetone.

Isopropanol and acetone are important chemical products and potential high-quality new fuels. Both of them are metabolites of isopropanol synthesis pathway, but they are toxic to most bacteria. In this study, toxicity tolerance of Escherichia coli strains was evaluated by detecting their growth rates under different concentrations of isopropanol and acetone. It was showed that isopropanol was more toxic to E. coli than acetone, and the native strain MG1655 had better tolerance over DH5α to either acetone or isopropanol of 300mM. Key genes of ethanol synthesis pathway, acetic acid metabolism pathway, and acetoacetic acid degradation pathway, including adhE, ackA-pta, and atoDA, were knocked out in MG1655 to form mutants MGΔadhE, MGΔackA-pta, and MGΔatoDA. The tolerance performances of the mutants to isopropanol and acetone were determined under various concentrations including 300mM, 500mM, and 700mM, respectively. The mutant MGΔatoDA exhibited excellent tolerance to both acetone and isopropanol of 500mM, and MGΔackA-pta could tolerate acetone at 500mM rather than isopropanol, while the deletion of adhE in MGΔadhE resulted in a severe cell growth defection. Although isopropanol and acetone at 700mM caused severe growth inhibition on each strain, cell growth could be restored to varying degrees with the prolongation of culture time. This phenomenon was suggested to be related to the volatilization of isopropanol and acetone based on volatilization tests. It was envisioned that MG1655 was a suitable host strain for isopropanol metabolic engineering research, and the acetoacetic acid degradation pathway gene atoDA, was probably the key optimizing point for isopropanol production.

Influence of Tandem Catalysis and Optimised Parameters on Syngas-Dimethyl Ether Co-fed Process for Ethanol Direct Synthesis in a Dual Bed Reactor

Ethanol use mainly as an alternative green fuel has attracted global attention, hence need for the development of more improved synthesis pathways. In this work, we proposed an in situ direct ethanol synthesis pathway, where dimethyl ether (DME) was co-fed with syngas (CO + H2) over a combination of metal impregnated zeolite (Cu-MOR) and ternary oxide CuO/ZnO/Al2O3 (CZA) catalysts, disjointly packed in a dual bed reactor. The route first transformed DME into an intermediate ester (methyl acetate) by carbonylation reaction on the upper modified zeolite catalyst, followed by hydrogenation of the intermediate ester into ethanol on the lower CZA catalyst. Dimethyl ether conversion reached 75.72% with an ethanol selectivity of 48.05% under Cu loading amount of 13 wt% of zeolite support.

Metal Oxide Mediated Extracellular NADPH Regeneration Improves Ethanol Production by Engineered Synechocystis sp. PCC 6803.

The ethanol synthesis pathway engineered Synechocystis sp. PCC 6803 (hereafter Synechocystis) was used to investigate the influence of metal oxide mediated extracellular NADPH regeneration on ethanol synthesis. The in-vitro studies proved that the metal oxides have the potential to generate the NADPH in the presence of electron donor, the usual components of photoautotrophic growth conditions. When the NADPH regeneration was applied in Synechocystsis, the strain showed improved growth and ethanol production. This improved ethanol synthesis is attributed to the increased availability of NADPH to the ethanol synthesis pathway and redirection of closely related carbon metabolism into the ethanol synthesis. Under optimized light intensity and NADP addition, the maximum ethanol production of 5,100 mg/L was observed in MgO mediated extracellular NADPH regeneration after 25 days of cultivation, which is 2-fold higher than the control. This study indicates the feasibility of metal oxide mediated extracellular NADPH regeneration of Synechocystis to increase the production of ethanol.

Systematic identification of a neutral site on chromosome of Synechococcus sp. PCC7002, a promising photosynthetic chassis strain

Cyanobacteria based photosynthetic biomanufacturing is supposed to be one of the alternative routes for sustainable production of biofuels and biochemicals. Synechococcus sp. PCC 7002 is a promising cyanobacterial chassis strain possessing desired properties for scaled cultivation in future. Development of cyanobacterial cell factories requires modifications of PCC7002 chromosome to expand the photosynthesis-based metabolism networks. Therefore, mining genomic neutral sites for stable integration of heterologous genes and pathways would be of great significance for expanding the toolbox for PCC7002 genome engineering. Here we demonstrate a paradigm for identification of potential neutral sites from chromosome of PCC7002 based on genomic and transcriptomics data. By refining the massive omics information from database, 51 putative sites with no significant physiological or metabolism effects were extracted as candidates. Combining genomic context analysis, a locus termed as NS0027 between two neighboring putative neutral genes was selected and evaluated as a neutral site for genetic integration. In addition, an ethanol synthesis pathway was introduced into the NS0027 site to assess the functionality of this site. The sites we identified and the strategy we adopted in this work would benefit the development of effective genetic toolbox and efficient photosynthetic cell factories based on PCC7002.

A novel strategy for production of ethanol and recovery of xylose from simulated corncob hydrolysate.

To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery. Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E. coli B0013-1030 (pta-ack, ldhA, pflB, xylH) by deletion of frdA, xylA and xylE. It exhibited robust growth on plates containing glucose, arabinose or galactose, but failed to grow on xylose. The ethanol synthesis pathway was then introduced into B0013-2012 to create an ethanologenic strain B0013-2012PA. In shaking flask fermentation, B0013-2012PA fermented glucose to ethanol with the yield of 48.4g/100g sugar while xylose remained in the broth. In a 7-l bioreactor, B0013-2012PA fermented glucose, galactose and arabinose in the simulated corncob hydrolysate to 53.4g/l ethanol with the yield of 48.9g/100g sugars and left 69.6g/l xylose in the broth, representing 98.6% of the total xylose in the simulated corncob hydrolysate. By using newly constructed strain B0013-2012PA, we successfully developed an efficient bioprocess for ethanol production and xylose recovery from the simulated corncob hydrolysate.

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP+ reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

Combinatory strategy for characterizing and understanding the ethanol synthesis pathway in cyanobacteria cell factories.

BackgroundPhotosynthetic production of chemicals and fuels by recycling CO2 in cyanobacteria is a promising solution facing energy shortage and resource declination. Ethanol is an attractive and demonstrative biofuel product, and ethanol synthesis in cyanobacteria has been achieved by assembling of a pathway consisting of pyruvate decarboxylase (PDCzm) and alcohol dehydrogenase II (slr1192). For enabling more powerful ethanol photosynthetic production, an optimized and balanced catalyzing route was required. In this work, we provided a paradigm for systematically characterizing and optimizing the PDCzm-slr1192 pathway from engineered cyanobacteria strains, combining in vitro reconstitution, genetic engineering and feeding-cultivation.ResultsWe reconstituted the PDCzm-slr1192 pathway in vitro and performed specific titration assays for enzymes, substrates, cofactors, and metal ions. In the in vitro system, K50 of PDCzm was 0.326 μM, with a Vmax of 2.074 μM/s; while for slr1192, the values were 0.109 μM and 1.722 μM/s, respectively. Titration response discrepancy indicated that PDCzm rather than slr1192 was the rate-limiting factor for ethanol synthesis. In addition, a 4:6 concentration ratio of PDCzm-slr1192 would endow the reaction with a maximal specific catalytic activity. Titration assays for other components were also performed. Km values for NADPH, pyruvate, TPP, Mg2+ and acetaldehyde were 0.136, 6.496, 0.011, 0.104, and 0.393 mM, respectively. We further constructed Synechocystis mutant strains with diverse PDCzm-slr1192 concentrations and ratios, and compared the growth and ethanol synthesis performances. The results revealed that activities of PDCzm indeed held control over the ethanol generation capacities. We performed pyruvate-feeding treatment with the newly developed Syn-YQ4 strain, and confirmed that improvement of pyruvate supply would direct more carbon flow to ethanol formation.ConclusionsWe systematically characterized and optimized the PDCzm-slr1192 pathway in engineered cyanobacteria for ethanol production. Information gained from in vitro monitoring and genetic engineering revealed that for further enhancing ethanol synthesis capacities, PDCzm activities needed enhancement, and the PDCzm-slr1192 ratio should be improved and held to about 1:1.5. Considering actual metabolites concentrations of cyanobacteria cells, enhancing pyruvate supply was also a promising strategy for further updating the current ethanol photosynthetic cell factories.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0367-z) contains supplementary material, which is available to authorized users.

Study of the role of anaerobic metabolism in succinate production by Enterobacter aerogenes.

Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain ΔadhE/PCK. The strain produced succinate from glucose with a 60.5% yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions.

10.2478/s11814-009-0323-3

Rhizopus oryzae mutants were isolated from the wild type strain ME-F01 after mutagenesis with UV coupled with nitrosoguanidine (NTG) and the following allyl alcohol resistance selection method. By analyzing the activities of alcohol dehydrogenase (ADH) involved in ethanol synthesis pathway and batch fermentation, one mutant, ME-UN-8, was isolated that produced 21.1% more fumaric acid than ME-F01 with the corresponding byproduct of ethanol decreased by 83.7%.

Strain improvement of Rhizopus oryzae for over-production of fumaric acid by reducing ethanol synthesis pathway

Rhizopus oryzae mutants were isolated from the wild type strain ME-F01 after mutagenesis with UV coupled with nitrosoguanidine (NTG) and the following allyl alcohol resistance selection method. By analyzing the activities of alcohol dehydrogenase (ADH) involved in ethanol synthesis pathway and batch fermentation, one mutant, ME-UN-8, was isolated that produced 21.1% more fumaric acid than ME-F01 with the corresponding byproduct of ethanol decreased by 83.7%.

The Effects of Feed and Intracellular Pyruvate Levels on the Redistribution of Metabolic Fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK–PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.