scholarly journals Respiratory Chain Analysis of Zymomonas mobilis Mutants Producing High Levels of Ethanol

2012 ◽  
Vol 78 (16) ◽  
pp. 5622-5629 ◽  
Author(s):  
Takeshi Hayashi ◽  
Tsuyoshi Kato ◽  
Kensuke Furukawa
Keyword(s):  
Ethanol Production ◽  
Dehydrogenase Activity ◽  
Respiratory Chain ◽  
Zymomonas Mobilis ◽  
Nadh Dehydrogenase ◽  
Nucleotide Sequence Analysis ◽  
Wild Type ◽  
Aerobic Growth ◽  
Content Type ◽  
Respiratory Chains

ABSTRACTWe previously isolated respiratory-deficient mutant (RDM) strains ofZymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochromebd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within thendhgene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.

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 Physiological relevance, localization and substrate specificity of the alternative (type II) mitochondrial NADH dehydrogenases of Ogataea parapolymorpha

2021 ◽  
Author(s):  
Hannes Juergens ◽  
Álvaro Mielgo-Gómez ◽  
Albert Godoy-Hernández ◽  
Jolanda ter Horst ◽  
Janine M. Nijenhuis ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Respiratory Chain ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Physiological Role ◽  
Proton Pumping ◽  
Type Ii ◽  
Respiratory Chains ◽  
Alternative Type ◽  
Nadh Dehydrogenases

AbstractMitochondria from Ogataea parapolymorpha harbor a branched electron-transport chain containing a proton-pumping Complex I NADH dehydrogenase and three alternative (type II) NADH dehydrogenases (NDH2s). To investigate the physiological role, localization and substrate specificity of these enzymes, growth of various NADH dehydrogenase mutants was quantitatively characterized in shake-flask and chemostat cultures, followed by oxygen-uptake experiments with isolated mitochondria. Furthermore, NAD(P)H:quinone oxidoreduction of the three NDH2s were individually assessed. Our findings show that the O. parapolymorpha respiratory chain contains an internal NADH-accepting NDH2 (Ndh2-1/OpNdi1), at least one external NAD(P)H-accepting enzyme and likely additional mechanisms for respiration-linked oxidation of cytosolic NADH. Metabolic regulation appears to prevent competition between OpNdi1 and Complex I for mitochondrial NADH. With the exception of OpNdi1, the respiratory chain of O. parapolymorpha exhibits metabolic redundancy and tolerates deletion of multiple NADH-dehydrogenase genes without compromising fully respiratory metabolism.ImportanceTo achieve high productivity and yields in microbial bioprocesses, efficient use of the energy substrate is essential. Organisms with branched respiratory chains can respire via the energy-efficient proton-pumping Complex I, or make use of alternative NADH dehydrogenases (NDH2s). The yeast Ogataea parapolymorpha contains three uncharacterized, putative NDH2s which were investigated in this work. We show that O. parapolymorpha contains at least one ‘internal’ NDH2, which provides an alternative to Complex I for mitochondrial NADH oxidation, albeit at a lower efficiency. The use of this NDH2 appeared to be limited to carbon excess conditions and the O. parapolymorpha respiratory chain tolerated multiple deletions without compromising respiratory metabolism, highlighting opportunities for metabolic (redox) engineering. By providing a more comprehensive understanding of the physiological role of NDH2s, including insights into their metabolic capacity, orientation and substrate specificity this study also extends our fundamental understanding of respiration in organisms with branched respiratory chains.

Mitochondrial NADH dehydrogenase activity and ability to tolerate acetaldehyde determine faster ethanol production in Saccharomyces cerevisiae

IUBMB Life ◽  
1996 ◽  
Vol 40 (1) ◽  
pp. 145-150 ◽  
Author(s):  
C. S. Shankar ◽  
P.Y. Aneez Ahamad ◽  
M. S. Ramakrishnan ◽  
S. Umesh-Kumar
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Dehydrogenase Activity ◽  
Nadh Dehydrogenase

scholarly journals Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase

Microbiology ◽  
2014 ◽  
Vol 160 (9) ◽  
pp. 2045-2052 ◽  
Author(s):  
Elina Balodite ◽  
Inese Strazdina ◽  
Nina Galinina ◽  
Samantha McLean ◽  
Reinis Rutkis ◽  
...  
Keyword(s):  
Electron Transport ◽  
Respiratory Chain ◽  
Peroxidase Activity ◽  
Zymomonas Mobilis ◽  
Alternative Oxidase ◽  
Oxygen Affinity ◽  
Aerobic Growth ◽  
Nadh Peroxidase ◽  
Kinetics Of

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc 1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc 1 complex via cytochrome c 552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc 1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc 1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase.

scholarly journals Cellulosic Ethanol Production by Recombinant Cellulolytic Bacteria Harbouring pdc and adh II Genes of Zymomonas mobilis

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
P. Sobana Piriya ◽  
P. Thirumalai Vasan ◽  
V. S. Padma ◽  
U. Vidhyadevi ◽  
K. Archana ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Cellulosic Ethanol ◽  
Zymomonas Mobilis ◽  
Ethanol Tolerance ◽  
Pyruvate Decarboxylase ◽  
Wild Type ◽  
Cellulolytic Bacteria ◽  
Cellulase Expression ◽  
Pretreated Bagasse ◽  
Industrial Level

The ethanol fermenting genes such as pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh II) were cloned from Zymomonas mobilis and transformed into three different cellulolytic bacteria, namely Enterobacter cloacae JV, Proteus mirabilis JV and Erwinia chrysanthemi and their cellulosic ethanol production capability was studied. Recombinant E. cloacae JV was found to produce 4.5% and 3.5% (v/v) ethanol, respectively, when CMC and 4% NaOH pretreated bagasse were used as substrates, whereas recombinant P. mirabilis and E. chrysanthemi with the same substrates could only produce 4%, 3.5%, 1%, and 1.5 % of ethanol, respectively. The recombinant E. cloacae strain produced twofold higher percentage of ethanol than the wild type. The recombinant E. cloacae strain could be improved further by increasing its ethanol tolerance capability through media optimization and also by combining multigene cellulase expression for enhancing ethanol production from various types of lignocellulosic biomass so that it can be used for industrial level ethanol production.

scholarly journals Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

2015 ◽  
Vol 82 (3) ◽  
pp. 972-978 ◽  
Author(s):  
Jesús G. Alvelo-Maurosa ◽  
Scott J. Lee ◽  
Samuel P. Hazen ◽  
Susan B. Leschine
Keyword(s):  
Ethanol Production ◽  
Filter Paper ◽  
Double Mutant ◽  
Plant Biomass ◽  
Cell Number ◽  
Growth Substrate ◽  
Wild Type ◽  
Content Type ◽  
Cell Numbers ◽  
Clostridium Phytofermentans

ABSTRACTA dual-fluorescent-dye protocol to visualize and quantifyClostridium phytofermentansISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-typeSorghum bicolor, or a reduced-ligninS. bicolordouble mutant (bmr-6 bmr-12double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.

scholarly journals Cell Aggregation and Aerobic Respiration Are Important forZymomonas mobilisZM4 Survival in an Aerobic Minimal Medium

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Sara E. Jones-Burrage ◽  
Timothy A. Kremer ◽  
James B. McKinlay
Keyword(s):  
Yeast Extract ◽  
Zymomonas Mobilis ◽  
Minimal Medium ◽  
Single Cells ◽  
Maximum Yield ◽  
Aerobic Respiration ◽  
Aerobic Growth ◽  
Poor Growth ◽  
Content Type ◽  
Multicellular Aggregates

ABSTRACTZymomonas mobilisproduces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to the yeastSaccharomyces cerevisiaefor industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle quickly and exhibit higher resistance to harmful chemicals than single cells. While spontaneous floc-formingZ. mobilismutants have been described, little is known about the natural conditions that induceZ. mobilisfloc formation or about the genetic factors involved. Here we found that wild-typeZ. mobilisforms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential for both floc formation and survival in a minimal aerobic medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in a minimal aerobic medium, providing a physiological role for this enzyme, which has previously been found to be disadvantageous in a rich aerobic medium. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation.IMPORTANCEThe bacteriumZymomonas mobilisis best known for its anaerobic fermentative lifestyle, in which it converts glucose into ethanol at a yield surpassing that of yeast. However,Z. mobilisalso has an aerobic lifestyle, which has confounded researchers with its attributes of poor growth, accumulation of toxic acetic acid and acetaldehyde, and respiratory enzymes that are detrimental for aerobic growth. Here we show that a majorZ. mobilisrespiratory enzyme and the ability to form multicellular aggregates, called flocs, are important for survival, but only during aerobic growth in a medium containing a minimum set of nutrients required for growth. Supplements, such as vitamins or yeast extract, promote aerobic growth and, in some cases, inhibit floc formation. We propose thatZ. mobilislikely requires aerobic respiration and floc formation in order to survive in natural environments that lack protective factors found in supplements such as yeast extract.

Sélection et analyse de mutants thermotolérants de Zymomonas mobilis, producteurs d'éthanol

1985 ◽  
Vol 31 (10) ◽  
pp. 934-937 ◽  
Author(s):  
Bénédicte Berthelin ◽  
Joseph Zucca ◽  
Jean-François Mescle
Keyword(s):  
Ethanol Production ◽  
Growth Kinetics ◽  
Growth Phase ◽  
Exponential Growth ◽  
Type Strain ◽  
Zymomonas Mobilis ◽  
Wild Type Strain ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Mutant Strains

Mutagenesis of Zymomonas mobilis ATCC 10988 induced by nitrosoguanidine and ethyl methanesulfonate leads to the acquisition of thermotolerant character. The nitrosoguanidine allows the obtention of the best mutants. At 38 °C, these mutant strains have growth kinetics and ethanol production rates similar to those of the wild-type strain grown at 30 °C. The mutants show delayed ethanol production when compared with Zymomonas mobilis ATCC 10988: this production occurs in part during the exponential growth phase and is still active at the beginning of the stationary phase.

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 Production of 4-Hydroxybenzoic Acid by an Aerobic Growth-Arrested Bioprocess Using Metabolically EngineeredCorynebacterium glutamicum

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Yukihiro Kitade ◽  
Ryoma Hashimoto ◽  
Masako Suda ◽  
Kazumi Hiraga ◽  
Masayuki Inui
Keyword(s):  
Aromatic Compound ◽  
Target Genes ◽  
Raw Material ◽  
No Production ◽  
Hydroxybenzoic Acid ◽  
Intestinal Bacterium ◽  
Wild Type ◽  
Aerobic Growth ◽  
Content Type ◽  
Gene Coding

ABSTRACTCorynebacterium glutamicumwas metabolically engineered to produce 4-hydroxybenzoic acid (4-HBA), a valuable aromatic compound used as a raw material for the production of liquid crystal polymers and paraben.C. glutamicumwas found to have a higher tolerance to 4-HBA toxicity than previously reported hosts used for the production of genetically engineered 4-HBA. To obtain higher titers of 4-HBA, we employed a stepwise overexpression of all seven target genes in the shikimate pathway inC. glutamicum. Specifically, multiple chromosomal integrations of a mutatedaroGgene fromEscherichia coli, encoding a 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthase, and wild-typearoCKBfromC. glutamicum, encoding chorismate synthase, shikimate kinase, and 3-dehydroquinate synthase, were effective in increasing product titers. The last step of the 4-HBA biosynthesis pathway was recreated inC. glutamicumby expressing a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from the intestinal bacteriumProvidencia rustigianii. To enhance the yield of 4-HBA, we reduced the formation of by-products, such as 1,3-dihydroxyacetone and pyruvate, by deletinghdpA, a gene coding for a haloacid dehalogenase superfamily phosphatase, andpyk, a gene coding for a pyruvate kinase, from the bacterial chromosome. The maximum concentration of 4-HBA produced by the resultant strain was 36.6 g/liter, with a yield of 41% (mol/mol) glucose after incubation for 24 h in minimal medium in an aerobic growth-arrested bioprocess using a jar fermentor. To our knowledge, this is the highest concentration of 4-HBA produced by a metabolically engineered microorganism ever reported.IMPORTANCESince aromatic compound 4-HBA has been chemically produced from petroleum-derived phenol for a long time, eco-friendly bioproduction of 4-HBA from biomass resources is desired in order to address environmental issues. In microbial chemical production, product toxicity often causes problems, but we confirmed that wild-typeC. glutamicumhas high tolerance to the target 4-HBA. A growth-arrested bioprocess using this microorganism has been successfully used for the production of various compounds, such as biofuels, organic acids, and amino acids. However, no production method has been applied for aromatic compounds to date. In this study, we screened for a novel final reaction enzyme possessing characteristics superior to those in previously employed microbial 4-HBA production. We demonstrated that the use of the highly 4-HBA-resistant UbiC from the intestinal bacteriumP. rustigianiiis very effective in increasing 4-HBA production.

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