Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.

AbstractPyruvate decarboxylase (PDC) from Zymobacter palmae (ZpPDC) has been reported to have a lower Km the Zymomonas mobilis PDC (ZmPDC). ZpPDC was combined with native slr1192 alcohol dehydrogenase (adh) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with Zmpdc. Native (Zppdc) and codon optimised (ZpOpdc) versions of the ZpPDC were cloned into a construct where the pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis PCC 6803. These constructs were transformed into wildtype Synechocystis PCC 6803. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL004) expressing the pdc from Zymomonas mobilis. The utilisation of a PDC with a lower Km from Zymobacter palmae did not result in enhanced ethanol production in Synechocystis PCC 6803.

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Zymobacter palmae Pyruvate Decarboxylase is Less Effective Than That of Zymomonas mobilis for Ethanol Production in Metabolically Engineered Synechocystis sp. PCC6803

Microorganisms ◽

10.3390/microorganisms7110494 ◽

2019 ◽

Vol 7 (11) ◽

pp. 494 ◽

Cited By ~ 1

Author(s):

Lorraine Quinn ◽

Patricia Armshaw ◽

Tewfik Soulimane ◽

Con Sheehan ◽

Michael P. Ryan ◽

...

Keyword(s):

Alcohol Dehydrogenase ◽

Ethanol Production ◽

Zymomonas Mobilis ◽

Pyruvate Decarboxylase ◽

Biomass Productivity ◽

Gene Cassette ◽

Major Effect ◽

Zymobacter Palmae ◽

Synechocystis Sp ◽

Pcc 6803

To produce bioethanol from model cyanobacteria such as Synechocystis, a two gene cassette consisting of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required to transform pyruvate first to acetaldehyde and then to ethanol. However the partition of pyruvate to ethanol comes at a cost, a reduction in biomass and pyruvate availability for other metabolic processes. Hence strategies to divert flux to ethanol as a biofuel in Synechocystis are of interest. PDC from Zymobacter palmae (ZpPDC) has been reported to have a lower Km then the Zymomonas mobilis PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The Zppdc gene was combined with the native slr1192 alcohol dehydrogenase gene (adhA) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (ZpOpdc) versions of the ZpPDC were cloned into a construct where pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis sp. PCC 6803. These constructs were transformed into wildtype Synechocystis sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from Zymomonas mobilis. All constructs demonstrated lower biomass productivity illustrating that the flux from pyruvate to ethanol has a major effect on biomass and ultimately overall biofuel productivity. Thus the utilization of a PDC with a lower Km from Zymobacter palmae unusually did not result in enhanced ethanol production in Synechocystis sp. PCC 6803.

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Consolidated Bioprocessing for Bioethanol Production by Metabolically Engineered Bacillus Subtilis Strains

10.21203/rs.3.rs-297375/v1 ◽

2021 ◽

Author(s):

Fatemeh Maleki ◽

Mohammad Changizian ◽

Narges Zolfaghari ◽

Sarah Rajaei ◽

Kambiz Akbari Noghabi ◽

...

Keyword(s):

Alcohol Dehydrogenase ◽

Ethanol Production ◽

Plant Biomass ◽

Fusion Gene ◽

Consolidated Bioprocessing ◽

Gene Dosage ◽

Pyruvate Decarboxylase ◽

Single Step ◽

Bioethanol Production ◽

Genes Encoding

Abstract Background: Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. Results: In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adhZ) and S. cerevisiae (adhS) were each used with Z. mobilis pyruvate decarboxylase gene (pdcZ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adhS caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdcZ and adhS and the other with a single pdcZ but double adhS genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdcZ and adhS in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adhS:pdcZ resulted in more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. Conclusion: In this study for the first time, Bacillus subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.

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Engineering lactic acid bacteria with pyruvate decarboxylase and alcohol dehydrogenase genes for ethanol production from Zymomonas mobilis

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-003-0055-z ◽

2003 ◽

Vol 30 (5) ◽

pp. 315-321 ◽

Cited By ~ 33

Author(s):

Nancy N. Nichols ◽

Bruce S. Dien ◽

Rodney J. Bothast

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Alcohol Dehydrogenase ◽

Ethanol Production ◽

Zymomonas Mobilis ◽

Pyruvate Decarboxylase

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Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks

World Journal of Microbiology and Biotechnology ◽

10.1007/s11274-014-1713-1 ◽

2014 ◽

Vol 30 (11) ◽

pp. 2871-2883 ◽

Cited By ~ 7

Author(s):

Mingfeng Yang ◽

Xuefeng Li ◽

Chunya Bu ◽

Hui Wang ◽

Guanglu Shi ◽

...

Keyword(s):

Escherichia Coli ◽

Alcohol Dehydrogenase ◽

Ethanol Production ◽

Pyruvate Decarboxylase ◽

Metabolic Enzyme ◽

High Ethanol

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Metal-catalyzed oxidation of Fe2+ dehydrogenases. Consensus target sequence between propanediol oxidoreductase of Escherichia coli and alcohol dehydrogenase II of Zymomonas mobilis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37413-6 ◽

1994 ◽

Vol 269 (9) ◽

pp. 6592-6597

Author(s):

E. Cabiscol ◽

J. Aguilar ◽

J. Ros

Keyword(s):

Escherichia Coli ◽

Alcohol Dehydrogenase ◽

Zymomonas Mobilis ◽

Target Sequence ◽

Metal Catalyzed Oxidation ◽

Metal Catalyzed ◽

Catalyzed Oxidation

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Specific Ethanol Production Rate in Ethanologenic Escherichia coli Strain KO11 Is Limited by Pyruvate Decarboxylase

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000111993 ◽

2008 ◽

Vol 15 (1) ◽

pp. 55-64 ◽

Cited By ~ 19

Author(s):

Gerardo Huerta-Beristain ◽

José Utrilla ◽

Georgina Hernández-Chávez ◽

Francisco Bolívar ◽

Guillermo Gosset ◽

...

Keyword(s):

Escherichia Coli ◽

Ethanol Production ◽

Production Rate ◽

Pyruvate Decarboxylase ◽

Ethanol Production Rate

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Effects of manipulation of pyruvate decarboxylase and alcohol dehydrogenase levels on the submergence tolerance of rice

Functional Plant Biology ◽

10.1071/pp00137 ◽

2001 ◽

Vol 28 (12) ◽

pp. 1231 ◽

Cited By ~ 13

Author(s):

Musrur Rahman ◽

Anil Grover ◽

W. James Peacock ◽

Elizabeth S. Dennis ◽

Marc H. Ellis

Keyword(s):

Alcohol Dehydrogenase ◽

Ethanol Production ◽

Rice Variety ◽

Pyruvate Decarboxylase ◽

Inducible Promoter ◽

Moderate Increase ◽

Sense Orientation ◽

Transgenic Lines ◽

Hybrid Lines ◽

Adh Activity

A transgenic approach was taken to manipulate the levels of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in rice, in order to investigate whether alteration of ethanol fermentation can affect anaerobic tolerance. A line transformed with an antisense Adh1 construct had only 4–8% of the ADH activity of untransformed plants. This line showed reduced ethanol production and coleoptile growth under anoxia. Mature plants had reduced survival when submerged in water and exposed to anoxia, suggesting that ADH plays an essential role in seed germination and plant survival in the absence of O2. A transgenic line transformed with a cotton Adh2 cDNA in the sense orientation relative to a constitutive promoter, showed 3–4-fold more ADH activity than either untransformed controls, or a flooding-tolerant rice variety (FR13A), both in air and under hypoxia. However, ethanol production by this line was only slightly higher than that of untransformed controls, and there was no increase in survival following anoxia treatments. Three independent transgenic lines containing the ricePdc1 cDNA driven by an anaerobically-inducible promoter (6XARE) showed an increase in PDC1 polypeptide in shoots, but not in roots or endosperm. A moderate increase in PDC activity and ethanol production was observed in shoots of these lines under anaerobic conditions, as well as decreased survival of shoots when submerged and exposed to anoxia. F1 plants containing both the PDC and ADH constructs showed levels of anoxia-tolerance similar to those of untransformed plants. These results suggest that over-production of PDC may be toxic to rice plants because of increased acetaldehyde. Consistent with this view, acetaldehyde levels were appreciably higher in plants over-producing PDC, compared with untransformed plants, or hybrid lines containing both the PDC and ADH constructs.

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