Genetic modifications and introduction of heterologous pdc genes in Enterococcus faecalis for its use in production of bioethanol

Rana, Nosheen Fatima; Gente, Stéphanie; Rincé, Alain; Auffray, Yanick; Laplace, Jean-Marie

doi:10.1007/s10529-012-0964-x

Cited by 6 publications

(9 citation statements)

References 20 publications

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“…Corresponding LDH deletion mutants of the E. faecalis clinical isolate V583 have also been constructed and characterized recently (2). The results showed that LDH-1 is the main enzyme in lactate production, which we confirmed for strain JH2-2 in our previous study (3). The growth rates of the V583 LDH deletion mutants and of the corresponding wild-type strain were comparable in chemically defined medium (2).…”

Section: Resultssupporting

confidence: 64%

“…Recently, ldh deletion mutants of E. faecalis have been constructed and characterized. Deficiency in either LDH-1, LDH-2, or both activities causes a significant increase in the levels of mixed acid fermentation end products in strain V583 (2,3,6). All LDH mutants showed growth rates similar to that of the parental strain (2,6,7).…”

mentioning

confidence: 96%

“…It is a homofermentative lactic acid bacterium that uses mainly the Embden-MeyerhoffParnas (glycolysis) pathway for the catabolism of glucose or related carbohydrates (1). The dominant end product of fermentation under these conditions is lactate (2,3). Reduction of the glycolytic end product pyruvate to lactate by lactate dehydrogenases (LDH) allows the regeneration of the NADH formed during glycolysis to NAD ϩ , which keeps glycolysis going.…”

mentioning

confidence: 99%

“…The amount of transcript was 2.2-fold lower in a ccpA mutant than in the wild-type strain, suggesting that CcpA activates ldh-1 transcription (4). Most of the activity is associated with LDH-1, and LDH-2 plays only a minor role (2,3). Recently, ldh deletion mutants of E. faecalis have been constructed and characterized.…”

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confidence: 99%

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Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis

et al. 2013

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e Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalis to maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken Enterococcus faecalis is an intestinal resident of humans and many animals and is a major cause of severe nosocomial infections that are difficult to treat due to intrinsic and acquired resistances to major classes of antibiotics. It is a homofermentative lactic acid bacterium that uses mainly the Embden-MeyerhoffParnas (glycolysis) pathway for the catabolism of glucose or related carbohydrates (1). The dominant end product of fermentation under these conditions is lactate (2, 3). Reduction of the glycolytic end product pyruvate to lactate by lactate dehydrogenases (LDH) allows the regeneration of the NADH formed during glycolysis to NAD ϩ , which keeps glycolysis going. E. faecalis possesses two cytosolic L-(ϩ)-lactate dehydrogenases encoded by the ldh-1 (EF_0255) and ldh-2 (EF_0641) genes. In the ldh-1 promoter region, a potential cre box was identified 114 bp upstream of the ATG start codon (4). cre boxes are cis-acting sequences targeted by CcpA, the major global transcription regulator of carbon catabolite repression in Firmicutes (5). Northern blot analysis showed that ldh-1 is expressed as a monocistronic transcript. The amount of transcript was 2.2-fold lower in a ccpA mutant than in the wild-type strain, suggesting that CcpA activates ldh-1 transcription (4). Most of the activity is associated with LDH-1, and LDH-2 plays only a minor role (2, 3). Recently, ldh deletion mutants of E. faecalis have been constructed and characterized. Deficiency in either LDH-1, LDH-2, or both activities causes a significant increase in the levels of mixed acid fermentation end products in strain V583 (2, 3, 6). All LDH mutants showed growth rates similar to that of the parental strain (2, 6, 7). This demonstrates that the loss of the characteristic metabolic reaction for lactic acid bacteria does not have an obvious impact on fitness under normal growth conditions in E. faecalis. Similar results have been obtained for Lactococcus lactis (8) and Streptococcus pyogenes (7), where little or no difference in the growth rate between the parent and ⌬ldh mutants has been observed. The growth yields were also comparable in E. faecalis when the strains grew under aerobic conditions, but final cell densities were even higher than those for the wild type when the ⌬ldh-1 mutant and the ⌬ldh-1 ⌬ldh-2 double mutant strain grew under anaerobic conditions (2). These combined results demonstrate the impressive metabolic flexibility of E. faecalis. Transcriptomic ...

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Section: Resultssupporting

confidence: 64%

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confidence: 96%

mentioning

confidence: 99%

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confidence: 99%

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Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis

et al. 2013

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“…LAB have an innate high tolerance to ethanol and are frequently found in bioethanol plants as contaminants (18). They are able to grow at low pH, and some also thrive at elevated temperatures (19), both properties that are important for avoiding contamination (18), and in addition they meet the requirements for simultaneous saccharification and fermentation (SSF), as the cellulases work optimally at around pH ϳ5 and 50°C.It has previously been attempted to turn LAB into ethanol producers, but these efforts have met with only limited success (15,17,(20)(21)(22). In this study, the target organism is Lactococcus lactis, which is the best-characterized LAB to date and one of the Grampositive model organisms.…”

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confidence: 99%

Rewiring Lactococcus lactis for Ethanol Production

Solem

Dehli

Jensen

2013

Appl Environ Microbiol

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Lactic 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 organism Lactococcus lactis toward ethanol production. Codon-optimized Zymomonas mobilis pyruvate decarboxylase (PDC) was introduced and expressed from synthetic promoters in different strain backgrounds. In the wild-type L. lactis strain 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 genes ldhX, ldhB, and ldh and introducing codon-optimized Z. mobilis alcohol 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). More than 85 billion liters of bioethanol is currently being produced globally each year (Renewable Fuels Association, 2012 [http://www.ethanolrfa.org/]), and ethanol is the main fermentation-derived biofuel on the market, with a well-established technology behind the process. Yeast has been and still is the major production organism, partly because of its robustness but also because it is able to grow on cheap industrial media (1, 2). But there are drawbacks to yeast. Being a eukaryote, it grows and ferments slowly, is capable of fermenting only a few sugars naturally, and tolerates high temperatures poorly (3). The desire to use lignocellulosic biomass (LCB) as a raw material for ethanol production also points to production organisms other than yeast. LCB is readily available (4, 5) and contains large amounts of carbohydrates (6), but the pretreatment process required releases or generates compounds, such as acetate, that are inhibitory to growth of microorganisms (7,8). Yeast is no exception, and especially acetate affects growth negatively (9). A huge effort has been put into developing the process, and novel genetically engineered microorganisms for ethanol fermentation have also been constructed (4). Common approaches have been to improve existing ethanol-producing microorganisms, e.g., by increasing the capability to metabolize various sugars (10) or the tolerance toward inhibitors (11-13). The ethanol pathway has also been introduced into hosts with advantageous properties such as ease of genetic manipulation (14), high ethanol tolerance (15), or the ability to degrade LCB-derived sugars (16). Lactic acid bacteria (LAB) as a group share many of these useful properties. Many LAB naturally fe...

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Enzymatic Decarboxylation—An Emerging Reaction for Chemicals Production from Renewable Resources

et al. 2014

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ChemCatChem 2014, 6, 689 -701 689 CHEMCATCHEM MINIREVIEWS Scheme 2. Fermentative production of styrene derivatives. This approach is applicable for the conversion of endogenously produced phenylalanine (R = H) and tyrosine (R = OH).Scheme 3. In bicarbonate buffer, PAD from Lactobacillus plantarum catalyzes the stereoselective addition of water on the vinyl substituent. [8] Scheme 4. Variants of AMD from Bordetella bronchiseptica catalyze the asymmetric synthesis of both enantiomers of a-arylpropionates. Variant G74C possesses racemizing activity on these compounds.

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Genetic modifications and introduction of heterologous pdc genes in Enterococcus faecalis for its use in production of bioethanol

Cited by 6 publications

References 20 publications

Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis

Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis

Rewiring Lactococcus lactis for Ethanol Production

Enzymatic Decarboxylation—An Emerging Reaction for Chemicals Production from Renewable Resources

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