Ethanol tolerance and carbohydrate metabolism in lactobacilli

Gold, Roger E.; Meagher, Michael M.; Hutkins, Robert W.; Conway, Tyrrell

doi:10.1007/bf01583633

Cited by 57 publications

(43 citation statements)

References 25 publications

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“…The production of succinic acid may be ascribed to citrate-fermenting LAB as well or to the conversion of fumaric acid and malic acid (36). The aciduric and ethanol-tolerant character of L. fermentum (3,7,68) may explain its survival and dominance of the whole cocoa bean fermentation process. In addition, strains of L. plantarum and L. fermentum are able to produce antimicrobial substances that play a role in bacterial ecology (17).…”

Section: Discussionmentioning

confidence: 99%

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria andAcetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans inGhana

Camu

Winter

Verbrugghe

et al. 2007

Appl Environ Microbiol

290

424

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The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).Cocoa beans are the principal raw material for chocolate production (39,43,73,81). These seeds are derived from the fruit pods of the cocoa tree (Theobroma cacao L.), which is cultivated in plantations in the equatorial zone, with the Ivory Coast, Brazil, and Ghana as the major producers (2). The cocoa beans are embedded in a mucilaginous pulp inside the pods. Raw cocoa beans have an astringent, unpleasant taste and flavor and have to be fermented, dried, and roasted to obtain the desired characteristic cocoa flavor and taste (26, 73). The final chocolate flavor is influenced by the origin and cultivar of the cocoa beans, the on-the-farm fermentation and drying process, and the roasting and further processing performed by the cocoa and chocolate manufacturer (4,10,32,33,57,75).After removal of the beans from the pods, the first step in cocoa processing is a spontaneous 3-to 10-day fermentation of beans and pulp in heaps, boxes, baskets, or trays, of which spontaneous heap fermentation is the most widely used method in Ghana ...

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Section: Discussionmentioning

confidence: 99%

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria andAcetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans inGhana

Camu

Winter

Verbrugghe

et al. 2007

Appl Environ Microbiol

290

424

View full text Add to dashboard Cite

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“…Until now, there has not been a PDC that has been expressed well in a recombinant Gram-positive bacterium (Gold et al, 1992(Gold et al, , 1996Barbosa et al, 1994;Nichols et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…The factors responsible for low-level production of PDC protein in recombinant Gram-positive bacteria are unknown (Gold et al, 1992(Gold et al, , 1996Barbosa et al, 1994;Nichols et al, 2003). In order to determine if transcript levels were limiting the production of PDC in Gram-positive hosts, we analysed the transcript levels specific for the various pdc genes when expressed in recombinant B.…”

Section: Analysis Of Pdc Transcript Levelsmentioning

confidence: 99%

“…While this strategy has been highly successful in engineering mesophilic Gramnegative bacteria (Ingram et al, 1998(Ingram et al, , 1999, advanced biocatalysts that withstand low pH, high temperature, high salt, high sugar, high ethanol and various other harsh conditions offer an opportunity to improve the commercial competitiveness of ethanol production (Ingram et al, 1999;Dien et al, 2003). Many of these qualities can be found in Gram-positive bacteria (Gold et al, 1992). Unfortunately, modifying Gram-positive bacteria for ethanol production has had limited success.…”

Section: Introductionmentioning

confidence: 99%

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Construction and expression of an ethanol production operon in Gram-positive bacteria

et al. 2005

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Pyruvate decarboxylase (PDC), an enzyme central to homoethanol fermentation, catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. PDC enzymes from diverse organisms have different kinetic properties, thermal stability and codon usage that are likely to offer unique advantages for the development of desirable Gram-positive biocatalysts for use in the ethanol industry. To examine this further, pdc genes from bacteria to yeast were expressed in the Gram-positive host Bacillus megaterium. The PDC activity and protein levels were determined for each strain. In addition, the levels of pdc-specific mRNA transcripts and stability of recombinant proteins were assessed. From this analysis, the pdc gene of Gram-positive Sarcina ventriculi was found to be the most advantageous for engineering high-level synthesis of PDC in a Gram-positive host. This gene was thus selected for transcriptional coupling to the alcohol dehydrogenase gene (adh) of Geobacillus stearothermophilus. The resulting Gram-positive ethanol production operon was expressed at high levels in B. megaterium. Extracts from this recombinant were shown to catalyse the production of ethanol from pyruvate.

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“…Wild-type E. coli strains are completely inhibited at 35 g/liter (4.4%, vol/vol) (41). Evolution experiments have resulted in strains able to grow at 50 g/liter (6.3%, vol/vol) (41), but this is still far from the 20% (vol/ vol) (158 g/liter) that has been reported for some LAB strains (42). We did a short time evolution experiment and were able to increase tolerance from 6 to 8% in just 2 months, which is not far from the 10 to 12% tolerated by the commonly used S. cerevisiae and Z. mobilis (43).…”

Section: Discussionmentioning

confidence: 98%

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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Ethanol tolerance and carbohydrate metabolism in lactobacilli

Cited by 57 publications

References 25 publications

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria andAcetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans inGhana

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria andAcetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans inGhana

Construction and expression of an ethanol production operon in Gram-positive bacteria

Rewiring Lactococcus lactis for Ethanol Production

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