Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineeredEscherichia coli strain KO11

Asghari, Ali; Bothast, Rodney J.; Doran, Joy B.; Ingram, L. O.

doi:10.1007/bf01569920

Cited by 100 publications

(54 citation statements)

References 25 publications

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“…Initial studies performed with Luria broth demonstrated that KO11 rapidly and efficiently converted sugars to ethanol, with yields approaching 95% of the theoretical maximum. However, the volumetric productivity and ethanol yields were considerably lower in mineral salts medium without complex nutrients (4,26,31,34,54,55).…”

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confidence: 98%

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Underwood

Buszko

Shanmugam

et al. 2002

Appl Environ Microbiol

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Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD ؉ . Expression of low-K m Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.The engineering of metabolic pathways for production of industrial chemicals from renewable carbohydrates offers an alternative to petroleum-based processes and chemical synthesis (3,12,22). Genetically altering metabolic pathways, however, often causes unanticipated changes which may limit utility. Undesirable changes, such as reduced growth, decreased glycolytic flux, and low volumetric productivity, are generally attributed to a lack of ATP (19, 50), creation of futile cycles (10,11,37), changes in intracellular metabolite pools, or a metabolic imbalance (2,7,9,13,28,51,53). Often, these detrimental effects are masked by abundant complex nutrients in laboratory media and are apparent only in more minimal media (7,10,11,31).Salmonella enterica serovar Typhimurium was engineered for succinate production by increasing expression of pyc encoding pyruvate carboxylase (50). Although succinate production increased, the growth rate declined by 18% and the glycolytic flux decreased by 40%. Similar results were reported for an analogous construction in Escherichia coli (19). Donnelly and cow...

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confidence: 98%

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Underwood

Buszko

Shanmugam

et al. 2002

Appl Environ Microbiol

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“…The use of raffinose-containing beet molasses as a substrate for the production of baker's yeast has prompted the recent development of recombinant strains which secrete ␣-galactosidase (12). Attempts to develop raffinose-fermenting strains of Zymomonas mobilis for ethanol production by using an enteric ␣-galactosidase gene have been partially successful (34).Enteric bacteria which express Z. mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) (4, 24, 33) are being considered for commercial ethanol production (1,14,17). In this study, raffinose fermentation was investigated by using three of these strains: E. coli KO11, Klebsiella oxytoca P2, and Erwinia chrysanthemi EC16(pLOI555).…”

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confidence: 99%

Extracellular melibiose and fructose are intermediates in raffinose catabolism during fermentation to ethanol by engineered enteric bacteria

et al. 1997

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Contrary to general concepts of bacterial saccharide metabolism, melibiose (25 to 32 g/liter) and fructose (5 to 14 g/liter) accumulated as extracellular intermediates during the catabolism of raffinose (O-␣-D-galactopyranosyl-1,6-␣-D-glucopyranosyl-␤-D-fructofuranoside) (90 g/liter) by ethanologenic recombinants of Escherichia coli B, Klebsiella oxytoca M5A1, and Erwinia chrysanthemi EC16. Both hydrolysis products (melibiose and fructose) were subsequently transported and further metabolized by all three organisms. Raffinose catabolism was initiated by ␤-fructosidase; melibiose was subsequently hydrolyzed to galactose and glucose by ␣-galactosidase. Glucose and fructose were completely metabolized by all three organisms, but galactose accumulated in the fermentation broth with EC16(pLOI555) and P2. MM2 (a raffinose-positive E. coli mutant) was the most effective biocatalyst for ethanol production (38 g/liter) from raffinose. All organisms rapidly fermented sucrose (90 g/liter) to ethanol (48 g/liter) at more than 90% of the theoretical yield. During sucrose catabolism, both hydrolysis products (glucose and fructose) were metabolized concurrently by EC16(pLOI555) and P2 without sugar leakage. However, fructose accumulated extracellularly (27 to 28 g/liter) at early stages of fermentation with KO11 and MM2. Sequential utilization of glucose and fructose correlated with a diauxie in base utilization (pH maintenance). The mechanism of sugar escape remains unknown but may involve downhill leakage via permease which transports precursor saccharides or novel sugar export proteins. If sugar escape occurs in nature with wild organisms, it could facilitate the development of complex bacterial communities which are based on the sequence of saccharide catabolism and the hierarchy of sugar utilization.is second only to sucrose as a soluble component in plant tissues (10,18,31) and beet molasses (19). Many members of the family Enterobacteriaceae can degrade this sugar (6). In a comparison of more than 300 Escherichia coli isolates from humans and farm animals (25, 30), half were able to metabolize raffinose and 15% were able to transfer this trait by conjugation. Subsequent studies established the presence of conjugal plasmids containing genes for raffinose utilization (7). One of these plasmids, pRSD2, has been investigated in considerable detail (2,3,28,29) and forms the basis for current models of raffinose utilization (21). Raffinose genes are organized into an operon (2, 3); rafR (regulatory protein), rafA (␣-galactosidase), rafB (raffinose permease), and rafD (sucrose hydrolase). According to the current model (2, 3, 28), intracellular raffinose is initially hydrolyzed by ␣-galactosidase into galactose and sucrose. Intracellular sucrose is subsequently cleaved into glucose and fructose by sucrose hydrolase.The rafA-encoded ␣-galactosidase is quite different from the native E. coli enzyme and has been used as a serological marker to compare 39 independently isolated raf plasmids (28). The cross-reactivity of ␣-galactos...

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“…The ethanol yield from hemicellulose acid hydrolysate by Pichia stipitis NRRL Y-7124 was 0.41 g g −1 , equivalent to 80.4% theoretical conversion efficiency (10), that by E. coli KO11 was 0.51 g g −1 (2), and that by Pachysolen tannophilus (6), Pichia stipitis NRRL Y-7124 (10), Saccharomyces cerevisiae (30), and Pichia stipitis (1) was less than 0.05 g g −1 (w/w). The maximum ethanol yield (0.509 g g −1 ), equivalent to 98.8% theoretical conversion efficiency, was similar to that of E. coli KO11.…”

Section: Discussionmentioning

confidence: 96%

“…Although after genetic manipulations such as gene knockout, some anaerobic thermophilic ethanogens could use higher concentrations of substrates and produce a stoichiometric amount of ethanol (2,32), among wild-type anaerobic thermophilic ethanogens, this strain had a higher ethanol yield when 0.5% xylose was used (Table 4).…”

Section: Discussionmentioning

confidence: 99%

Ethanol Production Efficiency of an Anaerobic Hemicellulolytic Thermophilic Bacterium, Strain NTOU1, Isolated from a Marine Shallow Hydrothermal Vent in Taiwan

et al. 2011

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A new extremely thermophilic, anaerobic, gram-negative bacterium, strain NTOU1, was enriched and isolated from acidic marine hydrothermal fluids off Gueishandao island in Taiwan with 0.5% starch and 0.5% maltose as carbon sources. This strain was capable of growth utilizing various sugars found in lignocellulosic biomass as well as xylan and cellulose, and produced ethanol, lactate, acetate, and CO 2 as fermentation products. The results of a 16S rRNA gene sequence analysis (1,520 bp) revealed NTOU1 to belong to the genus Thermoanaerobacterium. When tested for the ability to grow and produce ethanol from xylose or rice straw hemicellulosic hydrolysate at 70°C, the strain showed the highest levels of ethanol production (1.65 mol ethanol mol xylose −1 ) in a medium containing 0.5% xylose plus 0.5% yeast extract. Maximum ethanol production from the rice straw hemicellulose was 0.509 g g −1 , equivalent to 98.8% theoretical conversion efficiency. Low concentrations of inhibitors (derived from dilute acid hydrolysis) in the rice straw hemicellulose hydrolysate did not affect the ethanol yield. Thus, Thermoanaerobacterium strain NTOU1 has the potential to be used for ethanol production from hemicellulose.

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Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineeredEscherichia coli strain KO11

Cited by 100 publications

References 25 publications

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Extracellular melibiose and fructose are intermediates in raffinose catabolism during fermentation to ethanol by engineered enteric bacteria

Ethanol Production Efficiency of an Anaerobic Hemicellulolytic Thermophilic Bacterium, Strain NTOU1, Isolated from a Marine Shallow Hydrothermal Vent in Taiwan

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