Flux through Citrate Synthase Limits the Growth of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

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.     

Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Δxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.     

Ethanol production using nuclear petite yeast mutants

Two respiratory-deficient nuclear petites, FY23Δpet191 and FY23Δcox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23ρ⁰. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K _i, of 2.3% (w/v) and a specific rate of ethanol production, q _p, of 0.90 g ethanol g dry cells⁻¹ h⁻¹. FY23ρ⁰ was the most sensitive to ethanol, exhibiting a K _i of 1.71% (w/v) and q _p of 0.87 g ethanol g dry cells⁻¹ h⁻¹. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23Δpet191, having a K _i of 2.14% (w/v) and the 85% respiratory-deficient FY23Δcox5a, having a K _i of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23ρ⁰ is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation. Received: 22 September 1997 / Accepted: 7 December 1997     

An ethanol-tolerant recombinant Escherichia coli expressing Zymomonas mobilis pdc and adhB genes for enhanced ethanol production from xylose

An ethanol-tolerant mutant, ET1, was isolated by an enrichment method from Escherichia coli JM109. Strains JM109 and ET1 were transformed with expression vector pZY507bc containing Zymomonas mobilis alcohol dehydrogenase II (adhB) and pyruvate decarboxylase (pdc) genes, resulting in an ethanol-sensitive recombinant strain JMbc and an ethanol-tolerant recombinant strain, ET1bc. Alcohol dehydrogenase and pyruvate decarboxylase activities were 24 and 32% lower, respectively, in JMbc than in ET1bc. ET1bc fermented 10% (w/v) xylose to give 39.4 g ethanol/l (77%, theoretical yield), a 1.3-fold increase compared with the ethanol-sensitive strain JMbc.     

Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production

Genetically engineered Escherichia coli KO11 is capable of efficiently producing ethanol from all sugar constituents of lignocellulose but lacks the high ethanol tolerance of yeasts currently used for commercial starch-based ethanol processes. Using an enrichment method which selects alternatively for ethanol tolerance during growth in broth and for ethanol production on solid medium, mutants of KO11 with increased ethanol tolerance were isolated which can produce more than 60 g ethanol L⁻¹ from xylose in 72 h. Ethanol concentrations and yields achieved by the LY01 mutant with xylose exceed those reported for recombinant strains of Saccharomyces and Zymomonas mobilis, both of which have a high native ethanol tolerance. Received 18 September 1997/ Accepted in revised form 07 January 1998     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Engineering a native homoethanol pathway in Escherichia coli B for ethanol production

A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase (frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter. The resulting strain SZ420 (ΔfrdBC ΔldhA ΔackA ΔfocA-pflB ΔpdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90% under anaerobic conditions.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Betaine Tripled the Volumetric Productivity of d(-)-lactate by Escherichia coli Strain SZ132 in Mineral Salts Medium

Osmotic stress restricts glycolytic flux, growth (rate and yield), d-lactate productivity, and d-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mm betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of d-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l⁻¹ h⁻¹; 0.8 to 2.3 g l⁻¹ h⁻¹). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during d-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol d-lactate (90 g) per 100 g sugar (glucose or sucrose). Revisions requested 17 January 2006; Revisions received 7 February 2006     

Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non-aerated conditions in glucose-mineral medium

In this work, Escherichia coli MG1655 was engineered to produce ethanol and evolved in a laboratory process to obtain an acetate tolerant strain called MS04 (E. coli MG1655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, ΔldhA, PpflB::pdc _Zm -adhB _Zm , evolved). The growth and ethanol production kinetics of strain MS04 were determined in mineral medium, mainly under non-aerated conditions, supplemented with glucose in the presence of different concentrations of sodium acetate at pH?7.0 and at different values of acid pH and a constant concentration of sodium acetate (2?g/l). Results revealed an increase in the specific growth rate, cell mass formation, and ethanol volumetric productivity at moderate concentrations of sodium acetate (2–10?g/l), in addition to a high tolerance to acetate because it was able to grow and produce a high yield of ethanol in the presence of up to 40?g/l of sodium acetate. Genomic analysis of the ΔpflB evolved strain identified that a chromosomal deletion of 27.3?kb generates the improved growth and acetate tolerance in MG1655 ΔpflB derivative strains. This deletion comprises genes related to the respiration of nitrate, repair of alkylated DNA and synthesis of the ompC gene coding for porin C, cytochromes C, thiamine, and colonic acid. Strain MS04 is advantageous for the production of ethanol from hemicellulosic hydrolysates that contain acetate.     

Fermentation of xylose and hemicellulose hydrolysates by an ethanol-adapted culture ofBacteroides polypragmatus

Bacteroides polypragmatus type strain GP4 was adapted to grow in the presence of 3.5% (w/v) ethanol by successive transfers into 1% (w/v)d-xylose media supplemented with increasing concentrations of ethanol. The maximum specific growth rate of the ethanol-adapted culture (=0.30 h^-1) was not affected by up to 2% (w/v) ethanol but that of the non-adapted strain declined by about 50%. The growth rate of both cultures was limited by nutrient(s) contained in yeast extract. The ethanol yield of the adapted culture (1.01 mol/mol xylose) was higher than that (0.80 mol/mol xylose) of the non-adapted strain. The adapted culture retained the ability to simultaneously ferment pentose and hexose sugars, and moreover it was not inhibited by xylose concentrations of 7–9% (w/v). This culture also readily fermented hemicellulose hydrolysates obtained by mild acid hydrolysis of either hydrogen fluoride treated or steam exploded Aspen wood. The ethanol yield from the fermentation of the hydrolysates was comparable to that obtained from xylose.This paper is issued as NRCC No. 26338     

Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes

Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.     

Growth rate of a non-fermentative <Emphasis Type="Italic">Escherichia coli</Emphasis> strain is influenced by NAD<Superscript>+</Superscript> regeneration

By complementing a non-fermentative Escherichia coli (ldhA ⁻ pflB ⁻) strain with the recombinant Zymomonas mobilis ethanol pathway (pdc, adhB), we evaluated the effect of different levels of enzymatic activity on growth rate demonstrating that there is a direct relationship between anaerobic growth rate and the total specific activity of pyruvate decarboxylase, which is the limiting enzyme of this specific fermentative NAD⁺ regenerating pathway. This relationship was proved to be useful to establish a selection strategy based on growth rate for the analysis of lctE libraries, which encode lactate dehydrogenase from Bacillus subtilis.     

Fermentation of sugar cane bagasse hemicellulosic hydrolysate and sugar mixtures to ethanol by recombinant Escherichia coli KO11

Escherichia coli KO11, carrying the ethanol pathway genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) from Zymomonas mobilis integrated into its chromosome, has the ability to metabolize pentoses and hexoses to ethanol, both in synthetic medium and in hemicellulosic hydrolysates. In the fermentation of sugar mixtures simulating hemicellulose hydrolysate sugar composition (10.0 g of glucose/l and 40.0 g of xylose/l) and supplemented with tryptone and yeast extract, recombinant bacteria produced 24.58 g of ethanol/l, equivalent to 96.4% of the maximum theoretical yield. Corn steep powder (CSP), a byproduct of the corn starch-processing industry, was used to replace tryptone and yeast extract. At a concentration of 12.5 g/l, it was able to support the fermentation of glucose (80.0 g/l) to ethanol, with both ethanol yield and volumetric productivity comparable to those obtained with fermentation media containing tryptone and yeast extract. Hemicellulose hydrolysate of sugar cane bagasse supplemented with tryptone and yeast extract was also readily fermented to ethanol within 48 h, and ethanol yield achieved 91.5% of the theoretical maximum conversion efficiency. However, fermentation of bagasse hydrolysate supplemented with 12.5 g of CSP/l took twice as long to complete. This revised version was published online in November 2006 with corrections to the Cover Date.     

High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.     

Metabolic regulation analysis of an ethanologenic <Emphasis Type="Italic">Escherichia coli</Emphasis> strain based on RT-PCR and enzymatic activities

Background  

A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDC _Zm and ADH _Zm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C.     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

A novel assay for rapid in vivo determination of phenotypic stability of recombinant ethanol-producing microorganisms

A rapid empirical assay is presented for assessing the phenotypic stability of continuous cultures of recombinant bacteria containing transposed pdc and adh genes for ethanol production. The method measures spectrophotometrically the rate of colour formation when cells oxidize added ethanol to acetaldehyde in the presence of Schiff’s reagent. During chemostat cultures of the recombinant ethanologen Escherichia coli KO11 on 20 g/l glucose, assay activities were stable and high at ca 8 × 10⁻⁴ ΔOD₅₄₀/(s.OD₅₅₀), reflecting the high, stable ethanol yield (ca 95%). On 20 g/l and 50 g/l xylose, ethanol yields declined rapidly to about 60% and this was closely mirrored by the assay activities which fell to ca 1.5 ΔOD₅₄₀/(s.OD₅₅₀), only slightly higher than those measured for the parent strain. Typically taking only about an hour to perform, the assay provides a faster means of gauging the phenotypic stability of ethanol production than is possible by conventional methods.     

Ethanol production during D-xylose,L-arabinose,and D-ribose fermentation by Bacteroides polypragmatus

Summary The specific growth rate () during cultivation of Bacteroides polypragmatus in 2.51 batch cultures in 4–5% (w/v) l-arabinose medium was 0.23 h^-1 while that in either d-xylose or d-ribose medium was lower (=0.19 h^-1). Whereas growth on arabinose or xylose occurred after about 6–8 h lag period, growth on ribose commenced after a 30 h lag phase. The maximum substrate utilization rate for arabinose, ribose and xylose in media with an initial substrate concentration of 4–5% (w/v) was 0.77, 0.76, and 0.60 g/l/h respectively. In medium containing a mixture of glucose, arabinose, and xylose, the utilization of all three substrates occurred concurrently. The maximum amount of ethanol produced after 72 h growth in 4–5% (w/v) of arabinose, xylose, and ribose was 9.4, 6.5, and 5.3 g/l, respectively. The matabolic end products (mol/mol substrate) of growth in 4.4% (w/v) xylose medium were 0.73 ethanol, 0.49 acetate, 1.39 CO₂, 1.05 H₂, and 0.09 butyrate.National Research Council of Canada No. 23406     

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD⁺-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Δ and gpd2Δ were studied under alcoholic fermentation conditions. gpd1Δ mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Δ mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Δ) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Δ) but a slight decrease in the strain ANG2 (gpd2Δ). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Δ) and ANG2 (gpd2Δ) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.