Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli
© Munjal et al.; licensee BioMed Central Ltd. 2012
Received: 9 July 2012
Accepted: 29 October 2012
Published: 4 November 2012
E. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products.
The availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield.
The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.
KeywordsEscherichia coli Endogenous pathways Promoter engineering Pyruvate dehydrogenase Acetate kinase Ethanol
We are largely dependent upon fossil fuels for fulfilling our energy requirement . Fuels from renewable sources, such as agricultural and forest residues, hold promise in reducing our dependence on fossil fuel without competing with food. The agricultural and forestry waste mostly consist of lignocellulose, which is made-up of highly structured cellulose surrounded by hemicellulose and lignin . In principle, it is possible to breakdown lignocellulose into the monosaccharides and ferment them into ethanol. However, cost associated with this process is a major hurdle in terms of commercial application . One of the key advancement in the economy of ethanol production from lignocellulosic biomass will be to efficiently ferment both hexose and pentose sugars released after hydrolysis of lignocellulose into ethanol. Unfortunately, the conventional microorganisms used for ethanol fermentation, e.g., Saccharomyces cerevisiae and Zymomonas mobilis, do not have the capability to utilize pentose sugars . Attempts have been made to transfer genes for pentose degradation pathway from other organisms into S. cerevisiae and Z. mobilis. However, the disadvantages associated with foreign gene expression at large scale like instability, toxicity, containment, etc., prevent its wide usage. Escherichia coli, on the other hand, has the ability to ferment both hexose and pentose sugars and is being used to produce ethanol by various genetic manipulation . The genetic manipulation of E. coli that does not involve introduction of foreign gene has been attempted with some successes and these technologies certainly have advantages in the long-term genetic stability of the engineered strain [7, 8].
However, expression of PDH is repressed under anaerobic condition and remains active in the aerobically growing cells . To activate the expression of PDH under anaerobic condition, the promoter of PDH should be replaced with the one that is highly active under anaerobic condition. One such study showed that replacing PDH promoter with PFL promoter has enhanced the expression of PDH under anaerobic condition and increased the yield of ethanol . However, ethanol productivity was significantly lower in this study indicating sub-optimal flux through the PDH pathway.
In the study reported here, we have performed a systematic study of PDH expression under anaerobic condition by replacing its promoter with a number of promoters of the genes that are expressed at high level under anaerobic condition. A novel promoter-PDH operon combination was selected based on higher PDH enzyme activity and bioalcohol production and deletion mutants were generated to stop carbon flow to the competing byproducts. With the knowledge gained from the growth rate of various deletion mutants, we found a new way to improve cell growth and ethanol productivity by modulating expression of acetate kinase in the engineered cells. We further demonstrate that the engineered E. coli strain grown under microaerobic condition utilized xylose with the yield higher than reported before [7, 8]. Furthermore, to our knowledge we show for the first time fermentation of mixture of glucose and xylose into ethanol by the engineered E. coli strain without having any foreign gene for the ethanol production.
Results and discussion
Promoter replacement of pyruvate dehydrogenase (PDH) operon enhances its activity and ethanol yield under anaerobic condition
Strains, plasmids and primers used in the study
Reference or Source
E. coli B
E. coli B, ΔPDH-promoter::FRT-kan-FRT-ldhA gene promoter; promoter of pdh gene replaced with promoter of ldhA gene
E. coli B, ΔPDH-promoter::FRT-kan-FRT-frdA gene promoter
E. coli B, ΔPDH-promoter::FRT-kan-FRT-pflB gene promoter
E. coli B, ΔPDH-promoter::FRT-kan-FRT-adhE gene promoter
E. coli B, ΔPDH-promoter::FRT-kan-FRT-gapA gene promoter
SSY05 ΔldhA::FRT-kan-FRT; deletion mutant for ldhA gene in SSY05 host
SSY06 ΔfrdA::FRT-kan-FRT; deletion mutant for frdA gene in SSY06 host
SSY07 ΔackA::FRT-kan-FRT; deletion mutant for ackA gene in SSY07 host
SSY08 ΔpflB::FRT-kan-FRT; deletion mutant for pflB gene in SSY08 host
bla, cloning vector
bla, γ β exo (red recombinase), temperature-conditional replicon
bla, flp, temperature-conditional replicon
FRT-kan-FRT sequence from pKD4 was cloned into pUC19 at Eco RI and Bam HI sites
ldh A gene promoter from E. coli B was cloned into pSSY01 at Bam HI and Hind III site
frd A gene promoter from E. coli B was cloned into pSSY01 at Bam HI and Hind III sites
pfl B gene promoter from E. coli B was cloned into pSSY01 at Bam HI and Hind III sites
adh E gene promoter from E. coli B was cloned into pSSY01 at Bam HI and Hind III sites
gap A gene promoter from E. coli B was cloned into pSSY01 at Bam HI and Hind III sites
PLtetO1 expression vector, pSC101*origin, CmR
multiple cloning site derived from pET28a(+) cloned in pZSblank
ack gene cloned in pZS*mcs vector
ldh A promoter-F
ldh A promoter-R
frd A promoter-F
frd A promoter-R
pfl B promoter-F
pfl B promoter-R
adh E promoter-F
adh E promoter-R
gap A promoter-F
gap A promoter-R
The engineered strains SSY01 to SSY05 along with wild type E. coli B were grown in defined medium in filled Hungate tube with 2.5 g/l of either glucose or xylose as carbon source and cultures were analyzed for production of ethanol at different time intervals. Most engineered strains utilized complete 13.9 mM glucose in 12 h and produced ethanol in the range of 11.7 mM (for SSY01) to 17 mM (for SSY05) as compared to E. coli B which produced 12.2 mM ethanol. It took 24 h for the E. coli strains to utilize xylose (17 mM) with 0% (for SSY05) to 20% (for E. coli B and SSY01) residual sugar left at the end. Ethanol from xylose was produced in the range of 13 mM (for SSY01) to 21 mM (for SSY05) as compared to wild type E. coli B that produced 13 mM ethanol. These observations indicated that SSY01 with PldhPDH genotype behaved similar to wild type E. coli in terms of ethanol production as against Geobacillus thermoglucosidasius where it improved ethanol production . Possible explanation of these observations could be that ldh promoter in E. coli was either relatively weaker or regulated through complex mechanism under anaerobic condition. This hypothesis needs further exploration. Other engineered strains showed higher ethanol production as compared to the wild type strain. SSY05 performed best among all the engineered stains and therefore was considered for further strain improvement. Our approach of first optimizing the flux towards the PDH pathway through promoter engineering in the wild type strain before any deletion in the competing pathway, as against the previous report where the flux through PDH pathway was enforced by first deleting the competing pathway , had an advantage of finding optimal flux through PDH pathway even in presence of competing PFL pathway that is considered essential under anaerobic condition . This was likely to lower the adverse impact on cell growth upon pflB deletion.
Deletion of competing pathways improves ethanol yield in the engineered SSY05 strain
Fermentation parameters for cell growth, sugar utilization and product synthesis at the bioreactor level
Product Yield (mmol per mmol sugar)
% Theoretical yield of ethanolb
Max ethanol productivityC
E. coli B
Defined medium+20 g/l
Defined medium+20 g/l
Defined medium+20 g/l
Defined medium+20 g/l
Defined medium+20 g/l
E. coli B
LB medium+50 g/l
LB medium+50 g/l
E. coli B
Defined medium+20 g/l
Defined medium+20 g/l
E. coli B
LB medium+50 g/l
LB medium+50 g/l
LB medium+50 g/l (Microaerobic, pH 6.3)
LB medium+50 g/l (Microaerobic, pH 6.3)
Comparison of engineered strains for ethanol production at the bioreactor level in defined medium
Comparison of engineered strains for ethanol production at the bioreactor level in complex medium
We further tested utilization of mixture of glucose and xylose at 25 g/l each under micro-aerobic condition and found complete utilization of sugars in 55 hrs (Figure 6B). As evident from the fermentation profile, glucose was the preferred substrate and got utilized first in 15 h followed by xylose utilization in next 40 h. Ethanol yield from glucose and xylose was close to 0.43 g per g sugar (85% of the maximum theoretical yield) and ethanol was produced at the rate of 14.94 mmol/l/h (0.7 g/l/h) during glucose utilization phase and 8.17 mmol/l/h (0.38 g/l/h) during xylose utilization phase (Figure 6B). This rate of ethanol production from mixture of glucose and xylose was close to that of- recombinant E. coli KO11 strain that produced ethanol at overall 0.34 g/l/h from mixture of 10g/l glucose and 40 g/l xylose  and at 0.72 g/l/h h in the first 48 h from mixture of 70 g/l glucose and 30 g/l/h xylose of fermentation . None of the reports published before for the engineered E. coli without the foreign genes demonstrated utilization of mixture of glucose and xylose [7, 8].
The E. coli SSY09(pZSack) strain engineered for ethanol production in this study certainly has advantage over the other engineered E. coli strains such as KO11 for not having any foreign genes responsible for ethanol production. E. coli KO11 has been found to lose its ethanologenicity progressively when cultivated on hemicellulosic sugars in the chemostat culture, possibly due to the genetic instability . Since E. coli SSY09(pZSack) does not have any foreign genes for ethanol production, its ethanologenic property is expected to be stable for much longer generation and therefore this strain should be considered for further studies to evaluate ethanol production from lignocellulosic hydrolysates.
We replaced promoter of pyruvate dehydrogenase operon (PDH) in E. coli with promoters of various genes expressed under anaerobic condition and shown that PDH expression and ethanol yield was maximum under anaerobic condition when its promoter was replaced with gapA promoter. Deletion of pathways for competing products further increased the ethanol yield. However, there was significant drop in cell growth rate. Modulating expression of acetate kinase helped restoring the cell growth rate and improved ethanol productivity significantly. Microaerobic condition further improved the growth rate of the cells on both glucose and xylose. The strain reported here following engineering of endogenous pathway is likely to be genetically more stable and call for further study to evaluate ethanol production from hydrolysate of lignocellulosic biomass.
Bacterial strains, plasmids and genetic methods
List of bacterial strains, plasmids and primers used in the study has been provided in Table 1. E. coli DH5α strain (Invitrogen) was used for performing all the cloning work and E. coli B (Coli Genetic Stock Centre (CGSC), Yale University, USA) was used as parent strain for all the genomic manipulations. Recombinant DNA techniques were performed according to standard procedures . Restriction endonuclease and T4 DNA ligase were procured from New England Biolabs and DNA purification was performed using Qiagen kit. Custom oligonucleotides (primers) were synthesized from Sigma-Aldrich for PCR amplifications. DNA fragments were amplified by Phusion High Fidelity polymerase (Finnzymes) for cloning and template preparation for homologous recombination and Taq DNA polymerase (Bangalore Genei) was used for performing verification PCR of the engineered strains. Plasmids pKD4, pKD46 and pCP20 (CGSC, USA) were used as the source of FRT-kan-FRT fragment, lambda Red recombinase and flippase, respectively, for performing genetic manipulation.
For replacement of PDH operon promoter in the E. coli B genome, following procedure was adopted. FRT-kan-FRT sequence from pKD4 was amplified using FRT-kan-FRT-F and FRT-kan-FRT-R primers (Table 1), digested with Eco RI and Bam HI and ligated to the corresponding restriction endonuclease sites of pUC19 plasmid to generate the plasmid pSSY01. Promoters of the genes ldhA, adhE, frdA, gapA and pflB, along with their corresponding ribosome binding sites, were amplified from E. coli B genomic DNA using their respective primers listed in Table 1. The PCR products obtained were digested with Bam HI and Hind III and ligated at the 3’ end of FRT-kan-FRT in pSSY01 to produce plasmids pSSY02-06 (Table 1). A 45 bases homologous sequence for −202 to −157 bp upstream of pdhR coding region of PDH operon was added to the 20 bases of 5’ end of FRT-kan-FRT sequence to design primer H1 and a 45 bases homologous sequence corresponding to +1 to +45 coding region of ace E of PDH operon was added to 20–22 bases of 3’ end of each promoter to obtain primer H2 (Table 1). PCR was performed with the H1 and H2 primers and corresponding plasmid pSSY02-06 as template under following conditions: 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 15 sec, annealing at 59°C for 15 sec, extension at 72°C for 2 min and a final extension at 72°C for 10 min. The PCR product was gel eluted, digested with DpnI, re-purified and electroporated (2.5 KV, 25 μF and 200 Ω) into E. coli B carrying pKD46 (grown in LB broth with 1 mM L-arabinose at 30°C till OD600nm reaches ~ 0.3 - 0.4) to replace the promoter, RBS and pdhR gene of pyruvate dehydrogenase (PDH) operon with the heterologous promoter . Transformants were selected on kanamycin LB-agar plates. The engineered strains (SSY01-05) (Table 1) were verified for the PDH promoter replacement by performing two sets of colony PCR, one set using v-PDH-F (−372 bp upstream of pdhR) and v-PDH-R (+163 bp downstream of start of coding region of aceF) primers to verify native promoter deletion, and second set using forward primer of the heterologous promoter and v-PDH-R to verify introduction of heterologous promoter (data not shown). Before further manipulation, the kanamycin resistance marker gene was removed from the chromosome of the selected strain with the help of FLP recombinase by using the temperature sensitive helper plasmid, pCP20 .
Host gene deletions were achieved through P1 transduction method  using the single gene knockout Keio strains from CGSC, Yale University, USA . The kanamycin resistant marker gene was removed as described above and the resultant strain was used for sequential rounds of gene knockout.
For the construction of pZSack plasmid, the multiple cloning site (MCS) of pET28a(+) (Novagen) was amplified using pET28mcs-F and pET28mcs-R primers and cloned in pZSblank plasmid  to obtain pZS*mcs. The ack A gene encoding acetate kinase was amplified from E. coli B genome using pZS-ack-F and pZS-ack-R primers, digested with Bam HI and Sal I and the resultant fragment was ligated into the Bam HI-Sal I sites of pZS*mcs to produce pZSack. The pZSack plasmid was then electroporated into SSY09 for enhancement of growth rate.
Media and culture conditions
Bacterial strains were grown in either LB medium or Morpholino-propanesulfonic (MOPS) defined medium . Antibiotics were added as appropriate with ampicillin at 50 μg/ml, kanamycin at 30 μg/ml and chloramphenicol at 34 μg/ml. For checking production of metabolites by the engineered strains in the tube, the strains were grown overnight at 37°C on LB agar plates containing relevant antibiotic and an isolated colony was inoculated in Hungate tube filled until brim (17.5 ml) with 1X MOPS or LB medium supplemented with antibiotics and desired sugar as carbon source. In the study where engineered strains were transformed with pZSack or pZS*mcs plasmid, the cells were grown in the Hungate tube filled with media containing 0, 0.1 or 100 ng/ml of anhydrotetracycline as inducer and 34 μg/ml of chloramphenicol as antibiotic. The tubes were incubated at 37°C under rotating condition and harvested at different intervals. The optical density of the grown culture was recorded at 600 nm and supernatant was saved for metabolite analysis via HPLC as mentioned in analytical methods section.
The engineered strains were cultivated in the bioreactor to evaluate their performance under controlled environment at various stages of manipulation. Primary culture was prepared by incubating an isolated colony from agar plate into 17.5 ml MOPS medium containing 2.5 g/l glucose in Hungate tube for 24 hr at 37°C. In case of SSY09 where ack and pflB genes were deleted, primary culture was adapted to anaerobic condition in 100 ml medium in a 250 ml flask containing 2.5 g/l glucose or xylose for 48 hr at 37°C in anaerobic chamber (Bactron II, Shel Lab). Appropriate volume of the culture to achieve initial OD600nm of 0.05 in the bioreactor was centrifuged at 4000 rpm for 4 min and re-suspended in fresh medium. The culture was inoculated in one of the six 0.5 L vessels of Biostat Q plus fermentor (Sartorius) containing 350 ml of MOPS or LB medium having appropriate amount of sugar. The vessels were controlled independently at 37°C, 300 rpm and pH 6.8. High purity Argon gas was purged in the medium to create anaerobic environment at a rate of 0.02 L/min. In case of fermentation under microaerobic condition, compressed air was passed in the headspace of the vessel at the rate of 0.02L/min at which dissolved oxygen probe demonstrated zero reading throughout the fermentation. Samples collected from fermentor vessels at various time intervals were used to calculate cell growth, substrate utilization and product synthesis. All fermentations were performed in duplicate and data in the figures represented average of two bioreactor runs.
To check the activity of PDH enzyme under anaerobic conditions, engineered E. coli B strains with heterologous PDH promoter along with wild type strain as control were grown in Hungate tubes filled with MOPS medium + 2.5 g/l glucose for 12, 18, 24 and 36 hrs at 37°C. Cells were harvested by centrifugation (5 min, 5000 rpm), washed twice with 9 g/l NaCl and stored as cell pellets at −20°C. Cell pellets were resuspended in 0.1 M potassium phosphate buffer (pH 8.0) to obtain OD600 of 10 and were permeabilized with chloroform. The reaction was set-up in 1 ml in the cuvette containing 50 mM potassium phosphate buffer (pH 8.0), 2.0 mM sodium pyruvate, 2.5 mM NAD+, 0.2 mM thiamine pyrophosphate, 1.0 mM MgCl2, 0.13 mM CoA, 2.6 mM cysteine hydrochloride. Permeabilized cells (25 μl) were added to start the reaction and the pyruvate dehydrogenase activity was measured by detecting change in absorbance at 340 nm (Ultrospec 3100 pro, Amersham Biosciences) . Substrate blank where no sodium pyruvate was added served as control. Enzyme activity was calculated as nmol NADH formed/min/mg of cell protein. A protein content of 50% (wt/wt) with respect to dry cell mass was assumed in these calculations.
Extracellular metabolites of the grown culture were determined as follows. Culture of the grown cells was centrifuged at 13,200 rpm for 5 min. The aqueous supernatant was filtered and used for HPLC analysis. The metabolite separation was achieved using the HPLC system (Agilent technologies) attached with Aminex HPX-87 H anion exchange column (Bio-Rad). The filtered and degassed mobile phase (4 mM H2SO4) was used at a constant rate of 0.3 ml/min with column and RI detector temperatures maintained at 40°C and 35°C, respectively. Standards of the metabolites (Absolute Standards, USA) at 1 g/l were separated on HPLC column and areas obtained were used to calculate metabolite concentration in the text samples. Cell density was measured at an optical density 600 nm (OD600) in a spectrophotometer (BioRad). Dry cell mass was calculated by drying cell pellets of defined OD600 at 75°C in oven for 20 hr. The OD600 of 1.0 corresponded to 0.56 mg dry mass per ml of culture.
The values obtained for cell biomass, substrate utilization and product synthesis were used for calculation of biomass and product yields (mmol/mmol substrate), specific productivity (mmol/gcell/h) and volumetric productivity (mmol/L/h). For calculating biomass yield we used a molecular formula of cells as CH1.9O0.5N0.2 with an average molecular weight of 24.7 .
We are thankful to Dr. Ramon Gonzalez for providing pZSblank plasmid. The study was supported by the financial grants from Department of Biotechnology and Department of Science and Technology, Government of India. NM was supported by the fellowship from Council of Scientific and Industrial Research.
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