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Chemical and Biological Engineering Publications Chemical and Biological Engineering
2011
Engineering ethanologenic Escherichia coli forlevoglucosan utilizationDonovan S. LaytonIowa State University
Avanthi AjjarapuIowa State University
Dong Won ChoiTexas A & M University - Commerce
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AuthorsDonovan S. Layton, Avanthi Ajjarapu, Dong Won Choi, and Laura R. Jarboe
This article is available at Digital Repository @ Iowa State University: http://lib.dr.iastate.edu/cbe_pubs/177
Engineering ethanologenic Escherichia coli for levoglucosan utilization
Donovan S. Layton1, Avanthi Ajjarapu2, Dong Won Choi3, Laura R. Jarboe*1
1Chemical and Biological Engineering, Iowa State University
2CBiRC Young Engineers Program, Iowa State University
3Biological and Environmental Sciences, Texas A&M University - Commerce
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
ABSTRACT
Levoglucosan is a major product of biomass pyrolysis. While this pyrolyzed biomass,
also known as bio-oil, contains sugars that are an attractive fermentation substrate, commonly-
used biocatalysts, such as Escherichia coli, lack the ability to metabolize this anhydrosugar. It
has previously been shown that recombinant expression of the levoglucosan kinase enzyme
enables use of levoglucosan as carbon and energy source. Here, ethanologenic E. coli KO11 was
engineered for levoglucosan utilization by recombinant expression of levoglucosan kinase from
Lipomyces starkeyi. Our engineering strategy uses a codon-optimized gene that has been
chromosomally integrated within the pyruvate to ethanol (PET) operon and does not require
additional antibiotics or inducers. Not only does this engineered strain use levoglucosan as sole
carbon source, but it also ferments levoglucosan to ethanol. This work demonstrates that
existing biocatalysts can be easily modified for levoglucosan utilization.
Keywords: ethanol, levoglucosan, genomic integration, Escherichia coli
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
INTRODUCTION
While enormous progress has been made in engineering biocatalysts to produce a variety
of biorenewable fuels and chemicals from pure sugars (Clomburg and Gonzalez, 2010), the
economically viable use of biomass-derived sugars is still a challenge. Hydrolysis of biomass to
release fermentable sugars has been the focus of intense research; an alternative method of
extracting sugars from biomass is thermochemical processing, in which biomass is subjected to
rapid thermal decomposition by fast pyrolysis to yield syngas, bio-oil and bio-char (Brown,
2007). A recent comparative cost analysis showed fast pyrolysis to be an attractive means of
biofuels production relative to both enzymatic hydrolysis and gasification (Anex, 2010).
Bio-oil is a fluid that contains up to 20% water and a mixture of anhydrosugars, acids,
aldehydes, furans and phenols, with yields of up to 500 L of bio-oil per dry ton of biomass
(Brown, 2007). The anhydrosguar levoglucosan (1,6-anhydro-β-d-glucopyranose) is the most
abundant sugar in bio-oil and is thus the most attractive fermentation substrate. Pyrolysis of
untreated biomass can produce bio-oil that contains up to 12% levoglucosan (Patwardhan et al.,
2009; Patwardhan et al., 2010); pre-treatment of the biomass to remove cations can result in bio-
oil that contains up to 30% levoglucosan (Piskorz, 1997).
While levoglucosan can be converted to glucose by hydrolysis (Yu and Zhang, 2004) and
then used as a fermentative substrate (Chan and Duff, 2010), it is desirable to use biocatalysts
that can directly metabolize bio-oil into biorenewable chemicals with minimal processing steps.
The feasibility of this approach was previously demonstrated using fungal biocatalysts (Prosen et
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
al., 1993). However, our traditional workhorse biocatalysts, such as Escherichia coli, lack the
inherent ability to metabolize this compound.
Levoglucosan is naturally abundant where forest fires or other types of biomass burning
incidents have occurred. Several microorganisms have been identified that can use levoglucosan
as carbon and energy source (Nakagawa et al., 1984; Prosen et al., 1993; Zhuang and Zhang,
2002). For example, Aspergillus terreus K26 and Aspergillus niger CBX 209 can metabolize
levoglucosan to produce itaconic acid (Nakagawa et al., 1984) and citric acid (Zhuang and
Zhang, 2002), respectively. Biochemical studies have shown that the Mg-ATP-dependent
levoglucosan kinase (LGK) enzyme converts levoglucosan into glucose-6-phosphate (Kitamura
et al., 1991), routing it into the general glycolytic pathway. Given the status of E. coli as a
premier industrial workhorse and producer of biorenewable chemicals, previous attempts have
been made to engineer E. coli for levoglucosan utilization. The fungal LGK was cloned into E.
coli from A. niger CBX-209, but the resulting enzyme activity was low (Zhuang and Zhang,
2002). A more recent study isolated LGK from the yeast Lipomyces starkeyi YZ-215 and
expressed it in E. coli; the resulting strain utilized levoglucosan as sole carbon source in minimal
media (Dai et al., 2009). Here, codon-optimized L. starkeyi LGK is expressed in ethanologenic
E. coli KO11 (Ohta et al., 1991) and it is demonstrated that the engineered biocatalyst can utilize
levoglucosan as a sole carbon and energy source and for ethanol production (Figure 1).
MATERIALS AND METHODS
Strains and media: Ethanologenic E. coli KO11, a derivative of E. coli W engineered for
ethanol production (Jarboe et al., 2007; Ohta et al., 1991), was obtained from American Tissue
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
Type Collection (ATCC, strain 55124). Chloramphenicol was used when KO11 was maintained
on LB plates. Levoglucosan (1,6-anhydro-β-d-glucose) was obtained from Sigma. MOPS
minimal media was made according to (Wanner, 1994).
Genomic Integration of LGK: The L. starkeyi YZ-215 LGK sequence (GeneBank Accession #
EU751287) was codon optimized for E. coli (Table 1) and synthesized by GenScript
(Piscataway, New Jersey). The optimized sequence was amplified from its pUC57 construct for
genomic integration by PCR using Taq DNA polymerase (Qiagen), with integration and
verification primers as listed in Table 2. Two integration sites within the pyruvate to ethanol
(PET) genomic operon were utilized. Primer design for genomic integration was based on
original reports of strain construction (Conway et al., 1987; Conway et al., 1987) and our own
sequence analysis (data not shown). Genomic integration was performed using the helper
plasmid pKD46 (Datsenko and Wanner, 2000), with successful integrants selected on MOPS
minimal media with 0.5wt% levoglucosan and verified by PCR.
Growth Conditions: For small-small analysis, cells were grown in 3mL cultures in 5 mL
standing tubes with horizontal shaking at 80rpm for 24 or 48 hours. Cultures were inoculated to
an initial OD550 of 0.05 in MOPS media with filter-sterilized glucose or levoglucosan.
Fermentations were performed at 370C in 500mL fermentors in 350mL of Luria Broth (LB) with
filter-sterilized glucose or levoglucosan. Fermentations were maintained at pH 6.5 by addition of
2N KOH, with stirring at 150rpm. Three biological replicates for each for levoglucosan and
glucose were seeded at OD550 of 0.05 from a single culture grown in LB with no sugar.
Metabolite Analysis: Ethanol, levoglucosan and glucose were measured by HPLC on a Water
2424 Refractive Index Detector using a Bio-Rad Aminex HPX-87H column and Empower Pro
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
software for analysis. Samples were analyzed at 400C in 8 mM sulfuric acid at a flowrate of 0.6
mL/min.
RESULTS AND DISCUSSION
E. coli KO11 lacks the inherent ability to metabolize levoglucosan, as evidenced by its
inability to use levoglucosan as sole carbon source (Figure 2A). The goal of this project was to
engineer ethanologenic E. coli for levoglucosan utilization, and thus a LGK enzyme was needed
in order to provide a pathway for levoglucosan utilization. The LGK gene from L. starkeyi YZ-
215 was selected, given the previous success of this gene in E. coli (Dai et al., 2009). However,
in this work the gene was codon-optimized for E. coli, increasing the codon adaptation index
from 0.66 to 0.93. Codon optimization was performed by GenScript; the optimized sequence is
given in Table 1.
The pyruvate to ethanol (PET) operon was selected as our genomic integration site, since
these genes are expressed at a level sufficient to enable redox balanced production of ethanol as
the major fermentation product. The genomic integration strategy was such that the codon-
optimized LGK gene was genomically integrated into the PET operon either between pyruvate
decarboxylase (PDC) and alcohol dehydrogenase (ADH) (region 1) or between ADH and the
existing chloramphenicol resistance (CmR) gene (region 2). Construction of the PET operon
using Zymomonas mobilis genes and its genomic integration within E. coli were previously
described (Ohta et al., 1991).
Expression of the codon-optimized LGK gene in strain KO11 within either region 1 or
region 2 enabled the use of levoglucosan as sole carbon source (Figure 2B, 2C), with trends and
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
biomass yields comparable to the preferred carbon source glucose. These results confirm the
ability of ethanologenic E. coli to use levoglucosan as sole carbon source for growth upon
expression of LGK. The strain with the region 1 integration showed a slightly higher biomass
accumulation on both glucose and levoglucosan and was used for all other experiments.
As the primary interest in levoglucosan utilization is for the production of biorenewable
fuels and chemicals, such as ethanol, the fermentative performance of the engineered strain
relative to the preferred carbon source glucose was characterized. Fermentations with pH-,
temperature- and stir-controlled fermentations were performed for KO11 + lgkregion1. Given
KO11’s history of incomplete fermentations in minimal media (Jarboe et al., 2007), rich media
was used for these fermentations. As shown in Figure 3, the engineered KO11 strain ferments
levoglucosan to ethanol, though at a decreased titer (0.6 wt%) relative to the preferred carbon
source glucose (0.8 wt%). The yields at 48 hours were 0.43 g ethanol produced per g glucose
consumed and 0.35 g ethanol produced per g levoglucosan consumed.
The lower product titer can possibly be attributed to incomplete levoglucosan utilization:
approximately 0.25wt% (15mM) levoglucosan remained after 48 hours. This incomplete
levoglucosan utilization can in turn be possibly attributed to the relatively high substrate Km
(71.2mM) of the LGK enzyme (Zhuang and Zhang, 2002). It is also possible that levoglucosan
utilization is transport-limited; the transporter responsible for levoglucosan uptake by E. coli is
not known at this time.
CONCLUSIONS
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
This work shows that existing biocatalysts can be easily engineered for effective
levoglucosan utilization. Our metabolic engineering strategy yielded a biocatalyst that is not
dependent on additional antibiotics or inducers for levoglucosan utilization. While it is clear that
levoglucosan is a good fermentation substrate, it is not the only component of bio-oil. Many of
the other components are biocatalyst inhibitors: Prosen et al noted that several fungal species
could grow in bio-oil that had been treated with activated charcoal but not in the raw aqueous
extract (Prosen et al., 1993). Extraction with solvents (Chan and Duff, 2010) can also reduce
bio-oil toxicity and improve fermentability. Parallel efforts in decreasing toxicity and improving
biocatalyst tolerance are important steps towards biochemical utilization of this biomass-derived
carbon and energy.
ACKNOWLEDGEMENTS
This work was funded in part by Iowa State University’s Office of Biotechnology. A.A. was
funded in part by the NSF Center for Biorenewable Chemicals (CBiRC) Engineering Research
Center (award no. EEC-0813570). Special thanks for Meredith Breton and Sara Diamond Steffen
for their research contributions.
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
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NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
Table 1: Codon optimization of L. starkeyi YZ-215 LGK for E. coli. Codon optimization was
performed by GenScript. The top and bottom rows show the original and optimized sequence,
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
respectively. Altered codons are shown in bold.
1 ATG CCC ATC GCG ACT TCC ACT GGC GAC AAT GTG CTC GAC TTC ACC GTG CTC GGC CTC AAC 601 ATG CCG ATT GCG ACG TCT ACC GGC GAT AAC GTG CTG GAT TTT ACC GTT CTG GGC CTG AAT 60
61 TCG GGG ACG AGT ATG GAC GGC ATC GAC TGT GCG CTA TGC CAC TTT TAC CAA AAA ACT CCC 12061 AGT GGT ACG AGC ATG GAT GGT ATC GAT TGC GCA CTG TGT CAT TTC TAT CAGAAA ACC CCG 120
121 GAC GCG CCC ATG GAG TTT GAG CTG CTC GAG TAT GGA GAG GTC CCG CTT GCC CAG CCC ATC 180121 GAT GCG CCG ATG GAA TTT GAA CTG CTG GAA TAC GGC GAA GTG CCG CTG GCC CAG CCG ATT 180
181 AAG CAG CGA GTC ATG CGG ATG ATC TTG GAG GAC ACG ACA TCG CCG TCA GAG CTG TCC GAG 240181 AAA CAG CGT GTT ATG CGC ATG ATC CTG GAA GAT ACC ACGAGC CCG TCT GAA CTG AGC GAA 240
241 GTC AAC GTC ATT CTC GGG GAG CAC TTT GCC GAT GCT GTT CGA CAG TTT GCG GCC GAG CGC 300241 GTG AAC GTT ATT CTG GGT GAA CAT TTT GCC GAT GCA GTG CGT CAG TTC GCG GCC GAA CGC 300
301 AAC GTG GAC TTG AGC ACT ATC GAC GCG ATT GCA AGC CAC GGT CAG ACG ATC TGG CTG CTG 360301 AAT GTT GAT CTG AGC ACC ATT GAT GCG ATC GCC TCT CAC GGC CAG ACG ATT TGG CTG CTG 360
361 TCC ATG CCG GAG GAG GGA CAG GTC AAG TCG GCT CTG ACC ATG GCG GAA GGC GCG ATC CTC 420361 TCT ATG CCG GAA GAA GGT CAA GTG AAA AGT GCG CTG ACG ATG GCG GAA GGC GCG ATC CTG 420
421 GCA TCT CGC ACC GGC ATC ACG TCC ATC ACC GAC TTC CGA ATC TCC GAC CAG GCC GCC GGT 480421 GCC TCT CGT ACGGGT ATT ACC AGT ATC ACGGAT TTC CGT ATT AGC GAT CAG GCA GCA GGT 480
481 CGT CAG GGT GCT CCG CTG ATT GCC TTC TTC GAC GCT CTG CTC CTT CAC CAC CCG ACC AAG 540481 CGC CAG GGT GCA CCG CTG ATC GCG TTT TTC GAT GCC CTG CTG CTG CAT CAC CCG ACC AAA 540
541 CTG CGT GCG TGC CAG AAC ATC GGT GGT ATC GCA AAC GTC TGC TTC ATC CCT CCC GAC GTT 600541 CTG CGC GCG TGC CAG AAC ATT GGC GGT ATC GCC AAT GTG TGT TTT ATT CCGCCGGAT GTT 600
601 GAT GGC CGA CGC ACC GAC GAG TAC TAC GAC TTT GAC ACG GGA CCA GGC AAT GTC TTC ATA 660601 GAT GGC CGT CGC ACC GAT GAA TAT TAC GAT TTT GAT ACG GGT CCG GGC AAC GTG TTC ATC 660
661 GAT GCG GTC GTC CGA CAC TTC ACC AAC GGG GAG CAG GAG TAC GAC AAG GAT GGA GCG ATG 720661 GAT GCG GTG GTT CGT CAT TTT ACC AAT GGT GAA CAG GAA TAT GAT AAA GAT GGT GCG ATG 720
721 GGG AAG CGA GGC AAG GTG GAC CAG GAG CTC GTG GAT GAT TTC TTG AAG ATG CCA TAC TTC 780721 GGC AAA CGC GGT AAA GTG GAT CAG GAA CTG GTT GAT GAT TTT CTG AAA ATG CCG TAT TTC 780
781 CAA CTG GAC CCT CCC AAG ACT ACC GGT CGG GAG GTC TTC CGT GAT ACT CTG GCT CAC GAC 840781 CAG CTG GAC CCGCCGAAA ACC ACG GGT CGT GAA GTT TTT CGC GAT ACC CTG GCA CAT GAT 840
841 TTG ATC CGT CGC GCT GAG GCG AAA GGA CTG TCC CCC GAT GAC ATC GTT GCG ACG ACC ACC 900841 CTG ATT CGT CGC GCA GAA GCG AAA GGT CTG AGC CCG GAT GAT ATT GTG GCG ACC ACG ACC 900
901 AGG ATT ACC GCG CAA GCC ATT GTT GAC CAC TAC CGG CGC TAC GCT CCT AGC CAA GAG ATC 960901 CGT ATT ACGGCC CAGGCA ATC GTT GAT CAC TAT CGT CGC TAC GCC CCG AGC CAGGAA ATT 960
961 GAC GAG ATC TTC ATG TGC GGC GGA GGC GCG TAC AAC CCG AAC ATC GTC GAG TTC ATT CAG 1020961 GAT GAA ATC TTC ATG TGC GGC GGT GGC GCA TAT AAC CCG AAT ATT GTG GAA TTT ATC CAG 1020
1021 CAA AGC TAC CCT AAC ACC AAG ATC ATG ATG CTC GAC GAG GCT GGG GTC CCC GCT GGA GCA 10801021 CAG TCT TAC CCGAAC ACC AAA ATT ATG ATG CTG GAT GAA GCA GGC GTG CCGGCA GGT GCA 1080
1081 AAG GAG GCC ATC ACG TTC GCT TGG CAA GGA ATG GAA GCC CTT GTT GGC CGA TCC ATC CCT 11401081 AAA GAA GCA ATT ACG TTC GCG TGG CAGGGC ATG GAA GCA CTG GTT GGT CGT AGT ATC CCG 1140
1141 GTC CCC ACC CGC GTG GAG ACG CGA CAA CAC TAC GTG TTG GGC AAG GTG TCC CCG GGA CTG 12001141 GTG CCG ACC CGT GTT GAA ACG CGC CAG CAC TAT GTG CTG GGC AAA GTT AGT CCG GGT CTG 1200
1201 AAC TAC CGC AGC GTG ATG AAG AAG GGT ATG GCG TTC GGC GGA GAC GCG CAG CAG CTG CCG 12601201 AAT TAC CGC AGC GTG ATG AAA AAA GGC ATG GCC TTT GGT GGC GAT GCA CAG CAG CTG CCG 1260
1261 TGG GTC AGC GAG ATG ATT GTG AAG AAA AAG GGC AAG GTC ATT ACC AAC AAC TGG GCT TAA 13201261 TGG GTT AGC GAA ATG ATC GTG AAG AAA AAA GGC AAA GTT ATC ACC AAC AAC TGG GCG TAA 1320
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
Table 2: Primers used in this work.
LGK gene
forward verification primer
ATGCCGATTGCGACGTCTACCGGCG
reverse verification primer TTACGCCCAGTTGTTGGTGATAACT
Region 1: between PDC and ADH
forward knock-in primer CCTCTAGTTTTTGGGGATCAATTCGAGGAGGTATAATGCCGATTGCGACGTCTACCGGCG
reverse knock-in primer TACATACTAGTTTGGGTACCGAGCTTTACGCCCAGTTGTTGGTGATAACT
forward verification primer
GCCGTAAGCCTGTTAACAAGCT
reverse verification primer GAAGCCATAGCTATAACCTCACC
Region 2: between ADH and CmR
forward knock-in primer AACAATGCCTCCGATTTCTAATCGGAGGAGGTATAATGCCGATTGCGACGTCTACCGGCG
reverse knock-in primer TTGCAATAAACAAAAACAAATGCCTTTACGCCCAGTTGTTGGTGATAACT
forward verification primer
GGAAAACGGTTTTCCGTCCTGT
reverse verification primer TGGCAAATTATTTATGACGGTAGG
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
Figure 1: Metabolic pathway diagram for production of ethanol from levoglucosan. Ethanologenic E. coli KO11 has been previously engineered to produce ethanol as the major fermentation product via genomic integration of the Zymomonas mobilis PDC and ADH genes. Here a codon-optimized levoglucosan kinase (LGK) was chromosomally integrated within the PET operon and expressed for redox-balanced production of ethanol as the major fermentation product. .
ADH
PDC
acetaldehyde + CO2
ETHANOL
pyruvate
½ G6P
NADH3/2 ATP
NADH
½ glucose½ ATP equivalent
½ levoglucosan
½ ATP
LGK
Zym
omon
asm
obili
sho
moe
than
olpa
thw
ay
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
Figure 2: Ethanologenic E. coli KO11 was engineered for use of levoglucosan as sole carbon and energy source. Cells were grown for 48 hours in capped tubes at 370C in MOPS minimal media supplemented with glucose or levoglucosan, no antibiotics or inducers. (A) Unengineered KO11 does not utilize levoglucosan. KO11 with chromosomal integration of codon-optimized LGK within the PET operon (B) between PDC and ADH (region 1) or (C) between ADH and
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B: KO11 + lgkregion 1
levoglucosanglucose
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C: KO11 + lgkregion 2
levoglucosanglucose
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A: KO11
levoglucosanglucose
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
CmR (region 2) is able to utilize levoglucosan. Error bars show the standard deviation of three biological replicates.
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
Figure 3: Production of ethanol during fermentation of glucose or levoglucosan by engineered KO11 + lgkregion1. Cells were grown in rich media supplemented with 2.0 wt% glucose or levoglucosan at 370C, pH 6.5, 150 rpm. Data are the average of three biological replicates with error bars indicating standard deviation. Sugars and ethanol were measured by HPLC.
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Eth
anol
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time (hr)
Ethanol production
levoglucosan
glucose
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time (hr)
Sugar utilization
levoglucosan
glucose
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time (hr)
Growth
levoglucosan
glucose
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.
NOTICE: This is the author’s version of a work that was accepted for publication in Bioresource Technology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bioresource Technology, 102 (17)2011,doi: 10.1016/j.biortech.2011.06.011.