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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

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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


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


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


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


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


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


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.


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Brown, R. C., Daugaard, D. E., Platon, A., Kothandaraman, G., Hsu, D. D., Dutta, A. ,

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alcohol dehydrogenase-II gene from Zymomonas mobilis. Journal of Bacteriology, 169,

2591-2597.

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Cloning of a novel levoglucosan kinase gene from Lipomyces starkeyi and its expression

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Escherichia coli K-12 using PCR products. Proceedings of the National Academy of

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of ethanologenic bacteria. Adv Biochem Eng Biotechnol, 108, 237-61.

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glucopyranose) in microorganisms. Agricultural and Biological Chemistry, 55, 515-521.

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Ferment. Technol., 62, 201-203.

Ohta, K., Beall, D.S., Mejia, J.P., Shanmugam, K.T., Ingram, L.O., 1991. Genetic improvement

of Escherichia coli for ethanol production: chromosomal integration of Zymomonas

mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Appl

Environ Microbiol, 57, 893-900.

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and switch phoA gene, lacZ (op) and lacZ (pr) fusions. in: K.W. Adolph (Ed.) Methods in

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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,


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


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


GCCGTAAGCCTGTTAACAAGCT

reverse verification primer GAAGCCATAGCTATAACCTCACC

Region 2: between ADH and CmR

forward knock-in primer AACAATGCCTCCGATTTCTAATCGGAGGAGGTATAATGCCGATTGCGACGTCTACCGGCG

reverse knock-in primer TTGCAATAAACAAAAACAAATGCCTTTACGCCCAGTTGTTGGTGATAACT


GGAAAACGGTTTTCCGTCCTGT

reverse verification primer TGGCAAATTATTTATGACGGTAGG


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


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

0.0

0.2

0.4

0.6

0.8

0.0 0.5 1.0 1.5 2.0 2.5

OD

550

wt% sugar

B: KO11 + lgkregion 1

levoglucosanglucose

0.0

0.2

0.4

0.6

0.8

0.0 0.5 1.0 1.5 2.0 2.5

OD

550

wt% sugar

C: KO11 + lgkregion 2

levoglucosanglucose

0.0

0.2

0.4

0.6

0.8

0.0 0.5 1.0 1.5 2.0 2.5

OD

550

wt% sugar

A: KO11

levoglucosanglucose


CmR (region 2) is able to utilize levoglucosan. Error bars show the standard deviation of three biological replicates.


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.

0.0

0.2

0.4

0.6

0.8

1.0

0 12 24 36 48

Eth

anol

, wt%

time (hr)

Ethanol production

levoglucosan

glucose

0.0

0.5

1.0

1.5

2.0

0 12 24 36 48

Suga

r, w

t%

time (hr)

Sugar utilization

levoglucosan

glucose

0

1

2

3

4

5

0 12 24 36 48

OD

550

time (hr)

Growth

levoglucosan

glucose


engineering ethanologenic escherichia coli for ... · engineering ethanologenic escherichia coli...

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