Article Figures & Data
- FIG 1
Scheme for screening tolerance and increasing titer of isopentenol. (A) First, transcriptomics of E. coli DH1 expressing an inactive mevalonate pathway (MevT*; see Materials and Methods) in the presence of exogenous isopentenol was used to determine what genes are upregulated in response to isopentenol stress. (B) A library of those genes was overexpressed in the presence of exogenous isopentenol. Growth curves were used to measure enhanced tolerance. (C) Finally, genes that conferred tolerance were tested for their ability to increase the isopentenol titer in a production strain.
- FIG 2
Induction of tolerance genes is toxic above 10 µM IPTG. E. coli DH1 harboring each of the 40 genes cloned into pBbA5k was induced with 0, 10, 50, 100, and 250 µM IPTG and grown for 21 h. For each strain, the OD600 was measured and normalized to growth without IPTG. The only concentration of IPTG that did not decrease growth across all strains was 10 µM IPTG; thus, all subsequent tolerance assays were performed with 10 µM IPTG. For OD600 data for a specific strain, see Fig. S1 in the supplemental material.
- FIG 3
Addition of isopentenol decreases the growth rate and causes growth lag. (A) Maximum growth rates (μmax) of DH1 pBbA5k-rfp induced with 10 µM IPTG were determined with exposure to 0 to 0.3% (wt/vol) isopentenol. Error bars represent standard errors from at least 4 replicates. The MIC under these growth conditions is 0.24% (wt/vol) isopentenol. (B) Representative growth curves of E. coli DH1 harboring pBbA5k-rfp induced with 10 µM IPTG and grown in the presence of 0% (wt/vol), 0.10% (wt/vol), 0.15% (wt/vol), and 0.20% (wt/vol) isopentenol. The addition of 0.15% exogenous isopentenol caused a sufficient lag in growth recovery, and thus 0.15% isopentenol was chosen as the concentration of isopentenol for tolerance assays.
- FIG 4
Eight gene candidates demonstrated tolerance to 0.15% (wt/vol) isopentenol. Strains that demonstrated isopentenol tolerance in the initial screen were screened in triplicate for their tolerance to 0.15% (wt/vol) isopentenol (black or blue) in comparison to an rfp-expressing strain (red traces). The eight genes that conferred the greatest growth lag shortening, shown in blue, are metR, ibpA, nrdH, soxS, mdlB, fpr, gidB, and yqhD.
- FIG 5
MetR increases production of isopentenol by 55%. The eight tolerance genes were individually coexpressed with the isopentenol production pathway. The tolerance genes and the production pathway were simultaneously induced with 500 µM IPTG in triplicate, and the isopentenol concentration was measured 24, 48, and 72 h after induction. The production strain coexpressing metR (PS+MetR) improved the isopentenol titer 55% over that for the rfp-expressing strain (PS+RFP).
- FIG 6
The putative ABC transporter MdlB increases isopentenol production. The isopentenol titer was measured in triplicate in PS+MdlB and PS+RFP over a range of IPTG concentrations to determine if the increase in production is titratable. Isopentenol titer is significantly increased in PS+MdlB at 100 µM, 250 µM, and 500 µM IPTG (P value =8 × 10−4, 2 × 10−6, and 1 × 10−4, respectively, two-sided t test).
- TABLE 1
Isopentenol tolerance-enhancing genes
Gene Description Microarray log2, z score Isopentenol production (mg liter−1)a Improvement in titer (%)b Reference(s) soxS DNA-binding transcriptional dual regulator 3.51, 2.81 838 ± 29 0 28, 42, 43 nrdH Glutaredoxin-like protein 2.17, 2.08 860 ± 3 3 44 mdlB Predicted multidrug ABC transporter 3.75, 2.31 931 ± 16 12 25 ibpA Heat shock chaperone 4.80, 2.82 967 ± 23 16 26, 27 gidB Glucose-inhibited division protein B 5.32, 2.64 965 ± 1 16 29, 45, 46 fpr Ferredoxin-NADP reductase 2.30, 2.07 989 ± 10 19 28 yqhD Alcohol dehydro-genase, NAD(P) dependent 2.40, 2.39 994 ± 13 19 14, 30, 31 metR DNA-binding transcriptional activator 2.57, 2.55 1,290 ± 20 55 32, 33
Supplemental Material
Table S1
Description of microarray GEO GSE53138 Table S1, PDF file, 0.1 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Genes selected for cloning to investigate their abilities to confer isopentenol tolerance Table S2, DOCX file, 0.1 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S1
Expression of select tolerance library genes is toxic when induced with 50 µM or more IPTG. E. coli DH1 harboring plasmids of the 40 genes in pBbA5k was induced with 0, 10, 50, 100, and 250 µM IPTG and grown for 21 h. For each strain, the OD600 was measured and normalized to that for growth without IPTG. In this color map, genes are ordered in increasing OD600 for the 50 µM IPTG condition. The only condition that did not significantly change the final OD600 across all cultures was 10 µM IPTG, for which it ranged from 0.87 (mdtG) to 1.1 (yhcN). Thus, we performed all following experiments with pBbA5k at 10 µM IPTG to ensure that expression of the tolerance gene was not toxic. Download Figure S1, TIF file, 4 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S3
Plasmids and strains Table S3, DOCX file, 0.02 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
Growth curves of all 40 candidates reveal 8 potential targets for improved tolerance. The 40 genes were individually overexpressed in E. coli DH1 with 10 µM IPTG and grown in the presence of 0.15% (wt/vol) isopentenol. The growth of the strains (black) was monitored in two sets over 24 h (A) or 48 h (B). The tolerance strains were compared to the control strain expressing RFP (gray) from the same growth condition. Strains with growth lags shorter than that of the control were selected for secondary selection. Download Figure S2, TIF file, 12.5 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Inducing the eight selected tolerance genes in the low-copy-number pBbS5k vector with 500 µM IPTG does not cause significant toxicity. The growth curves of the selected eight tolerance-enhancing genes (black) show that overexpression with 500 µM IPTG does not significantly alter growth compared to that of the rfp-expressing strain (red). Only metR and mdlB are associated with a slight defect in growth. Swapping the tolerance-enhancing genes into the low-copy-number pBbS5k plasmid backbone allows for the high IPTG concentration necessary for isopentenol production without significant toxicity. Download Figure S3, TIF file, 1.5 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Dose-response relationship of inducer to isopentenol titer shows a plateau in titer after 500 µM IPTG. Production strains coexpressing tolerance genes were induced over a range of IPTG concentrations to determine the maximum titer for each strain. The titer plateaued for most strains between 500 and 1,000 µM IPTG after 48 h; thus, all subsequent production strains were induced with 500 µM IPTG. Download Figure S4, TIF file, 0.7 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S5
MetR and MdlB specifically confer tolerance to isopentenol. MdlB (blue), MetR (orange), and RFP (red) were overexpressed on pBbA5k in E. coli DH1 with 10 µM IPTG and grown in the presence of other alcohols in triplicate. The RFP growth curve with no alcohol added is shown in grey. (A) MdlB did not improve the growth rate when grown in the presence of any of the alcohols tested. (B) The growth rates of pBbA5k-rfp and pBbA5k-metR in the presence of ethanol are 0.151 ± 0.001 h−1 and 0.155 ± 0.001 h−1, respectively. The growth rates of pBbA5k-rfp and pBbA5k-metR in the presence of butanol are 0.103 ± 0.004 h−1 and 0.119 ± 0.007 h−1, respectively. MetR statistically improved the growth rate in the presence of ethanol by 2.5% and that in the presence of butanol by 15% (P = 0.008 and 0.03, respectively; two-sided t test). Growth of the RFP-expressing control strain in the absence of alcohol is indicated by “RFP no –OH.” Download Figure S5, TIF file, 6.7 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Text S1
Supplemental Materials and Methods. Download Text S1, DOCX file, 0.03 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S4
Sequences of oligonucleotides used in plasmid construction of tolerance genes Table S4, DOCX file, 0.02 MB.
Copyright © 2014 Foo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
Files in this Data Supplement:
- Table st1, PDF - Table st1, PDF
- Text s1, DOCX - Text s1, DOCX
- Figure sf1, TIF - Figure sf1, TIF
- Figure sf2, TIF - Figure sf2, TIF
- Figure sf3, TIF - Figure sf3, TIF
- Figure sf4, TIF - Figure sf4, TIF
- Figure sf5, TIF - Figure sf5, TIF
- Table st2, DOCX - Table st2, DOCX
- Table st3, DOCX - Table st3, DOCX
- Table st4, DOCX - Table st4, DOCX
