Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts

Article abstract of DOI:10.1039/c9gc03722e

Here, we demonstrate bioconversion of methane to muconic acid, a dicarboxylic acid that can be upgraded to an array of platform chemicals, by three gammaproteobacterial methanotrophs. All engineered methanotrophs expressing a heterologous dihydroxyshikimate dehydratase, protocatechuate decarboxylase, and catechol dioxygenase produced muconic acid from methane, with the highest titer (12.4 mg MA per L), yield (2.8 mg MA per g CH₄), and specific productivity (1.2 mg MA per g dcw, 48 hr) synthesized by Methylotuvimicrobium buryatense, Methylococcus capsulatus, and Methylotuvimicrobium alcaliphilium, respectively. Methylotuvimicrobium alcaliphilum genome-scale model-guided strain engineering predicted that disruption of the pyruvate dehydrogenase or shikimate dehydrogenase would significantly enhance flux to the heterologous muconic acid pathway in this organism. However, knock-out of these targets caused a growth defect, and coupled with similar muconic acid titers (～1 mg L^-1), resulted in minimal flux enhancement to muconic acid in these genetically-modified strains. The shikimate dehydrogenase mutant's ability to grow without aromatic amino acid supplementation revealed that M. alcaliphilum likely encodes an unidentified enzyme or pathway with shikimate biosynthetic capacity, which prevents maximal flux through the synthetic muconic acid pathway. This study expands the suite of products that can be generated from methane using methanotrophic biocatalysts, lays the foundation for green production of muconic acid-derived polymers from methane, and highlights the need for further analysis of methanotroph biosynthetic potential to guide refinement of metabolic models and strain engineering.

Full text of DOI:10.1039/c9gc03722e

Green Chemistry
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PAPER
Muconic acid production from methane using
rationally-engineered methanotrophic
biocatalysts†
Cite this: Green Chem., 2019, 21,
731
6
a
b,c
b
Calvin A. Henard, *‡ Ilya R. Akberdin,
Michael T. Guarnieri
§
Marina G. Kalyuzhnaya
and
a
*
Here, we demonstrate bioconversion of methane to muconic acid, a dicarboxylic acid that can be
upgraded to an array of platform chemicals, by three gammaproteobacterial methanotrophs. All engin-
eered methanotrophs expressing a heterologous dihydroxyshikimate dehydratase, protocatechuate de-
carboxylase, and catechol dioxygenase produced muconic acid from methane, with the highest titer
4
(12.4 mg MA per L), yield (2.8 mg MA per g CH ), and specific productivity (1.2 mg MA per g dcw, 48 hr)
synthesized by Methylotuvimicrobium buryatense, Methylococcus capsulatus, and Methylotuvimicrobium
alcaliphilium, respectively. Methylotuvimicrobium alcaliphilum genome-scale model-guided strain engin-
eering predicted that disruption of the pyruvate dehydrogenase or shikimate dehydrogenase would sig-
nificantly enhance flux to the heterologous muconic acid pathway in this organism. However, knock-out
of these targets caused a growth defect, and coupled with similar muconic acid titers (∼1 mg L− ), resulted
in minimal flux enhancement to muconic acid in these genetically-modified strains. The shikimate dehydro-
genase mutant’s ability to grow without aromatic amino acid supplementation revealed that M. alcaliphilum
likely encodes an unidentified enzyme or pathway with shikimate biosynthetic capacity, which prevents
maximal flux through the synthetic muconic acid pathway. This study expands the suite of products that can
be generated from methane using methanotrophic biocatalysts, lays the foundation for green production of
muconic acid-derived polymers from methane, and highlights the need for further analysis of methanotroph
biosynthetic potential to guide refinement of metabolic models and strain engineering.
1
Received 29th October 2019,
Accepted 8th November 2019
DOI: 10.1039/c9gc03722e
rsc.li/greenchem
upgrading strategies including lactonization, diolization, and
ketonization. As such, adipic acid can be readily converted
Introduction
1
Muconic acid (MA) is a dicarboxylic acid that can be upgraded to other large-market, high-value molecules, such as plastici-
to produce high-value commodity chemicals, including ter- zers, lubricants, engineering resins, and polyurethanes.
epthalic acid, which in turn serves as a precursor for the pro- Conventional adipic acid production has been estimated to
1
4
duction of polyester PET. Additionally, MA is readily con- account for nearly 10% of global N
2
O emissions. Though
verted to adipic acid, a “top 50” bulk chemical, via hydrogen- recent technologies have been deployed to help abate these
2
,3
5–8
ation, with high yield and specificity. Adipic acid represents emissions,
conventional routes to adipic acid production
a multi-billion-dollar global market, with nylon-6,6 accounting almost exclusively employ non-renewable, petroleum feed-
3
for >85% of global adipic acid utilization. The dicarboxylic stocks, specifically ketone/alcohol (KA) oil generated via oxi-
4
functionality of adipic acid also affords a wide variety of dation of cyclohexanone and cyclohexanol with nitric acid.
Thus, there is an increasing need to develop sustainable, green
routes to produce MA and derivatives thereof.
a
National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO,
Recently, a number of alternative bio-based MA production
USA. E-mail: Michael.Guarnieri@nrel.gov, Calvin.Henard@unt.edu
b
routes have been proposed, utilizing lignocellulosic aromatics
and sugars as substrates for wild-type and genetically modified
Biology Department, San Diego State University, San Diego, CA, USA
c
Federal Research Center Institute of Cytology and Genetics SB RAS, Novosibirsk,
Russia
1
–3,8
microbial biocatalysts.
MA biosynthesis naturally occurs
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9gc03722e
Current affiliation: Department of Biological Sciences and BioDiscovery
Institute, University of North Texas, Denton, TX, USA.
Current affiliation: BIOSOFT.RU, LLC, Novosibirsk, Russian Federation.
during the catabolism of aromatic compounds in a subset
of microorganisms, primarily saprophilic bacteria and fungi,
which have ready access to plant-derived aromatics.
Production of MA via heterologous pathway incorporation has
‡
§
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been demonstrated in heterotrophic bacteria and yeast by Methylotuvimicrobium buryatense 5G, and Methylococcus capsu-
exploiting naturally occurring intermediary metabolic path- latus Bath, industrially-promising gammaproteobacterial
ways involved in the detoxification or catabolism of aromatic methanotrophs with established metabolic engineering tools,
1
–3,9–11
26,28–30
compounds such as toluene and benzoate,
through cate- including genome-scale metabolic models.
Quantitative
chol and protocatechuate intermediates. Alternatively, MA can reverse-transcription PCR (qRT-PCR) analysis of the engin-
be synthesized in a three-step process from the 3-dehydroshiki- eered strains harboring the biosynthetic pathway under the
3
22
mate (DHS) intermediate produced in the shikimate pathway,
control of the inducible tetracycline promoter/operator indi-
which is essential for the synthesis of aromatic amino acids. cated that this promoter is functional in all three methano-
Lastly, MA can also be generated by converting the anthrani- trophic strains, with significant expression of AsbF, AroY, and
late intermediate of tryptophan biosynthesis into catechol by CatA-encoding genes detected after induction (Fig. 1B).
1
2
the anthranilate 1,2-dioxygenase. The above strategies have
been successfully deployed in an array of model heterotrophic strains in 0.5 L continuous-stirred tank reactors (CSTRs) with
microbes, however, to date, there has been no demonstration constant gas supply (20% CH v/v in air, 1 vvm; 2% CO was
of MA bioproduction from C1 carbon substrates. also supplied to M. capsulatus Bath). All strains excreted MA
We evaluated growth and MA production by the pMUC
4
2
Methanotrophic bacteria are promising hosts for carbon during active growth, coincident with carbon flux through the
sequestration and conversion of C1 carbon streams to shikimate pathway to provide proteinogenic aromatic amino
diverse target molecules, including fuel and chemical acids (Fig. 2). We did not detect the MA precursors proto-
1
3,14
intermediates.
regulators capable of utilizing CH
source and possess core metabolic machinery enabling meta- ing a similar heterologous MA biosynthetic pathway.
bolic engineering strategies paralleling those found in conven- M. alcaliphilum and M. capsulatus reached cell densities of
These bacteria are key global biochemical catechuic acid and catechol in the culture medium, contrary
4
as a carbon and energy to prior studies using yeast or bacterial biocatalysts express-
2
,3,31
1
5,16
−1
tional heterotrophic hosts.
These characteristics have led OD600 = 6–8 after 48 h, excreting MA at titers of 0.75 mg L
to intensified industrial interest for deployment of methano- and 0.97 mg L− MA, respectively (Fig. 2A and B, Table 1). We
trophs as production biocatalysts for bioconversion of gaseous previously increased the growth potential of M. buryatense in
waste streams, including anaerobic digestion-derived CSTR cultivation via media optimization, achieving maximum
1
1
7,18
21,22
biogas,
which is primarily comprised of CH4 and CO2. OD600 ∼ 50 culture densities.
Indeed, recent efforts have demonstrated the potential utility vation conditions, M. buryatense excreted 12.4 mg L MA into
Under these optimized culti-
−
1
of methanotrophic biocatalysts for production of platform the culture medium following 48 h of cultivation (Fig. 2C and
1
9–25
chemicals from diverse waste streams.
the first heterologous production of MA from CH
methanotrophic bacteria as a production chassis. Further, we methanotrophs
Here, we present Table 1), which is comparable with other proof-of-concept
−
1
4
using product titers (10–100 mg L ) produced by engineered
1
9,22–24,32
Although we were able to increase the
leveraged the M. alcaliphilum 20Z genome-scale model to MA titer by high-density cell cultivation, yields (0.65–2.7
guide rational metabolic engineering to increase carbon flux mg MA per g CH ) and specific productivities (0.68–1.2 mg MA
2
6
4
to the MA pathway, which revealed a yet unidentified shiki- per g DCW) were comparable between the engineered methano-
mate biosynthetic enzyme or pathway in this organism. These trophs, with M. capsulatus exhibiting the highest yield.
results underscore that further elucidation of methanotroph M. alcaliphilum exhibited the highest specific productivity
central metabolism is required to accurately model and predict (Table 1), which served as the basis for down-selection and
methanotrophic biosynthetic capacity.
further evaluation.
We leveraged our previously developed M. alcaliphilum
2
6
genome-scale model (GSM) to identify genetic targets for
increased carbon flux to the MA pathway. Based on the GSM,
the maximum theoretical specific productivity of MA pro-
Results and discussion
Methanotrophs are not known to naturally produce MA. duction is 0.2 g MA per g DCW. Disruption of the shikimate
However, these bacteria encode genes requisite for de novo aro- dehydrogenase (encoded by MALCv4_3465) and the pyruvate
matic amino acid biosynthesis and can be cultivated on dehydrogenase (encoded by MALCv4_1357-9) were predicted
minimal media lacking amino acid supplementation, indicat- by the model to significantly increase flux through the heter-
ing that the upstream metabolic machinery for shikimate ologous MA pathway (Table 2). This agrees with other studies
pathway-derived MA production is present. Leveraging this where disruption of the shikimate dehydrogenase is required
metabolic machinery, we incorporated a MA biosynthetic in heterotrophic hosts to achieve heterologous MA biosyn-
pathway encoding the Bacillus thuringiensis AsbF dihydroxy- thesis from aromatic amino acid precursors by inhibiting flux
shikimic acid dehydratase, Enterobacter cloacae AroY protocatech- through the shikimate pathway and increasing DHS substrate
1
,3,31
uate decarboxylase, and an Acinetobacter sp. CatAP76A catechol availability,
while disruption of pyruvate dehydrogenase
2
7
dioxygenase variant in order to shunt the aromatic amino would be predicted to increase availability of phosphoenolpyr-
acid precursor dihydroxyshikimic acid to MA in methano- uvate and erythrose-4-phosphate shikimate pathway precur-
trophic bacteria (Fig. 1A). The resultant vector, pMUC, was sors. We introduced the heterologous MA biosynthetic
R
introduced into Methylotuvimicrobium alcaliphilum 20Z , pathway into our previously generated M. alcaliphilum pyruvate
6732 | Green Chem., 2019, 21, 6731–6737
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Fig. 1 Metabolic engineering of methanotrophic bacteria for biogas conversion to muconic acid. (A) Schematic of the three-enzyme heterologous
biochemical pathway used to convert the dihydroxyshikimate (DHS) shikimate pathway precursor to muconic acid in methanotrophs. (B) qRT-PCR
R
analysis of muconic acid biosynthetic pathway genes in M. alcaliphilum 20Z , M. capsulatus Bath, and M. buryatense 5G engineered strains.
Fig. 2 Muconic acid production by methanotrophic bacteria in bioreactors with continuous biogas supply. (A) Methylotuviomicrobium alcaliphilum,
(
B) Methylococcus capsulatus, and (C) Methylotuvimicrobium buryatense growth (black circles) and muconic acid titer (MA, red circles) were
measured in continuously stirred tank bioreactors supplied with 20% CH in air at 1 volume of gas/volume of medium/minute. M. capsulatus reactors
were also supplemented with 2% CO
4
2
.
2
0
dehydrogenase knock-out strain and evaluated MA pro- the pMUC strain; thus a 1.5-fold increase in specific pro-
duction in CSTRs (Table 2). The pMUC Δpdh strain produced ductivity was observed, albeit much lower than the 23.2-fold
similar MA titers but displayed a growth defect compared to flux increase predicted by the GSM (Table 2). The discrepancy
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Table 1 Muconate titer, yield, and specific productivity in stirred bioreactors
Titer
CH
(g L /48 h)
4
consumed
Maximum yield
(mg MA per g CH
Specific productivity
(mg MA per g DCW, 48 h)
−
1
Strain
(mg MA per L/48 h)
4
, 24–48 h)
M. alcaliphilum pMUC
M. capsulatus pMUC
M. buryatense pMUC
0.75 ± 0.08
0.97 ± 0.21
12.4 ± 2.26
1.35 ± 0.33
0.74 ± 0.07
12.5 ± 0.14
0.65 ± 0.12
2.8 ± 0.04
1.2 ± 0.22
1.2 ± 0.01
0.68 ± 0.16
1.1 ± 0.20
The data represent the mean ± standard deviation of two biological replicate bioreactors.
R
(Table 2). The GSM predicted that removal of the shikimate
Table 2 M. alcaliphilum 20Z genome-scale model genetic alteration
flux predictions and engineered strain bioreactor relative productivity dehydrogenase would increase flux through the MA pathway
metrics for muconic acid (MA), lactic acid (LA), and biomass
by 29.4-fold and result in aromatic amino acid auxotrophy
with no carbon flux to biomass. Although deletion of
Predicted Observed
Observed
MALCv4_3465 decreased M. alcaliphilum growth, the ΔaroE
strain’s ability to grow in the absence of shikimate or aromatic
amino acid supplementation provides additional support for
the presence of an alternative shikimate biosynthetic enzyme
encoded by M. alcaliphilum. To confirm accurate gene annota-
tion of MALCv4_3465 as a shikimate dehydrogenase, we tested
the ability of MALCv4_3465 to complement an E. coli shiki-
mate dehydrogenase KO strain that does not grow on
M9 minimal medium without aromatic amino acid sup-
MA flux max MA flux max LA flux Biomass
Strain
ratio
1
ratio
ratio
(g DCW, 96 h)
R
2
2
2
0Z pMUC
0.91 ± 0.13 0.84 ± 0.22 0.69 ± 0.008
1.5 ± 0.07
1.0 ± 0.11
R
0Z pMUC Δpdh 23.2
0Z pMUC ΔaroE 29.4
4.0 ± 0.06
1.4 ± 0.53
0.35 ± 0.02
0.31 ± 0.04
R
The data represent the mean ± standard deviation of two biological
replicate bioreactors.
3
3
between predicted and actual flux values could be in part due plementation. Successful complementation of the E. coli
to the high lactic acid flux observed in this strain (Table 2), ΔaroE non growth phenotype in M9 medium was achieved via
presumably because of increased pyruvate availability and con- plasmid-based expression of the E. coli aroE gene or
version by the native lactate dehydrogenase.
MALCv4_3465, supporting that MALCv4_3465 encodes a func-
We hypothesized that knock-out of MALCv4_3465 would tional shikimate dehydrogenase (Fig. 3B), and provides an
result in an aromatic amino acid auxotrophic strain similar to additional line of evidence that M. alcaliphilum encodes an
that observed in Escherichia coli. We successfully removed the alternative shikimate biosynthetic enzyme. However, alterna-
3
4
locus via marker exchange mutagenesis (Fig. 3A). However, a tive AroE or YdiB homologs were not identified in the
R
MALCv4_3465 mutant (ΔaroE) was successfully isolated in the M. alcaliphilum 20Z genome via homology query. Based on
absence of shikimate or aromatic amino acid supplemen- previous metabolic engineering efforts to produce MA from
tation, suggesting an alternate shikimate biosynthetic pathway non-aromatic substrates in heterotrophic hosts, increasing flux
is present in M. alcaliphilum. To assess the potential for to the DHS substrate via gene disruption, pathway over-
increased DHS substrate availability in the M. alcaliphilum expression, and/or generation of feedback-insensitive shiki-
ΔaroE strain, we introduced pMUC and analyzed MA pro- mate pathway enzymes are requisite for efficient MA pro-
3
5
duction is CSTRs. Similar to the pMUC Δpdh strain, the pMUC duction from non-aromatic precursors. Thus, a complete
ΔaroE strain displayed a slight growth defect but a similar MA understanding of shikimate biosynthetic pathways is needed
specific productivity relative to the isogenic pMUC strain to more accurately predict competing metabolic pathways and
R
Fig. 3 M. alcaliphilum 20Z MALCv4_3465 rescues the auxotrophic phenotype of an E. coli shikimate dehydrogenase knockout. (A) Confirmation of
the M. alcaliphilum MALCv4_3465 knockout strain via PCR analysis (B) Auxotrophic complementation of an E. coli shikimate dehydrogenase (aroE)
knockout strain in M9 minimal medium via plasmid-based expression of E. coli AroE (pEcaroE) or M. alcaliphilum AroE (MALCv4_3465, p20ZaroE).
6734 | Green Chem., 2019, 21, 6731–6737
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guide future rational metabolic engineering pursuits to ations will need to be addressed to achieve economically-
increase MA production metrics in M. alcaliphilum. viable production. A primary biological driver is the identifi-
Notably, the highest yield observed in this study (2.7 mg cation and disruption of alternative shikimate biosynthetic
MA per g CH ) is comparable to initial MA yields produced by genes, which will enable increased flux to MA precursors.
heterotrophic biocatalysts expressing a similar heterologous Genome-wide mutagenic approaches offer a promising route
4
3
,31
MA biosynthetic pathway.
MA yields from glucose in these to identify auxotrophic phenotypes, implicating genes with
heterotrophic hosts have been significantly improved (12.9 mg redundant functionality. Lastly, conventional gas fermentation
MA per g glucose) via additional genetic modifications for challenges remain a hurdle; gas mass transfer limitations, in
increased flux to the MA pathway, including removal of aro- particular, will need to be addressed in order to achieve econ-
matic amino acid negative feedback regulation and disruption/ omically-viable production rates.
modification of the shikimate dehydrogenase that was
3
,35–38
attempted in M. alcaliphilum here.
These results indicate
a substantial yield enhancement can be achieved if an alterna- Materials and methods
tive shikimate biosynthetic route can be identified and dis-
rupted. Future strain engineering efforts for efficient MA pro-
Bacterial strains
Methylotuvimicrobium (previously Methylomicrobium) alcaliphi-
4
duction from CH will thus entail identification and disrup-
R 26
28
lum 20Z ,
Methylotuvimicrobium buryatense 5GB1,
and
tion of the unidentified shikimate biosynthetic enzyme, as
well as homologous overexpression of transketolase and/or
phosphoketolase for increased erythrose-4-phosphate substrate
availability, and expression of either a native or heterologous
feedback insensitive 2-dehydro-3-deoxyphosphoheptonate
Methylococcus capsulatus Bath (Table S1†) were cultivated in
either nitrate mineral salts (NMS) medium (Bath) or NMS
medium supplemented with 1.5% NaCl and carbonate buffer
4
1–43
as previously described.
Strains were routinely maintained
on solid medium in stainless-steel gas chambers (Schuett-
biotec GmbH) containing 20% (v/v) methane in air.
(DAHP) aldolase variant to increase flux through the shikimate
pathway.
Muconic acid biosynthetic pathway construction
Plasmids and primers used in this study are presented in
Table S2.† The muconic acid biosynthetic pathway was con-
Conclusions
CH
increased warming potential relative to CO . Nearly 60% of droshikimate dehydratase from Bacillus thuringensis, aroY-
4
is the second most abundant greenhouse gas (GHG), with structed by ligating codon-optimized asbF-encoding dehy-
2
CH
4
emissions are derived from anthropogenic sources, encoding protocatechuate decarboxylase from Enterobacter
including shale gas fracking, landfill gases, and industrial cloacae, and the catA-encoding catechol dioxygenase P76A
agriculture operations. Further, CH is the primary component variant from Acinetobacter baylyi (codon-optimized gene
4
of natural gas and anaerobic digestion-derived biogas, and sequences are in Table S3†) into the pCAH01 inducible
2
2
recent reports have indicated CH
low-cost substrate that is well-suited for biomanufacturing.
4
represents a high-volume, expression vector
to generate pMUC using 2× Gibson
1
3,39
Assembly Mix from New England Biolabs (Ipswich, MA), fol-
Importantly, the conversion of natural gas and biogas can sim- lowing the manufacturers protocol. The pMUC vector sequence
ultaneously offset petroleum usage while mitigated GHGs with was confirmed by sequence analysis (Genewiz, South
4
0
massive economic and environmental impacts. In contrast to Plainfield, NJ). This vector was transformed into the methano-
heterotrophic microbial conversion strategies employing trophic strains via biparental mating using E. coli S17 as pre-
2
8,42
biomass-derived substrates, the utilization of CH
version substrate does not compete with food production. medium containing 100 µg mL− of kanamycin were con-
Thus, strategies that convert CH
to high-value intermediates firmed by PCR and sequence analysis.
4
as a biocon- viously described.
Positive transformants selected on solid
1
4
present an opportunity to mitigate and sequester GHG emis-
sions, while concurrently valorizing CH . Additionally, CH
Quantitative reverse transcription PCR
4
4
biocatalysis offers a means to concurrently liquefy and Total RNA was isolated from cultures grown for 48 h on solid
−
1
upgrade CH , enabling its utilization in conventional transpor- medium with and without pMUC induction with 0.5 μg mL
4
tation and industrial manufacturing infrastructure. To this anhydrotetracycline inducer using the Monarch Total RNA
end, recent efforts have successfully generated methanotrophic miniprep kit (New England Biolabs) followed by removal of
biocatalysts with diverse production capacity for fuel and contaminating DNA by treatment with Turbo DNase (Life
1
7,19–25
chemical intermediates.
The work presented here provides proof-of-concept for pro- total RNA using the SuperScript VILO cDNA synthesis kit
duction of MA from CH . Methanotrophic production of MA (ThermoFisher) following the manufacturer’s protocol. Real-
Technologies, Carlsbad, CA. cDNA was synthesized from 1 μg
4
from CH presents a novel, green pathway to biosynthesize a time PCR was performed by using SYBR IQ master mix and a
4
key platform intermediate capable of displacing petroleum in QuantStudio Real-Time PCR System (ThermoFisher). The
the production of high-volume commodity chemicals and bio- primers used for the qRT-PCR are listed in Table S2.† The
polymers. However, a series of biological and process limit- cycle threshold (Ct) value for each gene was determined and
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normalized to the rpoD gene encoding the RNA polymerase tance marker. Sucrose-resistant, kanamycin-sensitive colonies
housekeeping sigma factor. Relative expression between were PCR-genotyped for removal of the MALCv4_3465 locus.
induced and uninduced control samples was calculated by
using the ΔΔCt method.
E. coli shikimate dehydrogenase mutant complementation
The E. coli shikimate dehydrogenase aroE and M. alcaliphilum
Methane fermentations
R
20Z MALCv4_3465 genes with native ribosomal binding sites
1
00 mL seed cultures were inoculated from plate biomass and
grown in 150 mL bubble columns with a continuous gas flow
20% CH , in air, 1 volume of gas per volume of liquid per
were amplified from the corresponding genomic DNA using
primers appending XbaI and HindIII restriction sites to the 5′
and 3′ ends (Table S2†), respectively. Genes were directionally
cloned into the pBluescript II KS+ vector. Expression con-
structs were sequence verified and transformed into the
JW3242 ΔaroE721::kan parent strain via electroporation.
Positive transformants selected on LB medium containing
kanamycin and ampicillin were grown in M9 minimal
medium supplemented with 0.5 mM IPTG for shikimate de-
hydrogenase induction. Bacterial growth of the complement-
ing strains in M9 medium was compared to wild-type E. coli
BW25113 and JW3242 ΔaroE721::kan parent strains, the latter
of which does not grow on M9 medium.
(
4
minute (vvm)). M. capsulatus Bath cultures were also supplied
with 2% CO in the gas mixture. Gas fermentations were per-
2
formed in a 0.5 L Biostat-Q plus bioreactor (Sartorius,
Gottingen, Germany) with a culture volume of 300 mL. The
M. buryatense bioreactor medium was supplemented with 8×
3
KNO (80 mM), 2× phosphate buffer (4.6 mM), and 4× trace
element solution to support high cell growth, as previously
2
2,44
described.
.1–0.5. Mixing was achieved by using a bottom marine impel-
ler at 500 rpm. A continuous flow rate of 1 vvm 20% CH in air
plus 2% CO for M. capsulatus Bath) was maintained for the
Seed cultures were diluted to a final OD600
=
0
4
(
2
duration of cultivation. At indicated intervals, growth was
measured spectrophotometrically at 600 nm and a 1 mL
sample was taken for HPLC analysis. The culture supernatant
was filtered using a 0.2 μm syringe filter and then a 0.1 mL
injection was separated using a model 1260 HPLC (Agilent,
Conflicts of interest
There are no conflicts to declare.
Santa Clara, CA) and a cation H HPx-87H column (Bio-Rad). A
.6 mL min− flow rate at 55 °C with 0.01 N sulfuric acid as the
1
0
Acknowledgements
mobile phase was used. Refractive index and diode array detec-
tors were used for compound detection. Muconic acid concen- We would like to thank Holly Rohrer of the National
trations were calculated by regression analysis compared to Renewable Energy Laboratory for technical assistance.
known standards with the minimum detection limit of 0.2 mg Funding provided by the U.S. Department of Energy Office of
−
1
L
muconic acid. The percent CH4 in the off-gas was Energy Efficiency and Renewable Energy Bioenergy
measured every 10 min for the duration of bioreactor fermen- Technologies Office DE-FOA-0001085. This work was authored
tations via mass spectrometry. Percent CH consumption was in part by Alliance for Sustainable Energy, LLC, the manager
converted to weight based on CH density (0.000656 g mL
4
−
1
)
and operator of the National Renewable Energy Laboratory for
per min). Dry Cell Weight (DCW) the U.S. Department of Energy under Contract No. DE-AC36-
4
and the flow rate (60 mL CH
4
was either measured directly after freeze-drying or estimated 08GO28308. The views expressed in the article do not necess-
from the final OD of the cell culture using the following arily represent the views of the DOE or the U.S. Government.
−
1 26
equation: DCW = OD × (0.35 ± 0.04) g L
.
Muconic acid The U.S. Government retains and the publisher, by accepting
yields were calculated using consumed substrate data and rep- the article for publication, acknowledges that the U.S.
resented as mg muconic acid produced per g CH consumed.
Government retains a non-exclusive, paid-up, irrevocable, world-
wide license to publish or reproduce the published form of this
work, or allow others to do so, for U.S. Government purposes.
4
M. alcaliphilum shikimate dehydrogenase mutant
construction
Plasmids and primers used for amplification of the shikimate
dehydrogenase flanking regions and the pK18mobsacB vector
for construction of the shikimate dehydrogenase (aroE) knock-
out are shown in Tables S1 and S2.† 1000 bp genomic frag-
ments flanking the shikimate dehydrogenase (MALCv4_3465)
gene were amplified by PCR and cloned into the
pK18mobsacB plasmid via Gibson assembly. The resulting
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