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Green Chemistry
Page 5 of 6
DOI: 10.1039/C5GC01347J
Journal Name
ARTICLE
pTipQ2-ligAB, giving absorbance changes of 0.25 ±0.02 after 60 jostii RHA1 in which the vanillate dehydrogenase gene was
min and a slight yellow colouration.
deleted, in a yield of 96 mg/litre culture media,6 hence
establishing that manipulation of the lignin breakdown
pathways of this organism could be used to generate
bioproducts from lignin breakdown. In this study we have used
Metabolite production
a
gene insertion approach to re-route the aromatic
Cultures (10 ml) of R. jostii pTipQ2-ligAB or R. jostii pTipQ2-
praA were grown for 24 hr at 30 oC in M9 minimal media (6 g/l
Na2HPO4, 3 g/l KH2PO4, 0.5 g/l NaCl, 1.0 g/l NH4Cl, 2 mM
MgSO4, 0.5 mM CaCl2) containing 50 µg/ml chloramphenicol
and either 0.1% (w/v) vanillic acid or 1.0% (w/v) wheat straw
lignocellulose, then induced with 1 µg/ml thiostrepton, and
then grown for a further 7-11 days at 30 oC, supplementing
with 1 µg/ml thiostrepton at 48 hr intervals. Aliquots (1 ml)
were removed, centrifuged (13000 rpm, microcentrifuge, 5
min), and the supernatant extracted into ethyl acetate (1 ml).
The ethyl acetate extract was injected onto a C18 Zorbax
Eclipse plus (Agilent) reverse phase HPLC column on an Agilent
1200 Series system, and analysed by LC-MS using a Bruker
HTC-Ultra ESI mass spectrometer. The HPLC solvents were
water/0.1% trifluoroacetic acid (solvent A) and methanol/0.1%
trifluoroacetic acid (solvent B). The applied gradient was 5% B
for 5 min; 5-15% B over 10 min; 15-25% B for 8 min; and 25-
100% B for 19 min, at a flow rate of 1.0 ml min-1. 2,4-PDCA
and 2,5-PDCA were detected by extracted ion analysis for
fragment m/z 168.0, in positive ion mode, and were compared
with authentic standards for 2,4-PDCA and 2,5-PDCA.
degradation pathways downstream from lignin oxidation.
Since the conversion of vanillin to protocatechuic acid appears
to be a central pathway involved in lignin metabolism,6 we
have re-routed the metabolism of key intermediate
protocatechuic acid to generate aromatic dicarboxylic acids
that could be used to manufacture polyester bioplastics.
Replacement of petroleum-derived terephthalic acid in
polybutyrate adipate terephthalate (PBAT) with 2,4-PDCA or
2,5-PDCA would generate a new bio-based plastic (see Note),
since adipic acid and butane-1,4-diol can both be derived from
renewable sources.9 Such a material could potentially have a
wide range of applications in packaging, durable goods, and
textiles,9 and is likely to be bio-degradable, since pyridine
derivatives are degraded by soil bacteria under aerobic and
anaerobic conditions.16
Utilising the known transformation of meta-ring fission
products with ammonia to generate picolinic acids,11,12 we
have inserted genes for protocatechuate 4,5-dioxygenase or
protocatechuate 2,3-dioxygenase into Rhodococcus jostii
RHA1, and have observed the anticipated 2,4-
pyridinedicarboxylic acid and 2,5-pyridinedicarboxylic acid
bioproducts, using either vanillic acid or wheat straw
lignocellulose as carbon source. The yields of 80-125 mg/litre
culture media are comparable with the yield of vanillin isolated
by gene deletion,6 suggesting that this is the amount of flux
that passes through the vanillic acid catabolic pathway from
lignin in R. jostii. Assuming that 20% of the wheat straw
lignocellulose is lignin, then the observed yields correspond to
approximately 5% of the lignin present in the fermentation. At
present the pathways by which each of the lignin structural
components are metabolised are not fully understood,17 hence
the vanillin catabolic pathway shown in Figure 1 is likely to be
only one of a number of pathways for metabolism of different
Bioreactor fermentation
Cultures of R. jostii pTipQ2-ligAB or R. jostii pTipQ2-praA were
o
grown at 30 C in 2.5 litres M9 minimal media (6 g/l Na2HPO4,
3 g/l KH2PO4, 0.5 g/l NaCl, 1.0 g/l NH4Cl, 2 mM MgSO4, 0.5 mM
CaCl2) containing 50 µg/ml chloramphenicol and either 0.1%
(w/v) vanillic acid or 1.0% (w/v) milled wheat straw
lignocellulose (1 mm mesh) or 0.5% (w/v) Kraft lignin in an
Electrolab FerMac 3010 bioreactor. Lignin/lignocellulose
feedstocks were added as solids prior to sterilisation.
Fermentations were induced by addition of
1 µg/ml
thiostrepton after 24 hr, and then grown for a further 3-8 days
at 30 oC, supplementing with 1 µg/ml thiostrepton at 48 hr
intervals. After fermentation, cultures were harvested by
centrifugation (6,000 g, 10 min), and the supernatant was
applied to an Amberlite IRA900 anion exchange column (100
ml volume), washed with water (200 ml), and then eluted with
0.5 M HCl (800 ml). 15 x 50 ml fractions were collected, and
analysed by UV-vis spectroscopy for the presence of 2,4-
lignin components (G, H,
S units) and sub-structures.
Therefore, improving our understanding of the microbial lignin
degradation pathways in the future may allow further
enhancement of this yield. The peak production of 2,5-PDCA is
somewhat earlier than that of 2,4-PDCA (see Figure 4),
perhaps reflecting a higher specific activity of the PraA
enzyme, compared to the LigAB enzyme.
= 3.1 x 103 M-1cm-1)
= 6.3 x 103 M-
This study provides
a novel biocatalytic route to
pyridinedicarboxylic acid (λmax 273 nm,
ε
pyridinedicarboxylic acids from a renewable feedstock lignin,
and highlights the opportunity for bioconversion of lignin into
aromatic products using biotechnology. Linger et al have
recently shown that Pseudomonas putida can also be used to
generate polyhydroxyalkanoate biopolyesters from lignin
breakdown, via metabolic conversion of lignin breakdown.18
Pathway engineering has also been used in P. putida to
generate ring cleavage product cis,cis-muconic acid, which can
be converted via chemocatalysis to adipic acid.19 Since lignin is
generated as a by-product of cellulosic bioethanol production
or 2,5-pyridinedicarboxylic acid (λmax 271 nm,
ε
1cm-1). Fractions containing the desired products were pooled,
and analysed by C18 reverse phase HPLC (gradient described
above).
Conclusions
Previously we have shown that is possible to accumulate
bioproduct vanillin in a gene deletion mutant of Rhodococcus
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J. Name., 2013, 00, 1-3 | 5
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