14 research outputs found
Engineered Corynebacterium glutamicum as an endotoxin-free platform strain for lactate-based polyester production
The first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D-lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by ÎČ-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester
<i>E</i>. <i>coli</i> strains used in this study.
<p><i>E</i>. <i>coli</i> strains used in this study.</p
Time course of P(LA-<i>co</i>-3HB) production in the <i>mtgA</i>-deleted <i>E</i>. <i>coli</i>.
<p><i>E</i>. <i>coli</i> BW25113 (wild type) (A) and JW3175 (Î<i>mtgA</i>) (B) harboring pTV118N<i>pctphaC1</i><sub><i>Ps</i></sub>(ST/QK)<i>AB</i> were grown on LB medium containing 20 g/l glucose. Triangle, glucose concentration in the medium. Square, cell dry weight. Gray bar, amount of 3HB unit in the polymer. White bar, amount of LA unit in the polymer. The data represent the average ± standard deviation of three independent trials. The cells were inoculated at time zero.</p
Incorporation of Glycolate Units Promotes Hydrolytic Degradation in Flexible Poly(glycolate-<i>co</i>-3-hydroxybutyrate) Synthesized by Engineered <i>Escherichia coli</i>
Glycolate
(GL)-based polyhydroxyalkanoate (PHA), PÂ[GL-<i>co</i>-3-hydroxybutyrate
(3HB)], was characterized with respect to its
physical properties and hydrolytic degradability. The copolymers were
produced from GL and xylose in recombinant <i>Escherichia coli</i> JW1375 (Î<i>ldhA</i>) expressing an engineered PHA
synthase and monomer supplying enzymes. The GL molar ratio in the
copolymer was regulated in the range of 0 to 16 mol % dependent on
the concentration of GL supplemented in the medium. Unlike PÂ(3HB)
homopolymers which are rigid and opaque, the transparency and elasticity
of PÂ(GL-<i>co</i>-3HB) films could be tuned dependent on
the GL molar ratio. For example, Youngâs modulus of the films
varied in the range of 1620 to 54 MPa. The hydrothermal treatment
of PÂ(GL-<i>co</i>-3HB)Âs resulted in the generation of water-soluble
oligomers, and their concentration was positively correlated with
the GL molar ratio in the polymer, indicating that the GL units in
the polymer chain promoted the hydrolytic degradation of the polymer.
The results of this study demonstrate that the GL molar ratio is a
potent determinant for regulating the elasticity and hydrolytic degradability
of PÂ(GL-<i>co</i>-3HB)
Model of polymer accumulation in fat <i>E</i>. <i>coli</i> cell with <i>mtgA</i> deletion.
<p>MtgA is a dispensable monofunctional glycosyltransferase catalyzing the polymerization of lipid II for the extension of glycan strands but not cross-linking. Penicillin-binding proteins (PBPs), which are bifunctional transpeptidases-transglycosylases and monofunctional transpeptidases, play a central role in the peptidoglycan formation. The <i>mtgA</i> deletion had no obvious effect on cell morphology without polymer accumulation, but generated a fat cell phenotype with polymer production. P(LA-<i>co</i>-3HB) production from glucose in <i>E</i>. <i>coli</i> was achieved by expressing four heterologous enzymes; ÎČ-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), propionyl-CoA transferase (PCT) and lactate-polymerizing engineered polyhydroxyalkanoate synthase [PhaC1(ST/QK)]. D-Lactate dehydrogenase (LDH) is an intrinsic enzyme. The polymer synthesis may elevate turgor pressure, which expands the cell to form the fat-cell and allowed to accumulate the additional amount of polymer.</p
Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters
Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_[Ps]) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_[Ps] that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol%). However, polymer content was often largely decreased (0.2 wt%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol% 3HB and 12 wt%) [Matsumoto et al. (2006) Biomacromolecules, 7:2436-2442]. In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_[Ps] was performed using single and double mutations at sites 130 and 325 as templates, to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_[Ps] mutants were screened based on enhanced P(3HB) content, and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol%) and also polymer content (18 wt%), indicating that mutation scrambling was effective for obtaining the desired mutants
In Vitro Analysis of dâLactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate-<i>co</i>-3-hydroxybutyrate) Syntheses
Engineered d-lactyl-coenzyme
A (LA-CoA)-polymerizing polyhydroxyalkanoate
synthase (PhaC1<sub>Ps</sub>STQK) efficiently produces polyÂ(lactate-<i>co</i>-3-hydroxybutyrate) [PÂ(LA-<i>co</i>-3HB]) copolymer
in recombinant Escherichia coli, while
synthesizing tiny amounts of polyÂ(lactate) (PLA)-like polymers in
recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, <i>in vitro</i> analysis of PhaC1<sub>Ps</sub>STQK was performed
using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA.
PhaC1<sub>Ps</sub>STQK polymerized LA-CoA as a sole substrate. However,
the extension of PLA chains completely stalled at a molecular weight
of âŒ3000, presumably due to the low mobility of the generated
polymer. The copolymerization of these substrates only proceeded with
a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration
in PÂ(LA-<i>co</i>-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that
the mobility of polymerized products and LA-CoA concentration are
dominant factors characterizing PLA and PÂ(LA-<i>co</i>-3HB)
biosynthetic systems
Biosynthesis of High-Molecular-Weight Poly(dâlactate)-Containing Block Copolyesters Using Evolved Sequence-Regulating Polyhydroxyalkanoate Synthase PhaC<sub>AR</sub>
Bacterial polyhydroxyalkanoate (PHA) synthase PhaCAR is a unique enzyme that can synthesize block copolymers.
In this
study, poly(d-lactate) (PDLA)-containing block copolymers
were synthesized using PhaCAR and its mutated variants.
Recombinant Escherichia coli harboring phaCAR and relevant genes were cultivated with
supplementation of the corresponding monomer precursors. Consequently,
PhaCAR synthesized poly(3-hydroxybutyrate)-b-2 mol % PDLA [P(3HB)-b-PDLA]. The incorporation
of the d-lactate (LA) enantiomer was confirmed by chiral
gas chromatography. Previously identified beneficial mutations in
PhaCAR, N149D (ND), and F314H (FH), which increased activity
toward a medium-chain-length substrate 3-hydroxyhexanoyl (3HHx)-CoA,
improved the incorporation of LA units. The combined pairwise mutation
NDFH synergistically increased the LA fraction to 21 mol % in P(3HB)-b-PDLA. Interestingly, a large amount of LA units (51 mol
%) was incorporated by copolymerization with 3HHx units, which yielded
P(3HHx)-b-PDLA. The block copolymerization of 3HHx
and D-LA units was confirmed by NMR analyses and solvent fractionation
of polymers. The PDLA crystal in P(3HHx)-b-PDLA was
detected using differential scanning calorimetry and wide-angle X-ray
diffraction. Its mass-average molecular weight was 8.6 Ă 105. Thus, block copolymerization utilized high-molecular-weight
PDLA as a component of PHAs
Dynamic Changes of Intracellular Monomer Levels Regulate Block Sequence of Polyhydroxyalkanoates in Engineered <i>Escherichia coli</i>
Biological
polymer synthetic systems, which utilize no template
molecules, normally synthesize random copolymers. We report an exception,
a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered <i>Escherichia coli</i>. Using an engineered PHA synthase, block
copolymers polyÂ[(<i>R</i>)-2-hydroxybutyrateÂ(2HB)-<i>b</i>-(<i>R</i>)-3-hydroxybutyrateÂ(3HB)] were produced
in <i>E. coli</i>. The covalent linkage between PÂ(2HB) and
PÂ(3HB) segments was verified with solvent fractionation and microphase
separation. Notably, the block sequence was generated under the simultaneous
consumption of two monomer precursors, indicating the existence of
a rapid monomer switching mechanism during polymerization. Based on <i>in vivo</i> metabolic intermediate analysis and the relevant <i>in vitro</i> enzymatic activities, we propose a model in which
the rapid intracellular 3HB-CoA fluctuation during polymer synthesis
is a major factor in generating block sequences. The dynamic change
of intracellular monomer levels is a novel regulatory principle of
monomer sequences of biopolymers
One-Pot Microbial Production, Mechanical Properties, and Enzymatic Degradation of Isotactic P[(<i>R</i>)â2-hydroxybutyrate] and Its Copolymer with (<i>R</i>)âLactate
PÂ[(<i>R</i>)-2-hydroxybutyrate]
[PÂ((<i>R</i>)-2HB)] is an aliphatic polyester analogous
to polyÂ(lactic acid) (PLA). However, little has been known for its
properties because of a high cost of commercially available chiral
2HB as a starting substance for chemical polymer synthesis. In this
study, PÂ[(<i>R</i>)-2HB] and PÂ[(<i>R</i>)-2HB-<i>co</i>-(<i>R</i>)-lactate] [PÂ((<i>R</i>)-2HB-<i>co</i>-(<i>R</i>)-LA)] with a new monomer combination
were successfully synthesized in recombinant Escherichia
coli LS5218 from less-expensive racemic 2HB using
an <i>R</i>-specific polyester synthase. The cells expressing
an engineered polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 and propionyl-CoA transferase from Megasphaera
elsdenii were grown on LB medium containing 2HB and
glucose in a shake flask and accumulated up to 17 wt % of PÂ[(<i>R</i>)-2HB] with optical purity of >99.1%. In addition, the
same cells cultured in a jar-fermentor produced PÂ(86 mol % 2HB-<i>co</i>-LA) copolymer. Notably, the molecular weights (<i>M</i><sub>w</sub>) of PÂ(2HB) (27000) and PÂ(2HB-<i>co</i>-LA) (39000) were 2- and 3-fold higher than that of PÂ(2HB) previously
synthesized by chemical polycondensation. PÂ(2HB) was processed into
a transparent film by solvent-casting and it had flexible properties
with elongation at break of 173%, which was contrast to the rigid
PLA. Regarding mechanical properties, PÂ(2HB-<i>co</i>-LA)
was tougher but less stretchy than PÂ(2HB). These results demonstrated
that PÂ(2HB) has useful properties and LA units in 2HB-based polymers
can act as a controllable modulator of the material properties. In
addition, PÂ[(<i>R</i>)-2HB] was efficiently degraded by
treatment of Novozym 42044 (lipase) but not Savinase 16L (protease),
indicating that the degrading behavior of the polymer was similar
to that of PÂ[(<i>R</i>)-LA]