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 β-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 (Δ<i>mtgA</i>) (B) harboring pTV118N<i>pctphaC1</i>_<i>Ps</i>(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

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; β-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

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), P­[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 (Δ<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 P­(3HB) homopolymers which are rigid and opaque, the transparency and elasticity of P­(GL-<i>co</i>-3HB) films could be tuned dependent on the GL molar ratio. For example, Young’s modulus of the films varied in the range of 1620 to 54 MPa. The hydrothermal treatment of P­(GL-<i>co</i>-3HB)­s 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 P­(GL-<i>co</i>-3HB)

Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaC_AR for the Biosynthesis of Poly[(<i>R</i>)‑3-hydroxybutyrate-<i>co</i>-6-hydroxyhexanoate]

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCAR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCAR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P­(3HB-co-6HHx) synthesis in Escherichia coli. As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P­(3HB-co-6HHx) decreased as the 6HHx fraction increased

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 d‑Lactyl-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_PsSTQK) efficiently produces poly­(lactate-<i>co</i>-3-hydroxybutyrate) [P­(LA-<i>co</i>-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly­(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, <i>in vitro</i> analysis of PhaC1_PsSTQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1_PsSTQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of ∼3000, 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 P­(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 P­(LA-<i>co</i>-3HB) biosynthetic systems

Biosynthesis of High-Molecular-Weight Poly(d‑lactate)-Containing Block Copolyesters Using Evolved Sequence-Regulating Polyhydroxyalkanoate Synthase PhaC_AR

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 × 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 poly­[(<i>R</i>)-2-hydroxybutyrate­(2HB)-<i>b</i>-(<i>R</i>)-3-hydroxybutyrate­(3HB)] were produced in <i>E. coli</i>. The covalent linkage between P­(2HB) and P­(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