Utilization of duckweed-derived biomass for polyhydroxybutyrate production in Escherichia coli via dual enzymatic saccharification using amylase and cellulase complex.

Duckweed is a rapid-growing plant with a high starch and low lignin content. The duckweed was saccharified via dual enzymatic treatment using amylase and cellulase complex. The duckweed-derived glucose was utilized for polyhydroxybutyrate (PHB) production in engineered Escherichia coli. In addition, the low concentration supplementation of the duckweed extract promoted cell growth compared to the analytical grade glucose.

In Vitro Analysis of d-Lactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate- co-3-hydroxybutyrate) Syntheses.

Engineered d-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaC1PsSTQK) efficiently produces poly(lactate- co-3-hydroxybutyrate) [P(LA- co-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, in vitro analysis of PhaC1PsSTQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1PsSTQK 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- co-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- co-3HB) biosynthetic systems.

Dynamic Changes of Intracellular Monomer Levels Regulate Block Sequence of Polyhydroxyalkanoates in Engineered Escherichia coli.

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 Escherichia coli. Using an engineered PHA synthase, block copolymers poly[(R)-2-hydroxybutyrate(2HB)-b-(R)-3-hydroxybutyrate(3HB)] were produced in E. coli. 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 in vivo metabolic intermediate analysis and the relevant in vitro 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.

Microbial production of poly(lactate-co-3-hydroxybutyrate) from hybrid Miscanthus-derived sugars.

P[(R)-lactate-co-(R)-3-hydroxybutyrate] [P(LA-co-3HB)] was produced in engineered Escherichia coli using lignocellulose-derived hydrolysates from Miscanthus × giganteus (hybrid Miscanthus) and rice straw. Hybrid Miscanthus-derived hydrolysate exhibited no negative effect on polymer production, LA fraction, and molecular weight of the polymer, whereas rice straw-derived hydrolysate reduced LA fraction. These results revealed that P(LA-co-3HB) was successfully produced from hybrid Miscanthus-derived sugars.

Advances and needs for endotoxin-free production strains.

The choice of an appropriate microbial host cell and suitable production conditions is crucial for the downstream processing of pharmaceutical- and food-grade products. Although Escherichia coli serves as a highly valuable leading platform for the production of value-added products, like most Gram-negative bacteria, this bacterium contains a potent immunostimulatory lipopolysaccharide (LPS), referred to as an endotoxin. In contrast, Gram-positive bacteria, notably Bacillus, lactic acid bacteria (LAB), Corynebacterium, and yeasts have been extensively used as generally recognized as safe (GRAS) endotoxin-free platforms for the production of a variety of products. This review summarizes the currently available knowledge on the utilization of these representative Gram-positive bacteria for the production of eco- and bio-friendly products, particularly natural polyesters, polyhydroxyalkanoates, bacteriocins, and membrane proteins. The successful case studies presented here serve to inspire the use of these microorganisms as a main-player or by-player depending on their individual properties for the industrial production of these desirable targets.

Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1.

Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVs) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVs for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVs generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVs cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVs but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVs exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.

Structures of AzrA and of AzrC complexed with substrate or inhibitor: insight into substrate specificity and catalytic mechanism.

Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyes via azoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P)H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P)H for direct hydride transfer to FMN. The structures of the AzrC-AR88 and AzrC-OI complexes showed two manners of binding for substrates possessing a hydroxy group at the ortho or the para position of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of ∼3.5 Što N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis.

Enhanced production of poly(lactate-co-3-hydroxybutyrate) from xylose in engineered Escherichia coli overexpressing a galactitol transporter.

Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (ΔpflA, Δpta, ΔackA, ΔpoxB, Δdld, and a dual mutant; ΔpflA + Δdld) and their parent strain, BW25113, were grown on 20 g l(-1) xylose for P(LA-co-3HB) production. The single deletions of ΔpflA, Δpta, and Δdld increased the LA fraction (58-66 mol%) compared to BW25113 (56 mol%). In particular, the ΔpflA + Δdld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in ΔpflA, Δpta, and Δdld mutants, and BW25113. The ΔpflA + gatC strain achieved a productivity of 8.3 g l(-1), which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l(-1) xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l(-1). On the other hand, the ΔpflA + Δdld strain grown on 30 g l(-1) xylose synthesized 6.4 g l(-1) P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000-114,000.

Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate) using a lactate-overproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme.

Xylose, which is a major constituent of lignocellulosic biomass, was utilized for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], having transparent and flexible properties. The recombinant Escherichia coli JW0885 (pflA(-)) expressing LA-polymerizing enzyme (LPE) and monomer supplying enzymes grown on xylose produced a copolymer having a higher LA fraction (34mol%) than that grown on glucose (26mol%). This benefit of xylose was further enhanced by combining it with an evolved LPE (ST/FS/QK), achieving a copolymer production having 60mol% LA from xylose, while glucose gave a 47mol% LA under the same condition. The overall carbon yields from the sugars to the polymer were similar for xylose and glucose, but the ratio of the LA and 3HB units in the copolymer was different. Notably, the P(LA-co-3HB) yield from xylose (7.3gl(-1)) was remarkably higher than that of P(3HB) (4.1gl(-1)), indicating P(LA-co-3HB) as a potent target for xylose utilization.

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase.

(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by ß-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L(-1) (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L(-1), with a productivity of 0.22 g L(-1) h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of (13)C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.

Flow cytometric analysis of the contributing factors for antimicrobial activity enhancement of cell-penetrating type peptides: case study on engineered apidaecins.

Contributing factors for the antimicrobial activity enhancement of N-terminally engineered mutants of cell-penetrating apidaecins were analyzed based on their cell-penetration efficiency. The flow cytometric analysis of the engineered apidaecins labeled with carboxyfluorescein (FAM) revealed their enhanced cell-penetrating efficiencies into Escherichia coli that should be one of key factors causing the enhanced antimicrobial activity. It is noteworthy that, for one mutant, the enhancement in antimicrobial activity (18-fold higher than wild type) was greater than that of cell penetration (5.9-fold), suggesting that the N-terminal mutation may reinforce both interaction with unidentified intracellular target(s) and cell-penetration efficiency.

Crystallization and preliminary X-ray studies of azoreductases from Bacillus sp. B29.

Azoreductases from Bacillus sp. B29 are NADH-dependent flavoenzymes which contain a flavin mononucleotide (FMN) as a prosthetic group and exist as homodimers composed of 23 kDa subunits. These enzymes catalyze the reductive degradation of various azo compounds by a ping-pong mechanism. In order to determine the structure-function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method. A crystal of SeMet-AzrA diffracted to 2.0 A resolution and was determined to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.9, b = 69.0, c = 105.4 A. The native crystals of AzrC belonged to space group C2, with unit-cell parameters a = 192.0, b = 56.6, c = 105.5 A, beta = 115.7 degrees , and diffracted to 2.21 A resolution.

Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus.

The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 degrees C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 degrees C and for 1 month at 30 degrees C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 degrees C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.

Synergy of valine and threonine supplementation on poly(2-hydroxybutyrate-block-3-hydroxybutyrate) synthesis in engineered Escherichia coli expressing chimeric polyhydroxyalkanoate synthase.

The engineered chimeric polyhydroxyalkanoate (PHA) synthase PhaCAR is composed of N-terminal portion of Aeromonas caviae PHA synthase and C-terminal portion of Ralstonia eutropha (Cupriavidus necator) PHA synthase. PhaCAR has a unique and useful capacity to synthesize the block PHA copolymer poly(2-hydroxybutyrate-block-3-hydroxybutyrate) [P(2HB-b-3HB)] in engineered Escherichia coli from exogenous 2HB and 3HB. In the present study, we initially attempted to incorporate the amino acid-derived 2-hydroxyalkanoate (2HA) units using PhaCAR and the 2HA-CoA-supplying enzymes lactate dehydrogenase (LdhA) and CoA transferase (HadA). Cells harboring the genes for PhaCAR, LdhA, and HadA, as well as for the 3HB-CoA-supplying enzymes ß-ketothiolase and acetoacetyl-CoA reductase, were cultivated with supplementation of four hydrophobic amino acids, i.e., leucine, valine (Val), isoleucine (Ile), and phenylalanine, in the medium. No hydrophobic amino acid-derived monomers were incorporated into the polymer, which was most likely because of the strict substrate specificity of PhaCAR; however, P(2HB-co-3HB) was unexpectedly produced with Val supplementation. The copolymer was likely P(2HB-b-3HB) based on proton nuclear magnetic resonance analysis. Based on the endogenous pathways in E. coli, 2HB units are likely derived from threonine (Thr) through deamination and dihydroxylation. In fact, dual supplementation with Thr and Val showed synergy on the 2HB fraction of the polymer. Val supplementation promoted the 2HB synthesis likely by inhibiting the metabolism of 2-ketobutyrate into Ile and/or activating Thr dehydratase. In conclusion, the LdhA/HadA/PhaCAR pathway served as the system for the synthesis of P(2HB-b-3HB) from biomass-derived carbon sources.

Biosynthesis of Random-Homo Block Copolymer Poly[Glycolate-ran-3-Hydroxybutyrate (3HB)]-b-Poly(3HB) Using Sequence-Regulating Chimeric Polyhydroxyalkanoate Synthase in Escherichia coli.

Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaC AR and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaC AR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1 P s STQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaC AR synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB).

A unique post-translational processing of an exo-beta-1,3-glucanase of Penicillium sp. KH10 expressed in Aspergillus oryzae.

To characterize an exo-beta-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-beta-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS-polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modification.

Comparative enzymatic analysis of azoreductases from Bacillus sp. B29.

We cloned and expressed two genes encoding azoreductase homologes, AzrB and AzrC, from Bacillus sp. B29. Purified recombinant AzrB and AzrC were homodimers with 23 kDa identical subunits, and were flavoproteins. NADH was preferred as electron donors for both azoreductases. The azoreductases showed optimal activities at 70 degrees C (AzrB) and 55 degrees C (AzrC), and retained activities up to 55 degrees C (AzrB) and 50 degrees C (AzrC) after incubation for 1 h. Other enzymatic properties, including the substrate specificities of both azoreductases, were also investigated.

The hydrophobicity in a chemically modified side-chain of cysteine residues of thanatin is related to antimicrobial activity against Micrococcus luteus.

The chemically modified thanatins with the methyl group (CH(3)), ethyl group (C(2)H(5)), and normal-octyl group (C(8)H(17)) at the side-chain of cysteine residues were synthesized. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteus than wild type thanatin. It was found that there was an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues in thanatin.

High-cell density culture of poly(lactate-co-3-hydroxybutyrate)-producing Escherichia coli by using glucose/xylose-switching fed-batch jar fermentation.

Poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] is produced in engineered Escherichia coli harboring the genes encoding an LA-polymerizing enzyme (LPE) and monomer-supplying enzymes. In this study, high cell-density fed-batch jar fermentation was developed using xylose and/or glucose as the carbon source. Fed-batch fermentation was initially performed with 20 g/L sugar during the batch phase for 24 h, and subsequent sugar feeding from 24 to 86 h. The feeding rate was increased in a stepwise manner. When xylose alone was used for cultivation, the cells produced the polymer at 11.6 g/L, which was higher than the 4.3 g/L obtained using glucose as the sole carbon source. However, in the first 24 h the growth in the glucose culture was greater than in the xylose culture. Based on these results, glucose was used for cell growth (at the initial stage) and xylose was used for polymer production (at the feeding stage). As expected, in the glucose/xylose switching fermentation method, polymer production was significantly enhanced, eventually reaching 26.7 g/L. The enhanced polymer production obtained by using xylose was presumably due to overflow metabolism. In fact, during xylose feeding, acetic acid excretion was greater than that in case of the glucose grown culture, suggesting the channeling of the metabolic flux from acetyl-CoA towards polymer production over into the tricarboxylic acid cycle in the xylose-fed cultures. Therefore, this sequential glucose/xylose feed strategy is potentially useful for production of acetyl-CoA derived compounds in E. coli.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-mediated de novo synthesis of glycolate-based polyhydroxyalkanoate in Escherichia coli.

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1PsSTQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (ΔglcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1PsSTQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms.