Optimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers Using Recombinant Escherichia coli.

Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction.

Enhanced Production of (R)-3-Hydroxybutyrate Oligomers by Coexpression of Molecular Chaperones in Recombinant Escherichia coli Harboring a Polyhydroxyalkanoate Synthase Derived from Bacillus cereus YB-4.

The biodegradable polyester poly-(R)-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a (R)-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by using recombinant Escherichia coli expressing PHA synthases. The 3HBO has potential value as an antibacterial substance and as a building block for various polymers. In this study, to construct an efficient 3HBO production system, the coexpression of molecular chaperones and a PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4) was examined. First, genes encoding enzymes related to 3HBO biosynthesis (phaRCYB4, phaA and phaB derived from Ralstonia eutropha H16) and two types of molecular chaperones (groEL, groES, and tig) were introduced into the E. coli strains BW25113 and BW25113ΔadhE. As a result, coexpression of the chaperones promoted the enzyme activity of PHA synthase (approximately 2-3-fold) and 3HBO production (approximately 2-fold). The expression assay of each chaperone and PHA synthase subunit (PhaRYB4 and PhaCYB4) indicated that the combination of the two chaperone systems (GroEL-GroES and TF) supported the folding of PhaRYB4 and PhaCYB4. These results suggest that the utilization of chaperone proteins is a valuable approach to enhance the formation of active PHA synthase and the productivity of 3HBO.

The influence of medium composition on the microbial secretory production of hydroxyalkanoate oligomers.

With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) in Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.

Microbial oversecretion of (R)-3-hydroxybutyrate oligomer with diethylene glycol terminal as a macromonomer for polyurethane synthesis.

Poly((R)-3-hydroxybutyrate) (P(3HB)) is a polyester that is synthesized and accumulated in many prokaryotic cells. Recently, a new culture method for the secretion of the intracellularly synthesized (R)-3-hydroxybutyrate oligomer (3HBO) from recombinant Escherichia coli cells was developed. In this study, we attempted to produce microbial 3HBO capped with a diethylene glycol terminal (3HBO-DEG) as a macromonomer for polymeric materials. First, we prepared recombinant E. coli strains harboring genes encoding various polyhydroxyalkanoate (PHA) synthases (PhaC, PhaEC or PhaRC) that can incorporate chain transfer (CT) agents such as DEG into the polymer's terminal and generate CT end-capped oligomers. To this end, each strain was cultivated under DEG supplemental conditions, and the synthesis of 3HBO-DEG was confirmed. As a result, the highest secretory production of 3HBO-DEG was observed for the PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4). To evaluate the usability of the secreted 3HBO-DEG as a macromonomer, 3HBO-DEG was purified from the culture medium and polymerized with 4,4'-diphenylmethane diisocyanate as a spacer compound. Characterization of the polymeric products revealed that 3HBO-based polyurethane was successfully obtained and was a flexible and transparent noncrystalline polymer, unlike P(3HB). These results suggested that microbial 3HBO-DEG is a promising platform building block for synthesizing polyurethane and various other polymers.

Microbial Secretion Platform for 3-Hydroxybutyrate Oligomer and Its End-Capped Forms Using Chain Transfer Reaction-Mediated Polyhydroxyalkanoate Synthases.

The biodegradable polyester 3-hydroxybutyrate (3HB) polymer [P(3HB)] is intracellularly synthesized and accumulated in recombinant Escherichia coli. In this study, native polyhydroxyalkanoate (PHA) synthases are used to attempt to microbially secrete 3HB homo-oligomers (3HBOs), which are widely distributed in nature as physiologically active substances. High secretory production is observed, especially for the two PHA synthases from Aeromonas caviae and Bacillus cereus YB4. Surprisingly, an ethyl ester at the carboxy terminus (ethyl ester form) of 3HBOs is identified for most of the PHA synthases tested. Next, 3HBOs with a functional carboxyl group (carboxyl form of 3HBO) are obtained by using the alcohol dehydrogenase gene (adhE)-deficient mutant strain, suggesting that the endogenous ethanol produced in E. coli acts as a chain transfer (CT) agent in the generation of 3HBOs. Furthermore, an in vitro polymerization assay reveals that CT agents such as ethanol and free 3HB are involved in the generation of ethyl ester and carboxyl form of 3HBO, respectively. The microbial platform established herein allows the secretion of 3HBOs with desirable end structures by supplementation with various CT agents. The obtained 3HBOs and their end-capped forms may be used as physiologically active substances and building blocks for polymeric materials.

PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective.

Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.

Low Carbon Concentration Feeding Improves Medium-Chain-Length Polyhydroxyalkanoate Production in Escherichia coli Strains With Defective ß-Oxidation.

Medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) of near homopolymeric composition are unnatural polymers, having almost identical repeating units throughout the polymer chain. These homopolymeric PHAs can be produced by ß-oxidation defective bacterial hosts. Escherichia coli is an attractive workhorse for the production of such genetically engineered PHAs; however, achieving efficient production of the near homopolymers by ß-oxidation defective strains is a major challenge because of a lack of process development studies. In order to address this issue, we investigated the optimization of carbon feeding for efficient production of MCL-PHAs by an E. coli strain with defective ß-oxidation, LSBJ. Engineered bacteria were cultured in shake-flasks with intermittent feeding of a fatty acid substrate [either decanoate (C10) or dodecanoate (C12)] at various concentrations together with a co-carbon source (glucose, glycerol, or xylose) in order to support cell growth. It was found that feeding low concentrations of both fatty acids and co-carbon sources led to an enhanced production of MCL-PHAs. Additionally, the supplementation of yeast extract improved cell growth, resulting in achieving higher titers of MCL-PHA. As a result, poly(3-hydroxydecanoate) [P(3HD)] and poly(3-hydroxydodecanoate) [P(3HDD)] were produced up to 5.44 g/L and 3.50 g/L, respectively, as near homopolymers by employing the developed feeding strategy. To the best of our knowledge, we record the highest titer of P(3HD) ever reported so far.

Increased synthesis of poly(3-hydroxydodecanoate) by random mutagenesis of polyhydroxyalkanoate synthase.

Poly(3-hydroxydodecanoate) [P(3HDD)], a medium-chain-length polyhydroxyalkanoate (PHA), is expected to be used as a novel type of bioplastic characterized by a soft and transparent nature. In this study, to achieve a high yield of P(3HDD), PHA synthase was modified through random mutagenesis of a region of the PHA synthase 1 gene from Pseudomonas putida KT2440 (phaC1Pp). Screening of the mutant library using a ß-oxidation-deficient Escherichia coli LSBJ was performed. As a result, four mutants, designated w10, w14, w309, and w311, were selected from 10,000 mutants. The w311 mutant had two amino acid replacements (E358G and N398S), and showed the highest production of P(3HDD) with increased polymer molecular weights when compared to the native enzyme. Saturation mutagenesis at the N398 position, which was found to be highly conserved among Pseudomonas PhaCs, revealed that amino acids with hydrophobic and smaller residues either retained or increased P(3HDD) production. This study demonstrates the benefit of using the PHA synthase mutants to enhance the production of P(3HDD).

Gas chromatography-mass spectrometry-based monomer composition analysis of medium-chain-length polyhydroxyalkanoates biosynthesized by Pseudomonas spp.

Medium-chain-length (mcl)-polyhydroxyalkanoates (PHAs), elastomeric polyesters synthesized by Genus Pseudomonas bacteria, generally have many different monomer components. In this study, PHAs biosynthesized by four type strains of Pseudomonas (P. putida, P. citronellolis, P. oleovorans, and P. pseudoalcaligenes) and a typical PHA producer (P. putida KT2440) were characterized in terms of the monomer structure and composition by gas chromatography-mass spectrometry (GC-MS) analysis. With a thiomethyl pretreatment of PHA methanolysis derivatives, two unsaturated monomers, 3-hydroxy-5-dodecenoate (3H5DD) and 3-hydroxy-5-tetradecenoate (3H5TD), were identified in mcl-PHAs produced by P. putida and P. citronellolis. The quantitative analysis of PHA monomers was performed by employing GC-MS with methanolysis derivatives, and the results coincided with those obtained by performing nuclear magnetic resonance spectroscopy. Only poly(3-hydroxybutyrate) was detected from the P. oleovorans and P. pseudoalcaligenes type strains. These analytical results would be useful as a reference standard for phenotyping of new PHA-producing bacteria.

Biosynthesis of polyhydroxyalkanoates containing 2-hydroxy-4-methylvalerate and 2-hydroxy-3-phenylpropionate units from a related or unrelated carbon source.

The discovery of the lactate-polymerizing enzyme (LPE) enabled the biosynthesis of a polyhydroxyalkanoate (PHA) containing 2-hydroxyalkanoate (2HA). Amino acids are potential precursors of 2HA with various side chain structures if appropriate enzymes are used to convert amino acids to 2HA-coenzyme A (CoA) as the substrate for LPE. In this study, the suitability and utility of (R)-2-hydroxy-4-methylvalerate (2H4MV) dehydrogenase (LdhA) and 2H4MV-CoA transferase (HadA) from Clostridium difficile as 2HA-CoA-supplying enzymes were investigated. By expressing LPE, LdhA, and HadA in Escherichia coli DH5α, we successfully produced poly(3-hydroxybutyrate-co-2HA) [P(3HB-co-2HA)] from a related or unrelated carbon source. The 2HA units incorporated into PHA from unrelated carbon sources were primarily 2H4MV and 2-hydroxy-3-phenylpropionate (2H3PhP), which were assumed to be derived from endogenous leucine and phenylalanine, respectively. Furthermore, P(3HB-co-22 mol% 2HA) synthesis was demonstrated by means of saccharified sugars, which are an abundant and renewable feedstock for polymer production from hemicellulosic biomass (Japanese cedar) as the carbon source. Our study shows that several types of 2HA units such as 2H4MV and 2H3PhP are endogenous monomers for PHA biosynthesis in E. coli expressing LdhA and HadA.

Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene.

Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ's function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJYB4) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJYB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJYB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJYB4, which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJYB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

Contribution of the distal pocket residue to the acyl-chain-length specificity of (R)-specific enoyl-coenzyme A hydratases from Pseudomonas spp.

(R)-Specific enoyl-coenzyme A (enoyl-CoA) hydratases (PhaJs) are capable of supplying monomers from fatty acid ß-oxidation to polyhydroxyalkanoate (PHA) biosynthesis. PhaJ1Pp from Pseudomonas putida showed broader substrate specificity than did PhaJ1Pa from Pseudomonas aeruginosa, despite sharing 67% amino acid sequence identity. In this study, the substrate specificity characteristics of two Pseudomonas PhaJ1 enzymes were investigated by site-directed mutagenesis, chimeragenesis, X-ray crystallographic analysis, and homology modeling. In PhaJ1Pp, the replacement of valine with isoleucine at position 72 resulted in an increased preference for enoyl-coenzyme A (CoA) elements with shorter chain lengths. Conversely, at the same position in PhaJ1Pa, the replacement of isoleucine with valine resulted in an increased preference for enoyl-CoAs with longer chain lengths. These changes suggest a narrowing and broadening in the substrate specificity range of the PhaJ1Pp and PhaJ1Pa mutants, respectively. However, the substrate specificity remains broader in PhaJ1Pp than in PhaJ1Pa. Additionally, three chimeric PhaJ1 enzymes, composed from PhaJ1Pp and PhaJ1Pa, all showed significant hydratase activity, and their substrate preferences were within the range exhibited by the parental PhaJ1 enzymes. The crystal structure of PhaJ1Pa was determined at a resolution of 1.7 Å, and subsequent homology modeling of PhaJ1Pp revealed that in the acyl-chain binding pocket, the amino acid at position 72 was the only difference between the two structures. These results indicate that the chain-length specificity of PhaJ1 is determined mainly by the bulkiness of the amino acid residue at position 72, but that other factors, such as structural fluctuations, also affect specificity.

Endogenous ethanol affects biopolyester molecular weight in recombinant Escherichia coli.

In biopolyester synthesis, polyhydroxyalkanoate (PHA) synthase (PhaC) catalyzes the polymerization of PHA in bacterial cells, followed by a chain transfer (CT) reaction in which the PHA polymer chain is transferred from PhaC to a CT agent. Accordingly, the frequency of CT reaction determines PHA molecular weight. Previous studies have shown that exogenous alcohols are effective CT agents. This study aimed to clarify the effect of endogenous ethanol as a CT agent for poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis in recombinant Escherichia coli, by comparing with that of exogenous ethanol. Ethanol supplementation to the culture medium reduced P(3HB) molecular weights by up to 56% due to ethanol-induced CT reaction. NMR analysis of P(3HB) polymers purified from the culture supplemented with (13)C-labeled ethanol showed the formation of a covalent bond between ethanol and P(3HB) chain at the carboxyl end. Cultivation without ethanol supplementation resulted in the reduction of P(3HB) molecular weight with increasing host-produced ethanol depending on culture aeration. On the other hand, production in recombinant BW25113(ΔadhE), an alcohol dehydrogenase deletion strain, resulted in a 77% increase in molecular weight. Analysis of five E. coli strains revealed that the estimated number of CT reactions was correlated with ethanol production. These results demonstrate that host-produced ethanol acts as an equally effective CT agent as exogenous ethanol, and the control of ethanol production is important to regulate the PHA molecular weight.

Characterization of polyhydroxyalkanoate (PHA) synthase derived from Delftia acidovorans DS-17 and the influence of PHA production in Escherichia coli.

Heterologous expression of polyhydroxyalkanoate (PHA) synthase from Delftia acidovorans DS-17 (PhaC(Da)) in Escherichia coli JM109 leads to effective production of high-molecular-weight poly[(R)-3-hydroxybutyrate] [P(3HB)]. This study examined the effect of PhaC(Da) expression on P(3HB) production in E. coli JM109 (Da strain) by comparing with the strain expressing PHA synthase (PhaC(Re)) from Ralstonia eutropha (Re strain). First, the kinetic properties of PhaC(Da) were investigated. Among the five detergents examined, Triton X-100 remarkably activated PhaC(Da), as well as PhaC(Re). The affinity of PhaC(Da) for its substrate was lower than that of PhaC(Re), whereas the maximum reaction rate of PhaC(Da) was higher than that of PhaC(Re). However, the kinetic differences were not likely to influence P(3HB) production in the cells. Under conditions of P(3HB) production, the translational levels of monomer-supplying enzymes (PhaA and PhaB) were similar in both the Da and Re strains, whereas PhaC exhibited different expression levels: the abundance of soluble PhaC(Da) was lower than that of soluble PhaC(Re). This observation suggests that the production of high-molecular-weight P(3HB) by the Da strain would be attributed to the low amounts of active PhaC(Da) in the cells.

Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli.

Ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.