Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava

A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.     

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).     

Size analysis of poly(3-hydroxybutyric acid) granules produced in recombinant Escherichia coli

Summary Size distributions of PHB granules synthesized in recombinant Escherichia coli are determined by photosedimentation. Mean granule Stokes diameters are in the range 1.13–1.25 m, which is larger than reported values for wild type microorganisms. Treatment with 1.5% hypochlorite and mild centrifugation did not affect granule size distribution. Treatment with 10% hypochlorite led to a significant reduction in mean diameter and total PHB.     

Synthesis and accumulation of poly(3-hydroxybutyric acid) by Rhizobium sp

Forty-two Rhizobium strains obtained from different culture collections were evaluated quantitatively for poly(3-hydroxy-butyric acid) [PHB] production in shake flask culture. The majority of the strains produced the maximum amount of PHB during the late exponential or stationary phase of growth. Synthesis and accumulation of PHB in different species of Rhizobium were found to vary between 1-38% of their dry biomass. Growth and PHB production by the Rhizobium strain TAL-640 were greatly influenced by the C-source and D-mannitol was fundamental to both processes. The identity and purity of PHB isolated from TAL-640 have also been confirmed by UV-, IR- and 1H-NMR spectroscopic analyses.     

Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts

Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18 days at 4 °C instead of storage at − 80 °C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum

Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Chromatium vinosum (PhaEC_Cv) was used to examine in vitro the specific synthase activity, turnover of R-(−)-3-hydroxybutyryl coenzyme A (3HB-CoA) and poly(3-hydroxybutyric acid) formation under various conditions. The 3HB-CoA consumption was terminated by a reaction-dependent inactivation of the PHA synthase. Salts (MgCl₂, CaCl₂, NaCl), proteins (bovine serum albumin, lysozyme, phasine) or detergent (Tween 20) increased the 3HB-CoA turnover to 2.5-fold. Specific PHA synthase activity was only partially affected by the added components. In general, a higher concentration of salt often inhibited the activity of PhaEC_Cv without affecting the yield according to 3HB-CoA turnover. NAD⁺ and NADP⁺ (2 mM) inhibited PhaEC_Cv completely, where-as NADH and NADPH did not. Macroscopic poly(3HB) granules were formed in vitro if PhaEC_Cv was incubated in the presence of sufficient amounts of 3HB-CoA and if MgCl₂ was present. The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protein as well as by bovine serum albumin and the GA24 protein, a poly(3HB)-granule-associated protein of Alcaligenes eutrophus. Scanning electron micrographs from the synthesized granules were obtained. The granules consisted of poly(3HB) that had a molar mass in the range (1–2) × 10⁶ g/mol. Received: 12 September 1997 / Received revision: 24 October 1997 / Accepted: 31 October 1997     

Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.

A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.     

喜盐鸢尾花色形成关键基因的克隆及表达分析

喜盐鸢尾(Iris halophila Pall.)及其变种蓝花喜盐鸢尾(I.halophila Pall.var.sogdiana(Bung)Grubov)因耐盐碱及其多种花色而具有盐碱地园艺开发价值。本文根据喜盐鸢尾内轮花被转录组测序结果,利用基因特异性引物从这2种植物中分别克隆了编码查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、类黄酮-3',5'-羟基化酶类(F3'5'H-like)等基因的部分片段,并对它们在内轮花被中的表达水平进行实时定量PCR分析。序列分析结果确认在喜盐鸢尾中所克隆的CHS(311 bp)、CHI(457 bp)、F3'5'H-like(496 bp)3个基因(部分)未见文献报道与NCBI等数据库记录。其中F3'5'H-like基因与经典的属于细胞色素P450CYP75A亚家族的F3'5'H不同,而与万带兰的F3'5'H-like同属于CYP76AB亚家族,为一类新的蓝花相关基因。实时定量PCR表达分析结果表明,与黄花的喜盐鸢尾相比,蓝花喜盐鸢尾中CHS与F3'5'H-like显著上调表达,可能是其花色不同于喜盐鸢尾的主要原因。     

Growth-associated production and characterization of poly (3-hydroxybutyric acid) of Azotobacter beijerinckii DAR-102

Production of poly (3-hydroxybutyric acid) [P(3HB)] by Azotobacter beijerinckii DAR-102 isolated in this laboratory has been optimized under batch-culture. The accumulatad polymer attained 58% of cell dry mass during mid-stationary phase with an yield of 0.58 g/l when grown in nitrogen-free medium. The optimum concentration of glucose and fructose for P(3HB) production was 3% (w/v) and 2% (w/v) respectively while that of casamino acid and tryptose was 0.1% (w/v). Phosphate at a concentration suboptimal for growth and limitation of oxygen in the medium favoured P(3HB) accumulation. The production of P(3HB) was maximum with an inoculum dose of 4% (v/v). The accumulated polymer was isolated by direct chloroform extraction of the dry cell mass and purified by precipitation with diethyl ether. The purified polymer has been characterized in terms of its solubility properties, melting temperature, and UV-, IR- and NMR-spectroscopic analyses.     

Intracellular degradation of poly(3-hydroxybutyric acid) accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [P(3HB)] accounting 69% of cell dry weight (CDW) from glucose during growth in nitrogen-free Stockdale medium. Degradation of the accumulated polymer by the organism was studied under carbon-free medium following two-step cultivation method. P(3HB) content of cells decreased rapidly from 69% to 4.8% of CDW after 35 h under carbon-deprived condition. Autodigestion of P(3HB) was evident from the estimation of intracellular P(3HB) depolymerase (i-depolymerase) activity in cell-free extract using artificial P(3HB) granules as substrate. Polymer content decreased rapidly along with the increase in i-depolymerase activity and rate of polymer degradation when medium was supplemented with (NH4)2SO4 at 0.1% (w/v) level. However, the effects were reverse when organic nitrogenous substrate, beef extract at similar concentration was present in the medium. The optimum temperature and pH for i-depolymerase activity were 35 degrees C and 7.7 respectively. The oxygen-limiting condition (culture volume per flask volume, 50%) decreased 10.7% activity of i-depolymerase over control resulting a slow P(3HB) degradation. The presence of NaCl (6 x 10(3) microg/ml) showed a positive effect on i-depolymerase whereas EDTA (40 microg/ml) resulted in 20% less activity. Furthermore, the intracellular degradation of P(3HB) decreased the intrinsic viscosity, molecular weight and tensile strength of the accumulated polymer.     

Enzymatic degradation processes of poly[(R)-3-hydroxybutyric acid] and poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid] single crystals revealed by atomic force microscopy: effects of molecular weight and second-monomer composition on erosion rates

Enzymatic degradation processes of poly[(R)-3-hydroxybutyric acid] (P(3HB)) and poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid] (P(3HB-co-3HV)) single crystals in the presence of PHB depolymerase from Ralstonia pickettii T1 were studied by real-time and static atomic force microscopy (AFM) observations. Fibril-like crystals were generated along the long axis of single crystals during the enzymatic degradation, and then the dimensions of fibril-like crystals were analyzed quantitatively. The morphologies and sizes of fibril-like crystals were dependent on the molecular weight and copolymer composition of polymers. For all samples, the crystalline thickness gradually decreased toward a tip from the root of a fibril-like crystal after enzymatic degradation for 1 h. The thinning of fibril-like crystals may be attributed to the destruction of chain-packing structure toward crystallographic c axis by the adsorption of enzyme. From the real-time AFM images, it was found that at the initial stage of degradation the enzymatic erosion started from the disordered chain-packing region in single crystals to form the grooves along the a axis. The generated fibril-like crystals deformed at a constant rate along the a axis with a constant rate after the induction time. The erosion rate at the grooves along the a axis increased with a decrease of molecular weight and with an increase of copolymer composition. On the other hand, the erosion rate along the a axis, at the tip of the fibril-like crystal, was dependent on only the copolymer composition, and the value increased with an increase in the copolymer composition. The morphologies and sizes of fibril-like crystals were governed by both the erosion rates along the a axis at the grooves and tip of fibril-like crystals. In addition, we were able to estimated the overall enzymatic erosion rate of single crystals by PHB depolymerase from the volumetric analysis.     

Stimulatory effects of amino acids and oleic acid on poly(3-hydroxybutyric acid) synthesis by recombinant Escherichia coli

Addition of cysteine, isoleucine, methionine, or proline promoted poly(3-hydroxybutyric acid) [PHB] synthesis by recombinant Escherichia coli more than two-fold. Oleic acid also enhanced PHB synthesis more than three-fold. A PHB concentration of 70 g/l could be obtained by fed-batch culture of recombinant E. coli in a defined medium supplemented with small amounts of isoleucine, methionine, and proline. The stimulatory effects of amino acids and oleic acid on PHB synthesis seems to be due to the availability of more acetyl-CoA and/or NADPH.     

Crystallization behavior and environmental biodegradability of the blend films of poly(3-hydroxybutyric acid) with chitin and chitosan

Crystallization behavior and environmental biodegradability were investigated for the films of bacterial poly(3-hydroxybutyric acid) (PHB) blends with chitin and chitosan. The blend films showed X-ray diffractive peaks that arose from the PHB crystalline component. It was suggested that the lamellar thickness of the PHB crystalline component in the blends was large enough to show detectable X-ray diffractive peaks, but this was too small to show observable melting endotherm in the DSC thermogram and the crystalline band absorption in the FT-IR spectrum. In the PHB/chitin and PHB/chitosan blends, thermal transition temperatures of PHB amorphous region observed by dynamic mechanical thermal analysis were almost the same as that of neat PHB. Both the PHB/chitin and the PHB/chitosan blend films biodegraded in an environmental medium. Several blend films showed faster biodegradation than the pure-state component polymers.     

Pseudomonas 2 F: Kinetics of growth and accumulation of poly - D(-)-3-hydroxybutyric acid (poly - HB)

Summary Kinetics of poly - HB accumulation in Pseudomonas 2F, a recently isolated and new strain, differ considerably from the behaviour found hitherto with other strains. After a period of carbon limitation of 1 hour, without the application of growth - limiting conditions, the accumulation of poly - HB is evidently accelerated (carbon - overcompensation).     

Biosynthesis of poly (3-hydroxybutyric acidco-3-hydroxy-4-pentenoic acid) from unrelated substrates byBurkholderia sp.

 From soil, two strains of Burkholderia sp. were isolated that synthesized and accumulated a copolyester of 3-hydroxybutyric acid and 3-hydroxy-4-pentenoic acid from single, unrelated carbon sources such as surcose or gluconate. Strain IPT77B, for instance, accumulated from gluconate poly(3-hydroxy- butyric acid co-3-hydroxy-4-pentenoic acid) up to 70% (w/w) of the cellular dry matter, and 3-hydroxy-4-pentenoic acid contributed up to 6.9 mol 100/mol constituents. The occurrence of the constituent with the vinyl pendant group in the polyester was confirmed by gas-chromatographic and nuclear magnetic resonance analysis. Received: 28 December 1994/Accepted: 4 January 1995     

Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.

We studied recovery of poly(3-hydroxybutyric acid) (PHB) from Alcaligenes eutrophus and a recombinant Escherichia coli strain harboring the A. eutrophus poly(3-hydroxyalkanoic acid) biosynthesis genes. The amount of PHB degraded to a lower-molecular-weight compound in A. eutrophus during the recovery process was significant when sodium hypochlorite was used, but the amount degraded in the recombinant E. coli strain was negligible. However, there was no difference between the two microorganisms in the patterns of molecular weight change when PHB was recovered by using dispersions of a sodium hypochlorite solution and chloroform. To understand these findings, we examined purified PHB and lyophilized cells containing PHB by using a differential scanning calorimeter, a thermogravimetric analyzer, and nuclear magnetic resonance. The results of our analysis of lyophilized whole cells containing PHB with the differential scanning calorimeter suggested that the PHB granules in the recombinant E. coli strain were crystalline, while most of the PHB in A. eutrophus was in a mobile amorphous state. The stability of the native PHB in the recombinant E. coli strain during sodium hypochlorite treatment seemed to be due to its crystalline morphology. In addition, as determined by the thermogravimetric analyzer study, lyophilized cell powder of the recombinant E. coli strain containing PHB exhibited greater thermal stability than purified PHB obtained by chloroform extraction. The PHB preparations extracted from the two microorganisms had identical polymer properties.