Application of the BPEC pathway for large-scale biotechnological production of poly(3-mercaptopropionate) by recombinant Escherichia coli, including a novel in situ isolation method

Metabolically engineered Escherichia coli JM109 harboring plasmid pBPP1 and expressing the nonnatural BPEC pathway for synthesis of thermoplastic polyhydroxyalkanoates (PHA) and novel polythioesters (PTE) to provide suitable substrates of PHA synthase was investigated with respect to biotechnological production of poly(3-mercaptopropionate) [poly(3MP)]. Fed-batch fermentation processes were established at the 30- and 500-liter scales in stirred tank bioreactors to produce kilogram amounts of poly(3MP). Cultivation was done in a modified M9 mineral salts medium containing glucose or glycerol as the carbon and energy source and with 3-mercaptopropionic acid (3MP) as the precursor substrate for poly(3MP) biosynthesis provided from the late exponential growth phase. Approximately 23 g of cell dry matter (CDM) per liter and poly(3MP) cell contents of up to 45% (wt/wt) were the highest cell densities and polymer contents obtained, respectively. At best, 69.1% (wt/wt) of 3MP was converted into poly(3MP), indicating that 3MP was mostly used for poly(3MP) biosynthesis. Furthermore, a novel in situ process for rapid and convenient isolation of poly(3MP) from the cells in the bioreactor was developed. This was achieved by addition of sodium dodecyl sulfate to the cultivation broth immediately after the fermentation, heating to 90 degrees C for 20 min with intensive stirring, and subsequent washing steps. The purity of such in situ isolated poly(3MP) was more than 98%, as revealed by gas chromatographic and elemental sulfur analyses of the material isolated.     

Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases

The biodegradability of microbial polythioesters (PTEs), a novel class of biopolymers which were discovered recently and can be produced by polyhydroxyalkanoate (PHA)-accumulating bacteria, was studied. Using poly(3-hydroxybutyrate-co-3-mercaptopropionate) [poly(3HB-co-3MP)] as sole carbon source for screening, 22 new bacterial strains were isolated and characterized. Interestingly, none of the PHA-degrading bacteria was able to utilize the homopolymer poly(3MP) as a carbon source for growth or to form clear zones on poly(3MP)-containing agar plates. The extracellular PHA depolymerases from two strains ( Schlegelella thermodepolymerans, Pseudomonas indica K2) were purified to electrophoretic homogeneity and biochemically characterized. The PHA depolymerase of S. thermodepolymerans exhibited a temperate optimum of about 75°C to 80°C and was stable at 70°C for more than 24 h. Regarding the substrate specificities of the PHA depolymerase of S. thermodepolymerans, enzyme activities decreased significantly with increasing 3MP content of the copolymer substrates. Interestingly, no activity could be detected with homoPTEs consisting only of 3MP or of 3-mercaptobutyrate. Similar results were obtained with the PHA depolymerases PhaZ2, PhaZ5 and PhaZ7 of Paucimonas lemoignei which were also investigated. The PHA depolymerase of Ps. indica K2 did not cleave any of the investigated polymers containing 3MP. Gas chromatography, infrared and ¹H-NMR spectrometry and matrix-assisted laser desorption/ionization time-of-flight analysis revealed that 3MPs containing oligomers were enriched in the water-insoluble fraction remaining after partial digestion of poly(3HB-co-3MP) by purified poly(3HB) depolymerase of S. thermodepolymerans. In contrast, 3HB was enriched in the water-soluble fraction, which also contained 3HB-co-3MP dimer obtained by partial digestion of this copolymer by the enzyme. This study clearly indicates that PHA depolymerases are specific for oxoester linkages of PHAs and that the thioester bonds of PTEs cannot be cleaved by this type of enzyme.This publication is dedicated to Prof. Dr. Hans G. Schlegel in honor of his 80th birthday     

From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.     

Poly(3-mercaptopropionate): a nonbiodegradable biopolymer?

Polythioesters (PTEs) represent a novel class of biopolymers, which basically can be synthesized with polyhydroxyalkanoate (PHA) biosynthesis systems. Albeit technical applications of PTEs have not been elucidated yet, biodegradability might be an important property of this new thermoplastic material. In this study, extensive approaches were employed to isolate microorganisms capable of degrading poly(3-mercaptopropionate), poly(3MP), as a model compound of PTEs. Screening of 74 different environmental samples using various enrichment techniques were applied, but neither bacteria nor fungi could be isolated hydrolyzing poly(3MP). Furthermore, microcosms such as soil, compost, or activated sludge were applied to search for poly(3MP) degrading microorganisms, considering microbial communities and/or nonculturable bacteria, and the poly(3MP) material was exposed for more than half a year. However, no poly(3MP) degrading organisms were found, indicating an unexpected persistence of this biologically produced polymer.     

Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Polyhydroxyalkanoates (PHA) have found widespread medical applications due to their biocompatibility and biodegradability, while further chemical modification requires functional groups on PHA. Halomonas bluephagenesis, a non-model halophilic bacterium serving as a chassis for the Next Generation Industrial Biotechnology (NGIB), was successfully engineered to express heterologous PHA synthase (PhaC) and enoyl coenzyme-A hydratase (PhaJ) from Aeromonas hydrophila 4AK4, along with a deletion of its native phaC gene to synthesize the short chain-co-medium chain-length PHA copolymers, namely poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhex-5-enoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyhex-5-enoate). After optimizations of the expression cassette and ribosomal binding site combined with introduction of endogenous acyl-CoA synthetase (fadD), the resulting recombinant strain H. bluephagenesis TDR4 achieved a remarkably high 3-hydroxyhexenoate (3HHxE) molar ratio of 35% when grown on glucose and 5-hexenoic acid as co-substrates. The total ratio of side chain consisting of 3HHx and 3HHxE monomers in the terpolymer can approach 44 mol%. H. bluephagenesis TDR4 was grown to a cell dry mass (CDM) of 30 g/L containing approximately 20% poly(3-hydroxybutyrate-co-22.75 mol% 3-hydroxy-5-hexenoate) in a 48-h of open and unsterile fermentation with a 5-hexenoic acid conversion efficiency of 91%. The resulted functional PHA containing 12.5 mol% 3-hydroxy-5-hexenoate exhibits more than 1000% elongation at break. The engineered H. bluephagenesis TDR4 can be used as an experimental platform to produce functional PHA.     

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.     

Engineering Escherichia coli for enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in larger cellular space

Polyhydroxyalkanoates (PHA) are intracellularly accumulated as inclusion bodies. Due to the limitation of the cell size, PHA accumulation is also limited. To solve this problem, Escherichia coli was enlarged by over-expression of sulA gene to inhibit the cell division FtsZ ring assembly, leading to the formation of filamentary E. coli that have larger internal space for PHA accumulation compared with rod shape E. coli. As a result, more than 100% increases on poly(3-hydroxybutyrate) (PHB) contents and cell dry weights (CDW) were achieved compared with its control strain under same conditions. The enlarged cell strategy was applied to the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or P(3HB-co-4HB) by sad, gabD, essential genes ispH and folK knockout E. coli harboring two addictives and thus stable plasmids consisting of P(3HB-co-4HB) producing genes, including phaCAB operon, orfZ, 4hbD, sucD, essential genes ispH and folK as well as the sulA. The so constructed E. coli grew in glucose to form filamentary shapes with an improved P(3HB-co-4HB) accumulation around 10% more than its control strain without addition of 4HB precursor, reaching over 78% P(3HB-co-4HB) in CDW. Importantly, the shape changing E. coli was able to precipitate after 20 min stillstand. Finally, the filamentary recombinant E. coli was not only able to produce more P(3HB-co-4HB) from glucose but also allow convenient downstream separation from the fermentation broth.     

Employing a recombinant strain of Advenella mimigardefordensis for biotechnical production of Homopolythioesters from 3,3'-dithiodipropionic acid

Advenella mimigardefordensis strain DPN7(T) was genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3'-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of the buk-ptb operon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, from Clostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaC(Am)). These measures yielded the potent PMP production strain A. mimigardefordensis strain SHX22. The deletion of mdo was required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-borne buk-ptb operon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase of A. mimigardefordensis (phaC(Am)) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.     

Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442

Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the beta-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.     

Characterization of a Novel Subgroup of Extracellular Medium-Chain-Length Polyhydroxyalkanoate Depolymerases from Actinobacteria

Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.     

Production of polyhydroxyalkanoates from inexpensive extruded rice bran and starch by Haloferax mediterranei

Low-cost raw materials can be used to reduce significantly the production cost of polyhydroxyalkanoates (PHA). In this study, extruded rice bran (ERB) and extruded cornstarch (ECS) were used as carbon sources to produce PHA by an archaea, Haloferax mediterranei, which cannot use native rice bran or cornstarch as a carbon source. By employing pH-stat control strategy to maintain pH at 6.9–7.1 in a 5-liter jar fermentor using ERB:ECS (1:8 g/g) as the major carbon source, we obtained a cell concentration of 140 g/L, PHA concentration of 77.8 g/L and PHA content of 55.6 wt.% in a repeated fed-batch fermentation. In contrast, when ECS was used as the major carbon source, we obtained 62.6 g/L cell concentration, 24.2 g/L PHA concentration and 38.7 wt.% PHA content. Under a hyper-saline condition and with no nitrogen-limitation restriction, the repeated fed-batch process can be sustained a long time for the mass production of PHA.     

Polymerase C1 levels and poly(R-3-hydroxyalkanoate) synthesis in wild-type and recombinant Pseudomonas strains.

A functional antibody highly specific for polymerase C1 of Pseudomonas oleovorans GPo1 was raised and used to determine polymerase C1 levels in in vivo experiments. The polymerase C1 antibodies did not show a cross-reaction with polymerase C2 of P. oleovorans. In wild-type P. oleovorans GPo1 and Pseudomonas putida KT2442, amounts of 0.075 and 0.06% polymerase relative to total protein, respectively, were found. P. oleovorans GPo1(pGEc405), which contained additional copies of the polymerase C1-encoding gene under the control of its native promoter, contained 0.5% polymerase C1 relative to total protein. Polymerase C1 reached 10% of total cell protein when the polymerase C1-encoding gene was overexpressed through the P(alk) promoter in P. oleovorans GPo1(pET702, pGEc74). Amounts of poly(R-3-hydroxyalkanoate) (PHA) increased significantly under non-nitrogen-limiting conditions when additional polymerase C1 was expressed in P. oleovorans. Whereas P. oleovorans produced 34% (wt/wt) PHA under these conditions, a PHA level of 64% (wt/wt) could be reached for P. oleovorans GPo1(pGEc405) and a PHA level of 52% (wt/wt) could be reached for P. oleovorans GPo1(pET702, pGEc74) after induction, compared to a PHA level of 13% for the uninduced control. All recombinant Pseudomonas strains containing additional polymerase C1 showed small changes in their PHA composition. Larger amounts of 3-hydroxyhexanoate monomer and smaller amounts of 3-hydroxyoctanoate and -decanoate were found compared to those of the wild type. Two different methods were developed to quantify rates of incorporation of new monomers into preexisting PHA granules. P. oleovorans GPo1 cells grown under nitrogen-limiting conditions showed growth stage-dependent incorporation rates. The highest PHA synthesis rates of 9.5 nmol of C8/C6 monomers/mg of cell dry weight (CDW)/min were found during the mid-stationary phase, which equals a rate of production of 80 g of PHA/kg of CDW/h.     

Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight

In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.     

High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40°C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47°C. At 43°C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43°C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50°C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

Swinging effect of salicylic acid on the accumulation of polyhydroxyalkanoic acid (PHA) in Pseudomonas aeruginosa BM114 synthesizing both MCLandSCL-PHA

A bacterium, Pseudomonas aeruginosa BM114, capable of accumulating a blend of medium-chain-length (MCL)- and short-chain-length (SCL)-polyhydroxyalkanoic acid (PHA), was isolated. Salicylic acid (SA), without being metabolized, was found to specifically inhibit only the accumulation of MCL-PHA without affecting cell growth. An addition of 20 mM SA selectively inhibited the accumulation of MCL-PHA in decanoate-grown cells by 83% of the control content in one-step cultivation, where overall PHA accumulation was inhibited by only approximately11%. Typically, the molar monomerunit ratio of the PHA for 25 mM decanoate-grown cells changed from 46:4:25:25 (=[3-hydroxybutyrate]:[3-hydroxycaproate]: [3-hydroxyoctanoate]:[3-hydroxydecanoate]) at 0 mM SA (dry cell wt, 1.97 g/l; PHA content, 48.6 wt%) to 91:1:4:4 at 20 mM SA (dry cell wt, 1.85 g/l; PHA content, 43.2 wt%). Thus, the stimulation of SCL-PHA accumulation was observed. Growth of P. aeruginosa BM114 on undecanoic acid also produced a PHA blend composed of 47.4% P(3HB-co-3- hydroxyvalerate) and 52.6% P(3-hydroxyheptanoate-co-3- hydroxynonanoate-co-3-hydroxyundecanoate). Similar to the case of even-carboxylic acids, SA inhibited the accumulation of only MCL-PHA, but stimulated the accumulation of SCLPHA. For all medium-chain fatty acids tested, SA induced a stimulation of SCL-PHA accumulation in the BM114 strain. SA could thus be used to suppress only the formation of MCL-PHA in Pseudomonas spp. accumulating a blend of SCL-PHA and MCL-PHA.     

Improved production of poly(3-hydroxybutyrate-co-4-hydroxbutyrate) copolymer using a combination of 1,4-butanediol and γ-butyrolactone

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was able to synthesize poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] when fed with the precursor carbon 1,4-butanediol using a two-stage cultivation process. When 1% (w/v) of 1,4-butanediol was used, 31 wt.% of P(3HB-co-4HB) copolymer with 41 mol.% of 4HB molar fraction was produced. Both the PHA content and 4HB composition of the copolymer increased as the concentration of 1,4-butanediol increased but the cell biomass did not show any significant changes. However, the 4HB fraction could be further increased using a combination of γ-butyrolactone and 1,4-butanediol. As high as 84 mol.% of 4HB composition was achieved with a combination of 0.35% (w/v) 1,4-butanediol and 1.4% (w/v) γ-butyrolactone. Nevertheless, it was found that Cupriavidus sp. USMAA2-4 cells were inhibited by high concentration of γ-butyrolactone. P(3HB-co-4HB) copolymer was also successfully synthesized using a simplified aerated tank.     

Biosynthesis and characterization of poly(3-hydroxydodecanoate) by β-oxidation inhibited mutant of Pseudomonas entomophila L48

A medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) producer Pseudomonas entomophila L48 was investigated for microbial production of 3-hydroxydodecanote homopolymer. Pseudomonas entomophila L48 was found to produce MCL PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxydodecanoate (3HDD) from related carbon sources fatty acids. In this study, some of the genes encoding key enzymes in β-oxidation cycle of P. entomophila such as 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase were deleted to study the relationship between β-oxidation and PHA synthesis in P. entomophila. Among the mutants constructed, P. entomophila LAC26 accumulated over 90 wt % PHA consisting of 99 mol % 3HDD. A fed-batch fermentation process carried out in a 6 L automatic fermentor produced 7.3 g L(-1) PHA consisting of over 97 mol % 3HDD fraction. Properties of MCL PHA were significantly improved along with increasing 3HDD contents. P(2.1 mol % 3HD-co-97.9 mol % 3HDD) produced by P. entomophila LAC25 had the widest temperature range between T(g) and T(m), which were -49.3 and 82.4 °C, respectively, in all MCL PHA reported so far. The new type of PHA also represented high crystallinity caused by side-chain crystallization compared with short side chain PHA. For the first time, P(3HDD) homopolymers were obtained.     

Application of random mutagenesis to enhance the production of polyhydroxyalkanoates by Cupriavidus necator H16 on waste frying oil

Using random chemical mutagenesis we obtained the mutant of Cupriavidus necator H16 which was capable of improved (about 35 %) production of poly(3-hydroxybuytrate) (PHB) compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably, due to the activation of these enzymes, we also observed an increase of NADPH/NADP⁺ ratio. It is likely that as a side effect of the increase of NADPH/NADP⁺ ratio the activity of PHB biosynthetic pathway was enhanced, which supported the accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for the utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of the utilization of waste frying oils and the production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for the development of innovative fermentation strategies for the production of PHA and also it might help to define novel targets for the genetic manipulations of PHA producing bacteria.     

Improving culture conditions for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam

The production of polyhydroxyalkanoate (PHA) by Bacillus sp. ND153, a bacterium strain isolated from a mangrove forest in Vietnam, was studied. Bacillus sp. ND153 was grown on HM-1 medium with different carbon sources (e.g. glucose, sucrose, maltose, dextrin, and starch). Glucose was found to be the most suitable carbon source for PHA accumulation, whereas starch and dextrin favored cell growth over PHA accumulation. Optimization of the culture medium for PHA production was investigated by applying factorial design, and a maximum PHA content of 79 % (w/w) was obtained with low concentrations of NH₄Cl and MgSO₄ and a high concentration of KH₂PO₄ in the medium. Propionate was used as the precursor for the production of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and the amount of 3-hydroxyvalerate (3HV) in the polymer showed an increasing linear trend with the increase in propionate concentration from 0.2 g l^?1 to 1.0 g l^?1. Thus, the production of PHBV by Bacillus sp. ND153, with 3HV fraction ranging from 1 mol% to 30 mol%, was noted to be high, and the characteristics of fast cell growth and accumulation of PHA exhibited by Bacillus sp. ND153 make it a promising choice for biopolyester production.