BackgroundOur study aimed to search for novel bacteria capable of producing polyhydroxyalkanoates (PHAs) using crude glycerol residue obtained from biodiesel production in which used cooking oils were the substrates.ResultsNewly isolated bacteria from soils in Thailand were screened for the efficient production of PHAs from crude glycerol. The bacterial strains were cultivated on glucose, refined glycerol, crude glycerol, or various cooking oils (canola oil, palm oil, soybean oil, sunflower oil, corn oil, grape seed oil, olive oil, rice bran oil, camellia seed oil) for growth and PHA production. The effects of the total organic carbon (TOC) concentration and the mole ratio of carbon to nitrogen were investigated in batch cultivation. 1H NMR, two dimensional-1H-correlation spectroscopy (2D-1H-COSY) and 13C NMR analyses confirmed four bacterial strains were capable of producing medium-chain-length PHAs (mcl-PHAs), consisting of 3-hydroxyoctanoate (3HO) and 3-hydroxy-5-cis-dodecanoate (3H5DD), from crude glycerol. On the basis of phenotypic features and genotypic investigations, the bacterial strains were assigned as: ASC1, Acinetobacter genus (94.9 % similarity); ASC2, Pseudomonas genus (99.2 % similarity); ASC3, Enterobacter genus (99.2 % similarity); ASC4, Bacillus genus (98.4 % similarity). The highest amount of mcl-PHAs, 17.5 ± 0.8 g/L (content 61.8 ± 3.3 % wt), with 3HO (14.7 ± 2.2 mol %), 3H5DD (85.3 ± 2.2 mol %), and a total biomass of 32.3 ± 0.3 g/L, was obtained from Pseudomonas sp. ASC2 in batch cultivation after 36 h. The mcl-PHAs recovered had a number-average molecular weight (MN) of 3.6 × 104 Da. Homopolymeric 3H5DD was obtained when the cultivation time was prolonged to 96 h.ConclusionsNovel PHA-producing strains were isolated and identified. These bacterial strains are able to produce mcl-PHAs from crude glycerol. The mcl-PHAs produced contained a high percentage of 3H5DD, which suggests their future application as softeners mixed with other biomaterials. The unsaturated side chain of 3H5DD monomers containing double bounds offers additional potential for improving the properties of the mcl-PHAs or extending their applications to the food industry.

Background: The present study attempted to increase PHB production by improving the functional expression of the PhaC gene using various types of promoters, and the effects on PhaC activity in terms of PHB productivity, yield coefficient (YP/S) and molecular weights were investigated.Results: Here, the PHB biosynthesis operon of Cupriavidus necator A-04, isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO and pUC19 (constitutive expression) and transformed into E. coli JM109. To alter the expression of phaCAB biosynthesis genes, we optimized parameters in flask experiments to obtain high expression of soluble PhaCA−04 protein with high YP/S and PHB productivity. pColdTF-phaCABA−04-expressing E. coli produced 2.5 ± 0.1 g/L (90.6±4.3%) PHB in 24 h, similar to pColdI-phaCABA−04-expressing E. coli. The amounts of phaC protein and PHB produced from pColdTF-phaCABA−04 and pColdI-phaCABA−04 were significantly higher than those from other promoters. Cultivation in a 5-L fermenter led to PHB production of 7.9±0.7 g/L with 90.0±2.3% PHB content in the cell dry mass (DCM), a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.26 g PHB/(L⋅h) using pColdTF-phaCABA−04. The PHB from pColdTF-phaCABA−04 had MW 5.79 × 105 Da, MN 1.86 × 105 Da and PDI 3.11 and the film exhibited high transparency, Young’s modulus and tensile strength, possibly due to the TF chaperones. Interestingly, when pColdI-phaCABA−04-expressing E. coli was used to produce PHB from crude glycerol and compared with constitutive pUC19-nativeP-phaCABA−04-expressing E. coli, the amounts of PHB were similar, but MW 1.1 × 106 Da, MN 2.6 × 105 Da and PDI 4.1 were obtained from constitutive pUC19-nativeP-phaCABA−04-expressing E. coli, indicating that slow and low expression could prolong and maintain phaC polymerization activity.Conclusions: The cspA promoter in a cold-inducible vector can improve PhaCA−04 expression levels, and TF chaperones show obvious effects on enhancing PhaCA−04 solubility. The high level of phaCA−04 resulted in a high amount of PHB, but the chain termination reaction of PhaC polymerization occurred faster than that with the retarded and low expression of phaCA−04 by the constitutive promoter pUC19, which in turn resulted in a low amount of PHB with a high molecular weight.

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaCA–04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA–04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA–04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA–04 protein with high YP/S and PHB productivity. Soluble phaCA–04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA–04 from pColdTF-phaCABA–04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCABA–04 enhanced soluble protein formation to approximately 3.09−4.1 times higher than that from pColdI-phaCABA–04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCABA–04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Background: The present study attempted to increase polyhydroxybutyrate (PHB) production by improving the functional expression of the PhaC gene using various types of promoters, and the effects on PhaC activity in terms of PHB productivity, yield coefficient (YP/S) and molecular weights were investigated.Results: Here, the PHB biosynthesis operon of Cupriavidus necator A-04, isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO and pUC19 (native promoter) and transformed into E. coli JM109. To alter the expression of phaCAB biosynthesis genes, we optimized parameters in flask experiments to obtain high expression of soluble PhaCA-04 protein with high YP/S and PHB productivity. pColdTF-phaCABA-04-expressing E. coli produced 2.5±0.1 g/L (90.6±4.3%) PHB in 24 h, similar to pColdI-phaCABA-04-expressing E. coli. The amounts of phaC protein and PHB produced from pColdTF-phaCABA-04 and pColdI-phaCABA-04 were significantly higher than those from other promoters. Cultivation in a 5-L fermenter led to PHB production of 7.9±0.7 g/L with 90.0±2.3% PHB content in the cell dry mass (CDM), a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L×h) using pColdTF-phaCABA-04. The PHB from pColdTF-phaCABA-04 had Mw 5.79×105 Da, Mn 1.86×105 Da and PDI 3.11 and the film exhibited high transparency, Young’s modulus and tensile strength, possibly due to the trigger factor (TF) chaperones. Interestingly, when pColdI-phaCABA-04-expressing E. coli was used to produce PHB from crude glycerol and compared with pUC19-nativeP-phaCABA-04-expressing E. coli, the amounts of PHB were similar, but Mw 1.1×106 Da, Mn 2.6×105 Da and PDI 4.1 were obtained from pUC19-nativeP-phaCABA-04-expressing E. coli, indicating that slow and low expression could prolong and maintain phaC polymerization activity.Conclusions: This is the first report to demonstrate that the cspA promoter in a cold-inducible vector can improve PhaCA-04 expression levels, and TF chaperones show obvious effects on enhancing PhaCA-04 solubility. The high level of PhaCA-04 resulted in a high PHB amount, but the chain termination reaction of PhaC polymerization occurred faster than that with the slowed and low expression of phaCA-04 by the native promoter pUC19, which resulted in a low amount of high-molecular-weight PHB produced from crude glycerol.