Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans.

The phaZ gene of Pseudomonas resinovorans codes for a poly(hydroxyalkanoates) (PHA) depolymerase. Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZ(FOAC001) (239 amino-acid residues) and PhaZ(FOAC002) (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZ(FOAC001) might still retain a partial PHA depolymerization activity, while PhaZ(FOAC002) is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source. The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of beta-hydroxyoctanoate and higher beta-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up. These phaZ mutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.

Poly(ethylene glycol)-mediated molar mass control of short-chain- and medium-chain-length poly(hydroxyalkanoates) from Pseudomonas oleovorans.

Three strains of Pseudomonas oleovorans, a well known poly(hydroxyalkanoate) (PHA) producer, were tested for the ability to control PHA molar mass and end group structure by addition of poly(ethylene glycol) (PEG) to the fermentation medium. Each strain of P. oleovorans - NRRL B-14682 (B-14682), NRRL B-14683 (B-14683), and NRRL B-778 (B-778) - synthesized a different type of PHA from oleic acid when cultured under identical growth conditions. Strain B-14682 produced poly(3-hydroxybutyrate) (PHB), while B-14683 synthesized a medium-chain-length PHA ( mcl-PHA) with a repeat unit composition ranging from C4 to C14 and some mono-unsaturation in the C14 alkyl side chains. Strain B-778 synthesized a mixture of PHB (95 mol%) and mcl-PHA (5 mol%). The addition of 0.5% (v/v) PEG (M(n) =200 g/mol, PEG-200) to the fermentation broth of strains B-14682 and B-778 resulted in chain termination through esterification at the carboxyl terminus of the PHB with PEG chain segments, thus reducing the molar mass by 54% and 23%, respectively. The molar mass of the mcl-PHA produced by strains B-14683 and B-778 also showed a 34% and 47% reduction in the presence of PEG-200, respectively, but no evidence of esterification was present. PEG-400 (M(n) =400 g/mol) had a reduced effect on PHA molar mass. In fact, the molar masses of the mcl-PHA derived from strain B-14683 and both the PHB and mcl-PHA from B-778 were unchanged by PEG-400. In contrast, the PHB produced by B-14682 showed a 35% reduction in molar mass in the presence of PEG-400.

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans.

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179 degrees C, enthalpies of fusion (AHf) of 20 cal/g and glasstransition temperatures (Tg) of 3-4 degrees C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the Tm values were 161 degrees C, deltaHf values were 7 cal/g and Tg values were - 3 degrees C.

Glucose/lipid mixed substrates as a means of controlling the properties of medium chain length poly(hydroxyalkanoates).

Glucose-triacylglycerol (TAG) mixed substrates were used to modulate the physical and mechanical properties of medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs). Pseudomonas resinovorans NRRL B-2649 grew and produced mcl-PHAs on glucose and TAGs (coconut oil, C; soybean oil, S) after 24 h in a shake flask culture. However, with the exception of coconut oil, maximum cell productivity was not reached in any of the cultures until 72 h post-inoculation. Here, 50:50 mixtures of glucose and coconut oil (glc/C) or glucose and soybean oil (glc/S) resulted in intermediate cell productivities with a maximum of 57% and 48% of the CDW at 72 h, respectively. In addition, mixed substrates resulted in mcl-PHAs with compositions that varied slightly over time. PHA-glc/C and PHA-glc/S were composed of 7 mol % and 8 mol % 3-hydroxydodecenoic acid (C(12:1)), respectively at 72 h. These concentrations were intermediate to the C(12:1) concentration of PHA-glc and respective PHA-TAG. Also, significant amounts of 3-hydroxytetradecanoic acid (C(14:0)), 3-hydroxytetradecenoic acid (C(14:1)), and 3-hydroxytetradecadienoic acid (C(14:2)) were present in PHA-glc/C and PHA-glc/S, which were derived from the respective TAG, as glucose resulted in almost no C(14:)(X) monomers. The molar masses of each of the polymers remained relatively constant between 24 and 96 h. At 72 h, the number-average molar masses (M(n)) of PHA-glc/C and PHA-glc/S were 178,000 and 163,000 g/mol, respectively, which were also intermediate to the M(n) of PHA-glc (225,000 g/mol) and the respective PHA-TAG (PHA-C = 153,000 g/mol; PHA-S = 75,000 g/mol). These physical differences caused variations in the mechanical properties of mcl-PHA films, thus providing a new and effective method of modifying their properties.

Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas.

Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l. The recombinant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.

Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing.

Medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers derived from linseed oil (PHA-L) have a relatively small molar mass and contain a high concentration of unsaturated side-chains. As such, these polymers are amorphous and take on the consistency of a viscous liquid at room temperature. In order to increase the application potential of this material, the side-chain olefinic groups of PHA-L were converted to epoxy derivatives (PHA-LE) using m-chloroperoxybenzoic acid (m-CPBA). Epoxidation resulted in a 37% conversion of olefinic to epoxy groups. The epoxy groups enhanced the PHA-LE film susceptibility to crosslinking upon exposure to air. PHA-LE films began to crosslink and stiffen in less than 25 days, whereas PHA-L films began to crosslink between days 50 and 75. The PHA-LE films showed an increase in tensile strength (TS, from 4.8 to 20.7 MPa) and Young's modulus (YM, from 12.9 to 510.6 MPa) between 25 and 100 days. In contrast, PHA-L had a TS of 25.0 MPa and YM of 767.8 MPa after 100 days. Epoxidation helped induce crosslink formation; however, aging for 100 days ultimately resulted in crosslinked films from both PHA-L and PHA-LE with higher strength and durability than the original materials.

Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction.

A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding for type II polyhydroxyalkanoate (PHA) synthases. The primer-pair, I-179L and I-179R, was based on the highly conserved sequences found in the coding regions of Pseudomonas phaC1 and phaC2 genes. Purified genomic DNA or lysate of colony suspension can serve equally well as the target sample for the PCR, thus affording a simple and rapid screening of phaC1/C2-containing microorganisms. Positive samples yield a specific 540-bp PCR product representing partial coding sequences of the phaC1/C2 genes. Using the PCR method, P. corrugata 388 was identified for the first time as a medium-chain-length (mcl)-PHA producer. Electron microscopic study and PHA isolation confirmed the production of mcl-PHA in P. corrugata 388. The mcl-PHA of this organism has a higher molecular weight than that of similar polymers produced by other pseudomonads.

Medium-chain-length poly(beta-hydroxyalkanoate) synthesis from triacylglycerols by Pseudomonas saccharophila.

Pseudomonas saccharophila NRRL B-628 is capable of utilizing agricultural lipids for growth. The organism exhibited good growth with triacylglycerol substrates that contained saturated fatty acyl moieties such as coconut oil (CO; C10-12 fatty acids) and tallow (T; C16-18 fatty acids). Electron micrographs of the triacylglycerol-grown cells showed the presence of intracellular granules indicative of poly(beta-hydroxyalkanoate) (PHA) production. Cells grown in a 250-ml CO-containing medium produced ca. 0.2 g of medium-chain-length (mcl)-PHA. Gas chromatographic analysis showed that beta-hydroxyoctanoic acid (30%), beta-hydroxydecanoic acid (40%), and beta-hydroxydodecanoic acid (16%) were the major monomer repeat-units of the CO-derived polymer. The estimated mean molecular mass of the CO-derived mcl-PHA as determined by gel permeation chromatography was 13.1 x 10(4) g/mol with a polydispersity of 3.16.

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction.

The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minusorigin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria.

Molecular characterization of the erythromycin resistance plasmid pPV142 from Staphylococcus simulans.

The 2.5-kb erythromycin resistance (EmR) plasmid pPV142 of Staphylococcus simulans 13044 was isolated and characterized. Sequence analysis identified ORF1 and ORF2 encoding a 158-residue replication protein (Rep142) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV142 shared homology with the EmR plasmid pPV141 (2.4 kb) of S. chromogenes 3688 and other EmR plasmids known to exist in staphylococci and bacilli. Based on the presence of a 61-bp repeat upstream of the ermM gene, pPV142 is apparently a unique member of the pSN2 family of EmR plasmid able to express erythromycin resistance constitutively.

Structural and functional properties of the hsp16.4-bearing plasmid pER341 in Streptococcus thermophilus.

The plasmid pER341 (2798 bp) of Streptococcus thermophilus ST134 was sequenced and its open reading frame (ORF) regions were characterized. Analysis of nucleotide sequences showed the putative translation product of ORF1 (rep) sharing a high level of homology with replication proteins of several small plasmids present in lactic acid bacteria and staphylococci. This and homology of regions of plus-strand (ORI) and minus-strand (ssoA) origin of replication with pC194-class plasmids indicated that pER341 replicates by the rolling-circle mechanism. ORF2 corresponded to a putative hsp gene that apparently encodes Hsp16.4, a 142-amino-acid heat stress protein. Hsp16.4 shared significant identity with other small, 18-kDa-class heat stress proteins from prokaryotic and eukaryotic sources. Hsp16.4 is apparently the first plasmidborne low-molecular-weight heat stress protein reported in dairy fermentation bacteria with a potential role in temperature-regulated functions in S. thermophilus.

STP2201, a chromosomal promoter sequence of Streptococcus thermophilus.

Analysis of the structural and functional properties of chromosomal DNA fragments of Streptococcus thermophilus ST128 delineated the promoter sequence STP2201 and identified its -35, -10 and Shine-Dalgarno regions. STP2201 was used in cloning vectors derived from small resident plasmids pER8 (2094 bp) and pER371 (2672 bp) of S. thermophilus strains to facilitate expression of a Streptomyces sp. marker gene (cholesterol oxidase) in lactic acid bacteria. Cell extracts of ST128 transformants converted up to 75% of cholesterol into 4-cholesten-3-one during 8 h of incubation.

Sth132I, a novel class-IIS restriction endonuclease of Streptococcus thermophilus ST132.

The Sth132I restriction endonuclease (R.Sth132I) was detected in Streptococcus thermophilus ST132 and purified to near homogeneity by heparin Sepharose CL-6B affinity chromatography. Fragments from Sth132I digestion of plasmid DNA were subcloned into pUC19 in Escherichia coli DH5alpha and sequenced. Sequence analysis of inserts and their ligation junction sites revealed that Sth132I is a novel class-IIS restriction endonuclease, which recognizes the non-palindromic sequence 5'-CCCG(N)4-3', 3'-GGGC(N) 8-5'.

Isolation and characterization of transcription signal sequences from Streptococcus thermophilus.

Streptococcus thermophilus (ST) chromosomal DNA fragments generated by partial Sau3A digestion were cloned into the unique BamHI site upstream from the promoterless chloramphenicol acetyltransferase (cat) gene of the Escherichia coli (EC)promoter-probe vector pKK520-3. Recombinant plasmids containing ST sequences with transcription-activation activity were isolated from chloramphenicol-resistant (CmR) EC transformants. A promoterless Streptomyces antibioticus melanin biosynthesis operon (melC) was inserted immediately downstream from the ST sequence to identify DNA with strong promoter activity. Several ST transcription-activation sequences, termed STPs, were isolated and subcloned, and their nucleic acid sequences determined. The -10 and -35 consensus sequences were identified in these putative ST promoters. Detailed analysis of STP3306 sequence data revealed two partial open reading frames (ORFs) with high degrees of homology to prokaryotic GTP-binding protein and DNA repair enzyme, thus providing valuable information for further study on DNA maintenance in this important lactic acid bacterium.

Molecular properties of the erythromycin resistance plasmid pPV141 from Staphylococcus chromogenes.

The 2.3-kb erythromycin resistance (EmR) plasmid pPV141 of Staphylococcus chromogenes 3688 was isolated and characterized. Nucleotide sequence analysis identified ORF1 and ORF2 separated by a 445-bp spacing, encoding a 158-residue replication protein (Rep141) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV141 shared homology with other known EmR plasmids. Based on sequence analysis, pPV141 was classified as a unique member of the pSN2 family of EmR plasmids.

Expression of Streptomyces melC and choA genes by a cloned Streptococcus thermophilus promoter STP2201.

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected in Escherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone STP2201 as a strong promoter in E. coli. Subcloning of a STP2201-melC DNA fragment into the pMEU-series S. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel+ phenotype to E. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase pro-anti-tyrosinase antiserum in S. thermophilus. Substituting melC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to an E. coli transformant at a level of (1.06 +/- 0.15) x 10(-7) units mg-1 protein. Introduction of this plasmid into S. thermophilus by electrotransformation yielded ChoA+ transformant that produced the enzyme at about 25% of the level found in E. coli.

Expression of cho and melC operons by a Streptococcus thermophilus synthetic promoter in Escherichia coli.

A 63-base-pair synthetic promoter, sP1, was synthesized on the basis of the nucleotide sequence of a putative Streptococcus thermophilus promoter. When inserted upstream from the Streptomyces cho operon in a recombinant plasmid, pUCO195P-36, sP1 activated the expression of the cho genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants. The sP1-driven cholesterol oxidase production in pUCO195P-36-transformed cells was estimated to be 40% of that produced by P(lac)-mediated cho expression in a pUCO193-containing host. The recombinant pUCO195P-36 appeared to be segregationally less stable in E. coli DH5 alpha than in HB101. Its non-expressing counterpart, pUCO195P-1, was stable in both E. coli strains. The activity of sP1 was further demonstrated in E. coli by the expression of a Streptomyces melC operon. When placed upstream from the test operon in the pMCU22aPa construct, sP1 activated the melC expression as shown by the production of tyrosinase at (3.0 +/- 0.3) x 10(-3) U/mg and (16.0 +/- 1.0) x 10(-3) U/mg protein equivalent of cell extract in the absence and presence of isopropyl beta-D-thiogalactopyranoside, respectively. The presence of a counter-oriented P(lac) at the 3' end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.

Native promoter-plasmid vector system for heterologous cholesterol oxidase synthesis in Streptococcus thermophilus.

The cholesterol oxidase gene (choA) of a streptomycete was used as a model for studying heterologous gene expression in Streptococcus thermophilus, an essential bacterium in dairy food fermentations. The vectors pER82 and pER82P were developed from the 2.2-kb indigenous plasmid (pER8) of S. thermophilus ST108, and sP1, a 51-bp synthetic promoter patterned after a chromosomal sequence of S. thermophilus. The presence of sP1 promoter in pER82PbCOb with the choA insert aligned with the cat gene was essential for the intracellular production of cholesterol oxidase. The pER82PbCOb was apparently stable in S. thermophilus with no detectable evidence of deletion mutational events.

Shuttle vectors developed from Streptococcus thermophilus native plasmid.

pER8 (2.2 kb), a native plasmid of Streptococcus thermophilus ST108, was used to develop pMEU-series shuttle vectors. In addition to the replication function of the pER8, these vectors contain origin of replication and beta-lactamase gene (bla) of Escherichia coli vector pUC18/19, the cat gene of pC194, and the pPV141-borne erm determinant of Staphylococcus hyicus ssp. chromogenes 3688. pMEU5a, pMEU5b, pMEU6a, and pMEU6b (all 5.7 kb in size) contain bla and erm markers, are capable of transforming E. coli and S. thermophilus at frequencies in the order of 10(5)-10(6) and 10(3) colony forming units (CFU)/microgram DNA, respectively, and are highly stable in the two host systems. pMEU9 and pMEU10 (both 6.9 kb) contain the cat marker in addition to the DNA elements found in pMEU5-6. These plasmids are also highly effective in transforming E. coli (at ca. 6 x 10(5) CFU/microgram DNA) and S. thermophilus (ca. 10(3) CFU/microgram DNA). Although expression of the resistance markers is not completely consistent, pMEU9 and pMEU10 remain important shuttle vectors for clonal selection by an insertional inactivation method.

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli.

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.