Title: Post-Synthetic Enzymatic and Chemical Modifications for Novel Sustainable Polyesters El-Malek FA, Steinbuchel A Ref: Front Bioeng Biotechnol, 9:817023, 2021 : PubMed
Because of their biodegradability, compostability, compatibility and flexible structures, biodegradable polymers such as polyhydroxyalkanoates (PHA) are an important class of biopolymers with various industrial and biological uses. PHAs are thermoplastic polyesters with a limited processability due to their low heat resistance. Furthermore, due to their high crystallinity, some PHAs are stiff and brittle. These features result sometimes in very poor mechanical characteristics with low extension at break values which limit the application range of some natural PHAs. Several in vivo approaches for PHA copolymer modifications range from polymer production to enhance PHA-based material performance after synthesis. The methods for enzymatic and chemical polymer modifications are aiming at modifying the structures of the polyesters and thereby their characteristics while retaining the biodegradability. This survey illustrates the efficient use of enzymes and chemicals in post-synthetic PHA modifications, offering insights on these green techniques for modifying and improving polymer performance. Important studies in this sector will be reviewed, as well as chances and obstacles for their stability and hyper-production.
Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host.
Title: Insights into the microbial degradation of rubber and gutta-percha by analysis of the complete genome of Nocardia nova SH22a Luo Q, Hiessl S, Poehlein A, Daniel R, Steinbuchel A Ref: Applied Environmental Microbiology, 80:3895, 2014 : PubMed
The complete genome sequence of Nocardia nova SH22a was determined in light of the remarkable ability of rubber and gutta-percha (GP) degradation of this strain. The genome consists of a circular chromosome of 8,348,532 bp with a G+C content of 67.77% and 7,583 predicted protein-encoding genes. Functions were assigned to 72.45% of the coding sequences. Among them, a large number of genes probably involved in the metabolism of xenobiotics and hardly degradable compounds, as well as genes that participate in the synthesis of polyketide- and/or nonribosomal peptide-type secondary metabolites, were detected. Based on in silico analyses and experimental studies, such as transposon mutagenesis and directed gene deletion studies, the pathways of rubber and GP degradation were proposed and the relationship between both pathways was unraveled. The genes involved include, inter alia, genes participating in cell envelope synthesis (long-chain-fatty-acid-AMP ligase and arabinofuranosyltransferase), beta-oxidation (alpha-methylacyl-coenzyme A [alpha-methylacyl-CoA] racemase), propionate catabolism (acyl-CoA carboxylase), gluconeogenesis (phosphoenolpyruvate carboxykinase), and transmembrane substrate uptake (Mce [mammalian cell entry] transporter). This study not only improves our insights into the mechanism of microbial degradation of rubber and GP but also expands our knowledge of the genus Nocardia regarding metabolic diversity.
Title: Unravelling the complete genome sequence of Advenella mimigardefordensis strain DPN7T and novel insights in the catabolism of the xenobiotic polythioester precursor 3,3'-dithiodipropionate Wubbeler JH, Hiessl S, Schuldes J, Thurmer A, Daniel R, Steinbuchel A Ref: Microbiology, 160:1401, 2014 : PubMed
Advenella mimigardefordensis strain DPN7(T) is a remarkable betaproteobacterium because of its extraordinary ability to use the synthetic disulfide 3,3'-dithiodipropionic acid (DTDP) as the sole carbon source and electron donor for aerobic growth. One application of DTDP is as a precursor substrate for biotechnically synthesized polythioesters (PTEs), which are interesting non-degradable biopolymers applicable for plastics materials. Metabolic engineering for optimization of PTE production requires an understanding of DTDP conversion. The genome of A. mimigardefordensis strain DPN7(T) was sequenced and annotated. The circular chromosome was found to be composed of 4,740,516 bp and 4112 predicted ORFs, whereas the circular plasmid consisted of 23,610 bp and 24 predicted ORFs. The genes participating in DTDP catabolism had been characterized in detail previously, but knowing the complete genome sequence and with support of Tn5: :mob-induced mutants, putatively involved transporter proteins and a transcriptional regulator were also identified. Most probably, DTDP is transported into the cell by a specific tripartite tricarboxylate transport system and is then cleaved by the disulfide reductase LpdA, sulfoxygenated by the 3-mercaptopropionate dioxygenase Mdo, activated by the CoA ligase SucCD and desulfinated by the acyl-CoA dehydrogenase-like desulfinase AcdA. Regulation of this pathway is presumably performed by a transcriptional regulator of the xenobiotic response element family. The excessive sulfate that is inevitably produced is secreted by the cells by a unique sulfate exporter of the CPA (cation : proton antiporter) superfamily.
Title: Acyltransferases in bacteria Rottig A, Steinbuchel A Ref: Microbiol Mol Biol Rev, 77:277, 2013 : PubMed
Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue.
Title: Involvement of two latex-clearing proteins during rubber degradation and insights into the subsequent degradation pathway revealed by the genome sequence of Gordonia polyisoprenivorans strain VH2 Hiessl S, Schuldes J, Thurmer A, Halbsguth T, Broker D, Angelov A, Liebl W, Daniel R, Steinbuchel A Ref: Applied Environmental Microbiology, 78:2874, 2012 : PubMed
The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search.
Title: Bacterial acyltransferases as an alternative for lipase-catalyzed acylation for the production of oleochemicals and fuels Stoveken T, Steinbuchel A Ref: Angew Chem Int Ed Engl, 47:3688, 2008 : PubMed
Bacterial acyltransferases are a new class of enzymes, and the first member was identified as WS/DGAT in Acinetobacter baylyi ADP1. Their unspecificity have been used in several biotechnological applications for lipid modification, a field that has been dominated by the use of lipases. Examples are the biosynthesis of jojoba-like wax esters and fatty-acid ethyl esters. In addition, these enzymes are also capable of synthesizing acylthioesters. Acyloxoesters and acylthioesters can thus be produced in vivo by whole-cell fermentations rather than in vitro in an enzyme reactor. In this Minireview, we focus on the biotechnological utilization of acyltransferases for the production of modified lipids from renewable resources.
The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.
Title: Characterization of the 101-kilobase-pair megaplasmid pKB1, isolated from the rubber-degrading bacterium Gordonia westfalica Kb1 Broker D, Arenskotter M, Legatzki A, Nies DH, Steinbuchel A Ref: Journal of Bacteriology, 186:212, 2004 : PubMed
The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1.
Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20.0, 20.2, 19.6 and 20.2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.
Title: Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases Phac1 and PhaC2. Hein S, Paletta JRJ, Steinbuchel A Ref: Applied Microbiology & Biotechnology, 58:229, 2002 : PubMed
Title: Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model Rehm BH, Antonio RV, Spiekermann P, Amara AA, Steinbuchel A Ref: Biochimica & Biophysica Acta, 1594:178, 2002 : PubMed
A threading model of the Ralstonia eutropha polyhydroxyalkanoate (PHA) synthase was developed based on the homology to the Burkholderia glumae lipase, whose structure has been resolved by X-ray analysis. The lid-like structure in the model was discussed. In this study, various R. eutropha PHA synthase mutants were generated employing random as well as site-specific mutagenesis. Four permissive mutants (double and triple mutations) were obtained from single gene shuffling, which showed reduced activity and whose mutation sites mapped at variable surface-exposed positions. Six site-specific mutations were generated in order to identify amino acid residues which might be involved in substrate specificity. Replacement of residues T323 (I/S) and C438 (G), respectively, which are located in the core structure of the PHA synthase model, abolished PHA synthase activity. Replacement of the two amino acid residues Y445 (F) and L446 (K), respectively, which are located at the surface of the protein model and adjacent to W425, resulted in reduced activity without changing substrate specificity and indicating a functional role of these residues. The E267K mutant exhibited only slightly reduced activity with a surface-exposed mutation site. Four site-specific deletions were generated to evaluate the role of the C-terminus and variant amino acid sequence regions, which link highly conserved regions. Deleted regions were D281-D290, A372-C382, E578-A589 and V585-A589 and the respective PHA synthases showed no detectable activity, indicating an essential role of the variable C-terminus and the linking regions between conserved blocks 2 and 3 as well as 3 and 4. Moreover, the N-terminal part of the class II PHA synthase (PhaC(Pa)) from Pseudomonas aeruginosa and the C-terminal part of the class I PHA synthase (PhaC(Re)) from R. eutropha were fused, respectively, resulting in three fusion proteins with no detectable in vivo activity. However, the fusion protein F1 (PhaC(Pa)-1-265-PhaC(Re)-289-589) showed 13% of wild type in vitro activity with the fusion point located at a surface-exposed loop region.
Title: Multiple evidence for widespread and general occurrence of type-III PHA synthases in cyanobacteria and molecular characterization of the PHA synthases from two thermophilic cyanobacteria: Chlorogloeopsis fritschii PCC 6912 and Synechococcus sp. strain MA19. Hai T, Hein S, Steinbuchel A Ref: Microbiology, 147:3047, 2001 : PubMed
Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for beta-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.
A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase.
Title: The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources Hoffmann N, Steinbuchel A, Rehm BH Ref: FEMS Microbiology Letters, 184:253, 2000 : PubMed
We recently identified the phaG(Pp) gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaG(Pa)) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaG(Pp)-negative mutant P. putida PhaG(N)-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaG(Pa) knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaG(Pa) mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty acid de novo synthesis towards PHA(MCL) synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.
Title: Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids Rodrigues MF, Valentin HE, Berger PA, Tran M, Asrar J, Gruys KJ, Steinbuchel A Ref: Applied Microbiology & Biotechnology, 53:453, 2000 : PubMed
Burkholderia sp. accumulates polyhydrox-yalkanoates (PHAs) containing 3-hydroxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media under limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source. Solvent fractionation and NMR spectroscopic characterization of these polyesters revealed the simultaneous accumulation of two homopolyesters rather than a co-polyester with random sequence distribution of the monomers [Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbuchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389-7395]. To understand the genetic requirements for such unusual polyester accumulation, we probed total genomic DNA from Burkholderia sp. by Southern hybridization experiments using phaC-specific probes. These experiments indicated the presence of more than one PHA synthase gene within the genome of Burkholderia sp. However, when total genomic DNA from Burkholderia sp. was used to complement a PHA-negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA synthase gene was obtained resembling the R. eutropha type of PHA synthases, based on amino acid sequence similarity. In addition to the PHA synthase gene, based on high sequence homology, genes encoding a beta-ketothiolase and acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene. The arrangement of the three genes is quite similar to the R. eutropha poly-beta-hydroxybutyrate biosynthesis operon.
Title: A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. The PHAG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme a transferase Rehm BH, Kruger N, Steinbuchel A Ref: Journal of Biological Chemistry, 273:24044, 1998 : PubMed
To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressing phaG gene under lac promoter control, was obtained. Heterologous expression of phaG in Pseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.
Title: Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates) Jaeger KE, Steinbuchel A, Jendrossek D Ref: Applied Environmental Microbiology, 61:3113, 1995 : PubMed
The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.
Title: Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophus Valentin HE, Zwingmann G, Schonebaum A, Steinbuchel A Ref: European Journal of Biochemistry, 227:43, 1995 : PubMed
Various aerobic Gram-negative bacteria have been examined for their ability to use 4-hydroxybutyrate and 1,4-butanediol as carbon source for growth. Alcaligenes eutrophus strains H16, HF39, PHB-4 and Pseudomonas denitrificans 'Morris' were not able to grow with 1,4-butanediol or 4-hydroxybutyrate. From A. eutrophus HF39 spontaneous primary mutants (e.g. SK4040) were isolated which grew on 4-hydroxybutyrate with doubling times of approximately 3 h. Tn5::mob mutagenesis of mutant SK4040 led to the isolation of two phenotypically different classes of secondary mutants which were affected in the utilization of 4-hydroxybutyrate. Mutants exhibiting the phenotype 4-hydroxybutyrate-negative did not grow with 4-hydroxybutyrate, and mutants exhibiting the phenotype 4-hydroxybutyrate-leaky grew at a significantly lower rate with 4-hydroxybutyrate. Hybridization experiments led to the identification of a 10-kbp genomic EcoRI fragment of A. eutrophus SK4040, which was altered in mutants with the phenotype 4-hydroxybutyrate-negative, and of two 1-kbp and 4.5-kbp genomic EcoRI fragments, which were altered in mutants with the phenotype 4-hydroxybutyrate-leaky. This 10-kbp EcoRI fragment was cloned from A. eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A. eutrophus H16 conferred the ability to grow with 4-hydroxybutyrate to the wild type. DNA-sequence analysis of this fragment, enzymic analysis of the wild type and of mutants of A. eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4-hydroxybutyrate dehydrogenase which was affected by transposon mutagenesis in five of six available 4-hydroxybutyrate-negative mutants. Enzymic studies also provided evidence for the presence of an active succinate-semialdehyde dehydrogenase in 4-hydroxybutyrate-grown cells. This indicated that degradation of 4-hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycle. NMR studies of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumulated from 4-hydroxy [1-13C]butyrate or 4-hydroxy[2-13C]butyrate as substrate gave no evidence for a direct conversion of 4-hydroxybutyrate into 3-hydroxybutyrate and therefore supported the results of enzymic analysis.
Title: Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway. Huang M, Oppermann FB, Steinbuchel A Ref: FEMS Microbiology Letters, 124:141, 1994 : PubMed
The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), apoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA, acoB, and acoC for E1 alpha (M(r) 34639), E1 beta (M(r) 37268), and E2 (M(r) 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M(r) 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.
Title: Purification and characterization of the poly(hydroxyalkanoic acid) synthase from Chromatium vinosum and localization of the enzyme at the surface of poly(hydroxyalkanoic acid) granules Liebergesell M, Sonomoto K, Madkour M, Mayer F, Steinbuchel A Ref: European Journal of Biochemistry, 226:71, 1994 : PubMed
A recombinant strain of Escherichia coli, which overexpressed phaC and phaE from Chromatium vinosum, was used to isolate poly(3-hydroxyalkanoic acid) synthase. The isolation was performed by a two-step procedure including chromatography on DEAE-Sephacel and Procion Blue H-ERD. The poly(3-hydroxyalkanoic acid) synthase consisted of two different kinds of subunit (PhaC, M(r) 39,500 and PhaE, M(r) 40.500). PhaC was separated from the poly(3-hydroxyalkanoic acid) synthase complex by chromatography on phenyl-Sepharose: PhaE was enriched by solubilization of protein inclusion bodies. The stoichiometry of PhaC and PhaE in the enzyme complex was not determined. The poly(3-hydroxyalkanoic acid) synthase (PhaEC) exhibited a native relative molecular mass of M(r) 400,000 and most probably consists of ten subunits. The Km value of the enzyme for D(-)-3-hydroxybutyryl-CoA was 0.063 mM. The enzyme synthesized poly(3-hydroxybutyric acid) in vitro from D(-)-3-hydroxybutyryl-CoA or, together with propionyl-CoA transferase in a coupled enzyme reaction, synthesized the same product from acetyl-CoA plus D(-)-3-hydroxybutyric acid. Antibodies were raised against both subunits of the poly(3-hydroxyalkanoic acid) synthase. By immunoelectron microscopy, the poly(3-hydroxyalkanoic acid) synthase was localized within the cytoplasm in cells of C. vinosum grown under non-storage conditions. In cells grown under poly(3-hydroxybutyric acid) storage conditions, the enzyme was observed to be located at the surface of the poly(3-hydroxybutyric acid) granules. Immunoblots with anti-PhaC, anti-PhaE IgG and crude extract proteins indicated that poly(3-hydroxyalkanoic acid) synthases with partial sequence similarities are widespread among purple sulphur bacteria.
Title: Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea Liebergesell M, Steinbuchel A Ref: Applied Microbiology & Biotechnology, 38:493, 1993 : PubMed
From a genomic library of Thiocystis violaceae strain 2311 in lambda L47, two adjacent EcoRI restriction fragments of 5361 base pairs (bp) and of 1978 bp were cloned. The 5361-bp EcoRI restriction fragment hybridized with a DNA fragment harbouring the Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthase operon (phbCAB) and restored the ability to synthesize and accumulate PHA in PHA-negative mutants derived from A. eutrophus. The nucleotide sequence analysis of both fragments revealed five open-reading frames (ORFs); at least three of them are probably relevant for PHA biosynthesis. The amino acid sequences of the putative proteins deduced from these genes indicate that they encode a beta-ketothiolase [phbATv, relative molecular mass (M(r)) 40850], which exhibited 87.3% amino acid identity with the beta-ketothiolase from Chromatium vinosum. The amino acid sequences of the putative proteins deduced from ORF2Tv (M(r) 41450) and phbCTv (M(r) 39550), which were located upstream of and antilinear to phbATv, exhibited 74.7% and 87.6% amino acid identity, respectively, with the corresponding gene products of C. vinosum. Downstream of and antilinear to phbCTv was located ORF5, which encodes for a protein of high relative molecular mass (M(r) 76428) of unknown function. With respect to the divergent organisation of ORF2Tv and phbCTv on one side and of phbATv on the other side and from the homologies of the putative gene products, this region of the T. violaceae genome resembled very much the corresponding region of C. vinosum, which was identified recently.
Title: Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene Valentin HE, Steinbuchel A Ref: Applied Microbiology & Biotechnology, 39:309, 1993 : PubMed
A cosmid gene bank of partially EcoRI-digested genomic DNA from Methylobacterium extorquens IBT no. 6 was screened for DNA fragments restoring polyhydroxyalkanoic-acid (PHA) accumulation in the PHA-negative mutant Alkaligenes eutrophus H16 PHB-4. The M. extorquens PHA-synthase structural gene phaCMex was mapped on a 23-kbp EcoRI fragment by complementation studies, by hybridization experiments with heterologous DNA probes from A. eutrophus H16 encoding for phaA, phaB and phaC and by nucleic acid sequence analysis. Evidence for the presence of genes for a beta-ketothiolase or an acetoacetyl-coenzyme A reductase on this fragment was not obtained. The nucleotide sequence of a 3.7-kbp region was obtained. It contained the entire 1.815-kbp phaCMex plus approximately each 900-bp upstream and downstream of phaCMex.PhaCMex encoded a protein of 605 amino acids with a relative molecular mass (M(r)) of 66742, which exhibited 38.1% amino acid identity with the A. eutrophus PHA synthase. Determination of the N-terminal amino acid sequence of an M(r) 65,000 protein, which was enriched concomitantly with the purification of PHA granules in sucrose gradients, revealed a sequence that was identical with the amino acid sequence deduced from the most probable translation start codon except for a valine, which was obviously removed post-translationally. Enzyme analysis, which was done with the native gene and a phaCMex'-'lacZ fusion gene, gave no evidence for expression of phaCMex in Escherichia coli.
Title: Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D Liebergesell M, Steinbuchel A Ref: European Journal of Biochemistry, 209:135, 1992 : PubMed
From a genomic library of Chromatium vinosum strain D in lambda L47, a 16.5-kbp EcoRI-restriction fragment was identified by hybridization with a DNA fragment harboring the operon for Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthesis. This fragment and subfragments thereof restored the ability to synthesize and accumulate PHA in PHA-negative mutants of A. eutrophus. A region of 6977 bp was sequenced; seven open reading frames (ORFs) were identified which probably represent coding regions; six of these are most probably relevant for PHA biosynthesis in C. vinosum. The structural genes for biosynthetic acetyl-CoA acyltransferase (beta-ketothiolase; phbACv, 1188 bp) and NADH-dependent acetoacetyl-CoA reductase (phbBCv, 741 bp) were separated by ORF4 (462 bp) and ORF5 (369 bp). Downstream of phbBCv ORF7 (471 pb) was identified which was not completed at the 3' terminus. The functions of ORF4, ORF5, and ORF7 are not known. The amino acid sequences of beta-ketothiolase and acetoacetyl-CoA reductase deduced from phbACv and phbBCv, exhibited a similarity of 68.2% and 56.4% identical amino acids, respectively, to the corresponding enzymes of A. eutrophus. Antilinear to and upstream of the genes mentioned above, two genes were identified which were transcribed from a sigma 70-dependent promoter. This promoter overlapped with and was divergent to the phbACv promoter; the transcriptional start sites were mapped by S1 nuclease protection assays. These genes were ORF2 (1074 bp), whose function is not known but whose presence in Escherichia coli is essential for expression of PHA synthase activity, and the structural gene for a PHA synthase of low M(r) (phbCCv, 1068 bp). The gene products of ORF2 and phbCCv, with M(r) of 40,525 and 39,730, respectively, were expressed in E. coli applying the T7 RNA polymerase/promoter system. Although the amino acid sequence of PHA synthase deduced from phbCCv exhibited only 24.7% overall similarity with the PHA synthase of A. eutrophus, highly conserved regions were identified.
Title: Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the gram-positive bacterium Rhodococcus ruber Pieper U, Steinbuchel A Ref: FEMS Microbiology Letters, 75:73, 1992 : PubMed
The first polyhydroxyalkanoic acid (PHA) synthase gene (phbCRr) of a Gram-positive bacterium was cloned from a genomic library of Rhodococcus ruber in the broad-host-range plasmid vector pRK404. The hybrid plasmid harboring phbCRr allowed the expression of polyhydroxybutyric acid (PHB) synthase activity and restored the ability of PHB synthesis in a PHB-negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phbCRr revealed an open reading frame of 1686 bp starting with the rare codon TTG and encoding a protein of relative molecular mass 61,371. The deduced amino acid sequence of phbCRr exhibited homologies to the primary structures of the PHA synthases of A. eutrophus and Pseudomonas oleovorans. Preparation of PHA granules by discontinuous density gradient centrifugation of crude cellular extracts revealed four major bands in an SDS polyacrylamide gel. A Mr 61,000 protein was identified as the PHA synthase of R. ruber by N-terminal amino acid sequence determination.
The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus, purple non-sulfur bacteria, such as Rhodospirillum rubrum, purple sulfur bacteria, such as Chromatium vinosum, pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens, and for the Gram-positive bacterium Rhodococcus ruber. Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.
Title: Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1 Timm A, Steinbuchel A Ref: European Journal of Biochemistry, 209:15, 1992 : PubMed
From genomic libraries, the polyhydroxyalkanoate gene locus of Pseudomonas aeruginosa PAO1 was cloned and characterised at the molecular level. Two genes coding for polyhydroxyalkanoate synthases, phaC1Pa and phaC2Pa, a polyhydroxyalkanoate depolymerase gene, phaDPa, and four adjacent open reading frames (ORF1, ORF2, ORF3 and ORF4) were identified from the nucleotide sequence. Two transcriptional start sites, which were preceded by sequences resembling the Escherichia coli consensus sequences for sigma 54 and sigma 70 promoters, were identified experimentally upstream of phaC1Pa, which was shown by Northern blot analysis to constitute an operon together with phaDPa. A third putative promoter resembling the E. coli consensus sequence for sigma 70-dependent promoters was proposed upstream of phaC2Pa, which is in a bicistronic operon with ORF3. Investigations of rpoN-negative mutants of related strains revealed that polyhydroxyalkanoate accumulation from gluconate required an intact rpoN locus in P. aeruginosa. Complementation experiments revealed multiple evidence that either polyhydroxyalkanoate synthase is involved in polyhydroylkanoate accumulation from gluconate as well as from octanoate. The P. aeruginosa PAO1 polyhydroxyalkanoate gene locus was expressed in the polyhydroxyalkanoate-negative mutant Alcaligenes eutrophus PHB-4 and in the poly(3-hydroxybutyrate)-accumulating strain P. oleovorans DSM1045. It conferred on the latter the ability to synthesize and accumulate polyhydroxyalkanoates consisting of medium-chain-length 3-hydroxyalkanoic acids from unrelated substrates in addition to poly(3-hydroxybutyrate). The sequence of the putative translational product of ORF1 was similar to those of the leukotoxin repressor of Pasteurella haemolytica and to the ORF9 product of Azotobacter vinelandii, and that of ORF4 was similar to the algP product of P. aeruginosa and to eukaryotic histone H1 proteins. The proteins of ORF2 and ORF3 appear to be previously unidentified.
Title: Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter Schubert P, Kruger N, Steinbuchel A Ref: Journal of Bacteriology, 173:168, 1991 : PubMed
Molecular methods have been applied to analyze the expression of the Alcaligenes eutrophus poly(3-hydroxybutyrate) (PHB) synthase gene (phbC). The translational initiation codon was identified by analysis of the amino acid sequence of a PHB synthase-beta-galactosidase fusion protein. This protein was purified to almost gel electrophoretic homogeneity by chromatography on DEAE-Sephacel and on aminophenyl-beta-D-thiogalactopyranoside-Sepharose from cells of A. eutrophus which harbored a phbC'-'lacZ fusion gene. A sequence (TTGACA-18N-AACAAT), exhibiting striking homology to the Escherichia coli sigma 70 promoter consensus sequence, was identified approximately 310 bp 5' upstream from the translation initiation codon. An S1 nuclease protection assay mapped the transcription start point of phbC 6 bp downstream from this promoter. The location of the promoter was confirmed by analyzing the expression of active PHB synthase in clones of E. coli harboring 5' upstream deletions of phbC ligated to the promoter of the lacZ gene (lacZp) in a Bluescript vector. Plasmids do181 and do218, which were deleted for the first 108 or 300 bp of the phbC structural gene, respectively, conferred the ability to synthesize large amounts of different truncated PHB synthase proteins to the cells. These proteins contributed to approximately 10% of the total cellular protein as estimated from sodium dodecyl sulfate-polyacrylamide gels. The modified PHB synthase encoded by plasmid do181 was still active. Clones in which the lacZp-'phbC fusion harbored the complete phbC structural gene plus the phbC ribosome binding site did not overexpress PHB synthase.
