Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (phaC) Gene fromChromobacterium violaceum

ABSTRACT The polyhydroxyalkanoic acid synthase gene fromChromobacterium violaceum (phaC Cv) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaC Cv and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaA Cv). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes andRalstonia eutropha (formerly Alcaligenes eutrophus), harboring phaC Cv, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. colinor recombinant Pseudomonas putida harboringphaC Cv accumulated significant levels of PHA. Sequence analysis of the phaC Cv product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no. AAD10274).

Download Full-text

Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4897-4903.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4897-4903 ◽

Cited By ~ 79

Author(s):

Jong-il Choi ◽

Sang Yup Lee ◽

Kyuboem Han

Keyword(s):

Escherichia Coli ◽

Ralstonia Eutropha ◽

Pha Synthase ◽

Wild Type ◽

High Productivity ◽

E Coli ◽

Alcaligenes Latus ◽

Enhanced Production ◽

Wide Range ◽

High Level

ABSTRACT Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. RecombinantEscherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinantE. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. RecombinantE. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinantE. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

Download Full-text

Analysis of 4-Phosphopantetheinylation of Polyhydroxybutyrate Synthase from Ralstonia eutropha: Generation of β-Alanine Auxotrophic Tn5 Mutants and Cloning of thepanD Gene Region

Journal of Bacteriology ◽

10.1128/jb.181.5.1429-1435.1999 ◽

1999 ◽

Vol 181 (5) ◽

pp. 1429-1435 ◽

Cited By ~ 12

Author(s):

Astrid Hoppensack ◽

Bernd H. A. Rehm ◽

Alexander Steinbüchel

Keyword(s):

Escherichia Coli ◽

Ralstonia Eutropha ◽

Open Reading Frame ◽

Pha Synthase ◽

Gene Region ◽

Amino Acid Sequence Similarity ◽

Reading Frame ◽

E Coli ◽

Strong Similarity ◽

Link Type

ABSTRACT The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutrophaby 4-phosphopantetheine was investigated. Four β-alanine auxotrophic Tn5-induced mutants of R. eutropha HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the panDgene from Escherichia coli, encodingl-aspartate-1-decarboxylase (EC 4.1.1.15 ), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)+ transhydrogenase (EC 1.6.1.1 ) alpha 1 subunit, encoded by the pntAA gene fromEscherichia coli. The panD gene was cloned by complementation of the panD mutant of R. eutropha Q20. DNA sequencing of the panD gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from E. coli and Bacillus subtilis, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS− led to complementation of the panD mutants of R. eutropha and E. coli SJ16, respectively. panD-encodedl-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of panD, an ORF with strong similarity to pntAA from E. coli, encoding NAD(P)+ transhydrogenase subunit alpha 1 was found; downstream of panD, two ORFs with strong similarity topntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization ofpan and pnt genes was found in R. eutropha. Labeling experiments using one of the R. eutropha panD mutants and [2-14C]β-alanine provided no evidence that R. eutropha PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the E. coli panD mutant exhibit detectable labeling of functional PHA synthase from R. eutropha.

Download Full-text

Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.185.18.5391-5397.2003 ◽

2003 ◽

Vol 185 (18) ◽

pp. 5391-5397 ◽

Cited By ~ 64

Author(s):

Si Jae Park ◽

Sang Yup Lee

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Chain Length ◽

Biosynthetic Pathway ◽

Pha Synthase ◽

Enzymatic Analysis ◽

E Coli ◽

Medium Chain ◽

Medium Chain Length ◽

Enoyl Coa Hydratase

ABSTRACT The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

Download Full-text

Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

Microorganisms ◽

10.3390/microorganisms10010184 ◽

2022 ◽

Vol 10 (1) ◽

pp. 184

Author(s):

Izumi Orita ◽

Gento Unno ◽

Risa Kato ◽

Toshiaki Fukui

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Ralstonia Eutropha ◽

Negative Impact ◽

Value Added ◽

Pha Synthase ◽

Growth Impairment ◽

Oxidation Pathway ◽

Methylotrophic Growth ◽

Value Added Products

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Download Full-text

Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

Metabolites ◽

10.3390/metabo10060228 ◽

2020 ◽

Vol 10 (6) ◽

pp. 228

Author(s):

R. Axayacatl Gonzalez-Garcia ◽

Lars K. Nielsen ◽

Esteban Marcellin

Keyword(s):

Escherichia Coli ◽

Natural Products ◽

Carbon Source ◽

Sole Carbon Source ◽

Structural Diversity ◽

Heterologous Production ◽

E Coli ◽

Anticancer Properties ◽

Defined Media ◽

Extender Unit

Polyketides are a remarkable class of natural products with diverse functional and structural diversity. The class includes many medicinally important molecules with antiviral, antimicrobial, antifungal and anticancer properties. Native bacterial, fungal and plant hosts are often difficult to cultivate and coax into producing the desired product. As a result, Escherichia coli has been used for the heterologous production of polyketides, with the production of 6-deoxyerythronolide B (6-dEB) being the first example. Current strategies for production in E. coli require feeding of exogenous propionate as a source for the precursors propionyl-CoA and S-methylmalonyl-CoA. Here, we show that heterologous polyketide production is possible from glucose as the sole carbon source. The heterologous expression of eight genes from the Wood-Werkman cycle found in Propionibacteria, in combination with expression of the 6-dEB synthases DEBS1, DEBS2 and DEBS3 resulted in 6-dEB formation from glucose as the sole carbon source. Our results show that the Wood-Werkman cycle provides the required propionyl-CoA and the extender unit S-methylmalonyl-CoA to produce up to 0.81 mg/L of 6-dEB in a chemically defined media.

Download Full-text

Dissemination of the blaNDM-5 Gene via IncX3-Type Plasmid among Enterobacteriaceae in Children

mSphere ◽

10.1128/msphere.00699-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Dongxing Tian ◽

Bingjie Wang ◽

Hong Zhang ◽

Fen Pan ◽

Chun Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Drug Resistance ◽

Gene Transfer ◽

Horizontal Gene Transfer ◽

Klebsiella Pneumoniae ◽

Pediatric Patients ◽

Transmission Mechanism ◽

Klebsiella Aerogenes ◽

Content Type ◽

E Coli

ABSTRACT The continuous emergence of novel New Delhi metallo-β-lactamase-5 (NDM-5)-producing Enterobacteriaceae isolates is receiving more and more public attention. Twenty-two NDM-5-producing strains were identified from 146 carbapenemase-producing Enterobacteriaceae (CRE) strains isolated from pediatric patients between January and March 2017, indicating that the blaNDM-5 gene has spread to children. All 22 isolates, including 16 Klebsiella pneumoniae strains, four Klebsiella aerogenes strains, and two Escherichia coli strains, showed significantly high resistance to β-lactam antibiotics (except aztreonam) but remained susceptible to tigecycline and colistin. K. pneumoniae and K. aerogenes strains were respectively defined as homologous clonal isolates by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) results confirmed the genetic relatedness with all K. pneumoniae strains belonging to sequence type (ST) 48. Two E. coli isolates (ST617 and ST1236) were considered genetically unrelated. Twenty-two blaNDM-5 plasmids were positive for the IncX3 amplicon and showed almost identical profiles after digestion with HindIII and EcoRI. Four representative strains (K. pneumoniae K725, K. aerogenes CR33, E. coli Z214, and E. coli Z244) were selected for further study. Plasmids harboring blaNDM-5 showed strong stability in both clinical isolates and transconjugants, without apparent plasmid loss after 100 serial generations. S1-PFGE followed by Southern blot analysis demonstrated that the blaNDM-5 gene was located on an ∼46-kb plasmid. Plasmid sequences of pNDM-K725, pNDM-CR33, and pNDM-Z214 were almost identical but were slightly different from that of pNDM-Z244. Compared with pNDM-Z244, ΔISAba125 and partial copies of IS3000 were missing. The genetic backgrounds of the blaNDM-5 gene in four strains were slightly different from that of the typical pNDM_MGR194. This study comprehensively characterized the horizontal gene transfer of the blaNDM-5 gene among different Enterobacteriaceae isolates in pediatric patients, and the IncX3-type plasmid was responsible for the spread. IMPORTANCE The emergence of CRE strains resistant to multiple antibiotics is considered a substantial threat to human health. Therefore, all the efforts to provide a detailed molecular transmission mechanism of specific drug resistance can contribute positively to prevent the further spread of multidrug-resistant bacteria. Although the new superbug harboring blaNDM-5 has been reported in many countries, it was mostly identified among E. coli strains, and the gene transfer mechanism has not been fully recognized and studied. In this work, we identified 22 blaNDM-5-positive strains in different species of Enterobacteriaceae, including 16 Klebsiella pneumoniae strains, four Klebsiella aerogenes strains, and two Escherichia coli strains, which indicated the horizontal gene transfer of blaNDM-5 among Enterobacteriaceae strains in pediatric patients. Moreover, blaNDM-5 was located on a 46-kb IncX3 plasmid, which is possibly responsible for this widespread horizontal gene transfer. The different genetic contexts of the blaNDM-5 gene indicated some minor evolutions of the plasmid, based on the complete sequences of the blaNDM-5 plasmids. These findings are of great significance to understand the transmission mechanism of drug resistance genes, develop anti-infection treatment, and take effective infection control measures.

Download Full-text

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000495752 ◽

2018 ◽

Vol 28 (5) ◽

pp. 225-235

Author(s):

Edmar Ramos Oliveira-Filho ◽

Linda P. Guamán ◽

Thatiane Teixeira Mendonça ◽

Paul F. Long ◽

Marilda Keico Taciro ◽

...

Keyword(s):

Carbon Source ◽

Aeromonas Hydrophila ◽

Sole Carbon Source ◽

Ralstonia Eutropha ◽

Pha Synthase ◽

Medium Chain Length ◽

Marker Free ◽

Pha Copolymers ◽

First Time ◽

Induced Mutant

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHAMCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HAMCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HAMCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Download Full-text

Utilization of chymotrypsin as a sole carbon and (or) nitrogen source by Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/m92-048 ◽

1992 ◽

Vol 38 (4) ◽

pp. 290-295 ◽

Cited By ~ 1

Author(s):

Arthur S. Brecher ◽

Timothy A. Moehlman ◽

William D. Hann

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Nitrogen Source ◽

Degradation Products ◽

Defined Medium ◽

Host Protein ◽

Mammalian Host ◽

Sole Nitrogen Source ◽

E Coli ◽

Nitrogen Nutrients

α-Chymotrypsin serves as a sole carbon source, sole nitrogen source, and as sole carbon plus nitrogen source for wild-type Escherichia coli in a totally defined medium. Hence, a mammalian host for E. coli may supply the necessary carbon and nitrogen nutrients for the microorganism. Growth is most rapid when chymotrypsin is a sole nitrogen source,and least rapid with chymotrypsin as a carbon source. The approximate doubling times for E. coli utilizing chymotrypsin as a nitrogen source, carbon plus nitrogen source, and carbon source are 1.6, 4.6, and 11.3 h, respectively. The activity of the residual enzyme in the culture supernates falls off asymptotically over the course of time, as followed by cleavage of glutaryl-L-phenylalanine-p-nitroanilide. Chymotrypsin hydrolyzes succinyl-L-ala-L-ala-L-ala-p-nitroanilide, the elastase substrate, to some extent. Peptidases do not appear to be secreted that hydrolyze such model substrates as benzoyl-DL-arginine-p-nitroanilide, the tryptic and cathepsin B substrate, L-leucine-p-nitroanilide, the leucine aminopeptidase substrate, or L-lysine-p-nitroanilide, the aminopeptidase B substrate. Growth of E. coli is generally directly related to the loss of chymotryptic activity in the medium. Hence, autolysis of chymotrypsin, i.e., self-degradation, is an important factor for the availability of degradation products of the enzyme to the bacterium for growth purposes. Accordingly, the degradation of a host protein by autolysis presents an opportunity for E. coli to survive during periods of host nutritional crisis by utilization of the degradation peptides that are produced during autolysis. Key words: chymotrypsin, Escherichia coli, growth, nutrition, peptide source.

Download Full-text

Rearrangement of Gene Order in thephaCABOperon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(R)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.07715-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3177-3184 ◽

Cited By ~ 71

Author(s):

Ayaka Hiroe ◽

Kenji Tsuge ◽

Christopher T. Nomura ◽

Mitsuhiro Itaya ◽

Takeharu Tsuge

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Gene Order ◽

Ralstonia Eutropha ◽

Genetically Engineered ◽

Pha Synthase ◽

Limiting Factor ◽

Expression Plasmid ◽

Content Type ◽

Ultrahigh Molecular Weight

ABSTRACTUltrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineeredEscherichia coliis an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized asphaCAB) encodes the essential proteins for the production of P(3HB) in the native producer,Ralstonia eutropha. The three genes of thephaCABoperon arephaC, which encodes the polyhydroxyalkanoate (PHA) synthase,phaA, which encodes a 3-ketothiolase, andphaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of thephaCABoperon (phaABC,phaACB,phaBAC,phaBCA,phaCAB, andphaCBA) on an expression plasmid in genetically engineeredE. coliwas examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of thephagenes relative to the promoter. The most balanced production result was achieved in the strain harboring thephaBCAexpression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.

Download Full-text

Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02666-14 ◽

2014 ◽

Vol 80 (24) ◽

pp. 7702-7709 ◽

Cited By ~ 14

Author(s):

Jessica Eggers ◽

Alexander Steinbüchel

Keyword(s):

Escherichia Coli ◽

Ralstonia Eutropha ◽

Model Organism ◽

Dry Weight ◽

Growth Behavior ◽

Carbon Limitation ◽

Content Type ◽

Phb Depolymerase ◽

E Coli ◽

The Impact

ABSTRACTThe model organism for polyhydroxybutyrate (PHB) biosynthesis,Ralstonia eutrophaH16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinantEscherichia coliBL21(DE3) strains were used to study the impact of selected PHB depolymerases ofR. eutrophaH16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinantE. coliBL21(DE3) strains were constructed, which harbored a plasmid carrying thephaCABoperon fromR. eutrophaH16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase fromR. eutrophaH16. It is shown in this study that the growth behavior of the respective recombinantE. colistrains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboringphaZ7reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed ifphaZ1was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.

Download Full-text