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1.  Perspectives on biotechnological applications of archaea 
Archaea  2002;1(2):75-86.
Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.
PMCID: PMC2685559  PMID: 15803645
biotechnology; extremozymes; high density cultivation; recombinant DNA technology
2.  RNA-Seq Analysis Provides Insights for Understanding Photoautotrophic Polyhydroxyalkanoate Production in Recombinant Synechocystis Sp. 
PLoS ONE  2014;9(1):e86368.
The photosynthetic cyanobacterium, Synechocystis sp. strain 6803, is a potential platform for the production of various chemicals and biofuels. In this study, direct photosynthetic production of a biopolymer, polyhydroxyalkanoate (PHA), in genetically engineered Synechocystis sp. achieved as high as 14 wt%. This is the highest production reported in Synechocystis sp. under photoautotrophic cultivation conditions without the addition of a carbon source. The addition of acetate increased PHA accumulation to 41 wt%, and this value is comparable to the highest production obtained with cyanobacteria. Transcriptome analysis by RNA-seq coupled with real-time PCR was performed to understand the global changes in transcript levels of cells subjected to conditions suitable for photoautotrophic PHA biosynthesis. There was lower expression of most PHA synthesis-related genes in recombinant Synechocystis sp. with higher PHA accumulation suggesting that the concentration of these enzymes is not the limiting factor to achieving high PHA accumulation. In order to cope with the higher PHA production, cells may utilize enhanced photosynthesis to drive the product formation. Results from this study suggest that the total flux of carbon is the possible driving force for the biosynthesis of PHA and the polymerizing enzyme, PHA synthase, is not the only critical factor affecting PHA-synthesis. Knowledge of the regulation or control points of the biopolymer production pathways will facilitate the further use of cyanobacteria for biotechnological applications.
doi:10.1371/journal.pone.0086368
PMCID: PMC3899235  PMID: 24466058
3.  Isolation and Screening of Polyhydroxyalkanoates Producing Bacteria from Pulp, Paper, and Cardboard Industry Wastes 
Background. Polyhydroxyalkanoates (PHAs) are storage materials that accumulate by various bacteria as energy and carbon reserve materials. They are biodegradable, environmentally friendly, and also biocompatible bioplastics. Unlike petrochemical-based plastics that take several decades to fully degrade, PHAs can be completely degraded within a year by variety of microorganisms into CO2 and water. In the present study, we aim to utilize pulp, paper, and cardboard industry sludge and waste water for the isolation and screening of polyhydroxyalkanoates (PHAs) accumulating bacteria and production of cost-effective PHB using cardboard industry waste water. Results. A total of 42 isolates showed black-blue coloration when stained with Sudan black B, a preliminary screening agent for lipophilic compounds, and a total of 15 isolates showed positive result with Nile blue A staining, a more specific dye for PHA granules. The isolates NAP11 and NAC1 showed maximum PHA production 79.27% and 77.63% with polymer concentration of 5.236 g/L and 4.042 g/L with cardboard industry waste water. Both of the selected isolates, NAP11 and NAC1, were classified up to genus level by studying their morphological and biochemical characteristics and were found to be Enterococcus sp., Brevundimonas sp. and, respectively. Conclusion. The isolates Enterococcus sp. NAP11 and Brevundimonas sp. NAC1 can be considered as good candidates for industrial production of PHB from cardboard industry waste water. We are reporting for the first time the use of cardboard industry waste water as a cultivation medium for the PHB production.
doi:10.1155/2013/752821
PMCID: PMC3830821  PMID: 24288534
4.  Crystallization and preliminary X-ray analysis of a novel thermoalkalophilic poly(3-hydroxybutyrate) depolymerase (PhaZ7) from Paucimonas lemoignei  
A novel thermoalkalophilic depolymerase, PhaZ7, from P. lemoignei was crystallized by the microdialysis technique. Crystals belong to space group C2 and diffract to 2.75 Å resolution at a synchrotron source.
Polyhydroxyalkanoates (PHA) are biodegradable polyesters that have attracted commercial and academic interest as environmentally friendly materials. A number of enzymes are able to degrade polyhydroxyalkanoates to water-soluble products. PhaZ7 poly(3-hydroxybutyrate) (PHB) depolymerase (EC 3.1.1.75), a 342-amino-acid hydrolase from the PHA-degrading bacterium Paucimonas lemoignei, has been found to possess substrate specificity for amorphous PHA. PhaZ7 was crystallized by the microdialysis method. Thin rod-like crystals were grown in low ionic strength solution and found to belong to the monoclinic space group C2, with unit-cell parameters a = 225.8, b = 46.5, c = 171.3, β = 128.9°. A complete data set was collected to 2.75 Å resolution at 100 K using synchrotron radiation.
doi:10.1107/S174430910501016X
PMCID: PMC1952301  PMID: 16511073
biopolymers; biodegradation; Paucimonas lemoignei; serine hydrolases; depolymerase
5.  From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae 
Applied and Environmental Microbiology  2013;79(12):3582-3589.
In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.
doi:10.1128/AEM.00161-13
PMCID: PMC3675910  PMID: 23542629
6.  Future of microbial polyesters 
Numerous microorganisms accumulate polyesters classified as polyhydroxyalkanoates (PHAs) as carbon and energy storage material when the growth condition is unfavorable in the presence of excess carbon source. Natural PHAs typically consist of various (R)-hydroxycarboxylic acids, and exhibit different material properties depending on the monomer composition. Such diversity comes from different metabolic pathways operating in the cell, and thus generating different monomers. Even more diverse PHAs can be produced by metabolically engineered microorganisms, which leads to the biosynthesis of non-natural polyesters containing lactate as a monomer. In order to make PHAs as useful polymers in our daily life, their production cost should be significantly lowered and material properties should be compatible with those produced by petrochemical industries. Metabolic engineering can address these issues by developing microbial strains capable of producing PHAs of desired material properties with high productivity and yield from inexpensive carbon sources. This commentary aims at peeking into the future of PHAs, focusing on the possible metabolic engineering strategies to be taken to achieve these goals.
doi:10.1186/1475-2859-12-54
PMCID: PMC3680211  PMID: 23714196
Polyester; Polyhydroxyalkanoate; Metabolic engineering
7.  PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium 
Journal of Bacteriology  2001;183(14):4235-4243.
Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to the phaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaCBm) and PhaR (PhaRBm) for activity in vivo and in vitro. PhaCBm showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaRBm had no obvious homology to PhaE. Previously we showed that PhaCBm, and here we show that PhaRBm, is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaCBm to produce an active PHA synthase.
doi:10.1128/JB.183.14.4235-4243.2001
PMCID: PMC95313  PMID: 11418564
8.  Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing 
PLoS ONE  2013;8(6):e66588.
The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species.
doi:10.1371/journal.pone.0066588
PMCID: PMC3689743  PMID: 23805243
9.  Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum 
Journal of Bacteriology  2012;194(20):5522-5529.
This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C5 and C6 monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.
doi:10.1128/JB.01111-12
PMCID: PMC3458679  PMID: 22865850
10.  Polyester production by halophilic and halotolerant bacterial strains obtained from mangrove soil samples located in Northern Vietnam 
MicrobiologyOpen  2012;1(4):395-406.
This research article reports halophilic and halotolerant bacteria isolated from mangrove forests located in Northern Vietnam. Several of these bacteria were able to synthesize polyhydroxyalkanoates (PHAs). PHAs are polyesters stored by microorganisms under the presence of considerable amounts of a carbon source and deficiency of other essential nutrient such as nitrogen or phosphorous. Mangrove forests in Northern Vietnam are saline coastal habitats that have not been microbiologically studied. Mangrove ecosystems are, in general, rich in organic matter, but deficient in nutrients such as nitrogen and phosphorus. We have found about 100 microorganisms that have adapted to mangrove forests by accumulating PHAs. The production of polyesters might therefore be an integral part of the carbon cycle in mangrove forests. Three of the strains (ND153, ND97, and QN194) isolated from the Vietnamese forests were identified as Bacillus species, while other five strains (QN187, ND199, ND218, ND240, and QN271) were phylogenetically close related to the α-proteobacterium Yangia pacifica. These strains were found to accumulate PHAs in noticeable amounts. Polymer inclusions and chemical structure were studied by transmission electron microscopy and proton nuclear magnetic resonance (NMR) spectroscopy analyses, respectively. Strains ND153, ND97, QN194, QN187, ND240, and QN271 synthesized poly(3-hydroxybutyrate) (PHB) from glucose, whereas strains ND199 and ND218 synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from this carbohydrate. With the exception of strain QN194, the strains accumulated PHBV when a combination of glucose and propionate was included in the culture medium. The polymer yields and cell growth reached by one Bacillus isolate, strain ND153, and one Gram-negative bacterium, strain QN271, were high and worth to be researched further. For experiments performed in shake flasks, strain ND153 reached a maximum PHBV yield of 71 wt% and a cell dry weight (CDW) of 3.6 g/L while strain QN271 attained a maximum PHB yield of 48 wt% and a CDW of 5.1 g/L. Both strain ND153 and strain QN271 may only represent a case in point that exemplifies of the potential that mangrove forests possess for the discovery of novel halophilic and halotolerant microorganisms able to synthesize different types of biopolyesters.
doi:10.1002/mbo3.44
PMCID: PMC3535385  PMID: 23233461
Biopolyesters; halophilic bacteria; halotolerant bacteria; mangrove forests; polyhydroxyalkanoates
11.  Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium 
BMC Microbiology  2001;1:23.
Background
Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium.
Results
A semi-defined polysaccharide medium was developed and evaluated on six strains of Streptococcus thermophilus. The EPSs were analysed using a novel protocol incorporating ultracentrifugation for the removal of interfering sugars, hydrolysis and analysis of the monomer composition by High Performance Anion-Exchange Chromatography with pulsed amperometric detection. The medium and analysis method allowed accurate quantification and monomer analysis of 0.5 ml samples of EPSs from tube cultures.
Conclusions
The presented medium should be useful for physiological studies of S. thermophilus, and, in combination with the method of analysis of EPS, will allow downscaling of physiological studies and screening for EPSs.
PMCID: PMC57807  PMID: 11602017
12.  New PHA products using unrelated carbon sources 
Brazilian Journal of Microbiology  2011;42(4):1354-1363.
Polyhydroxyalkanoates (PHA) are natural polyesters stored by a wide range of bacteria as carbon source reserve. Due to its chemical characteristics and biodegradability PHA can be used in chemical, medical and pharmaceutical industry for many human purposes. Over the past years, few Burkholderia species have become known for production of PHA. Aside from that, these bacteria seem to be interesting for discovering new PHA compositions which is important to different industrial applications. In this paper, we introduce two new strains which belong either to Burkholderia cepacia complex (Bcc) or genomovar-type, Burkholderia cepacia SA3J and Burkholderia contaminans I29B, both PHA producers from unrelated carbon sources. The classification was based on 16S rDNA and recA partial sequence genes and cell wall fatty acids composition. These two strains were capable to produce different types of PHA monomers or precursors. Unrelated carbon sources were used for growth and PHA accumulation. The amount of carbon source evaluated, or mixtures of them, was increased with every new experiment until it reaches eighteen carbon sources. As first bioprospection experiments staining methods were used with colony fluorescent dye Nile Red and the cell fluorescent dye Nile Blue A. Gas chromatography analysis coupled to mass spectrometry was used to evaluate the PHA composition on each strain cultivated on different carbon sources. The synthesized polymers were composed by short chain length-PHA (scl-PHA), especially polyhydroxybutyrate, and medium chain length-PHA (mcl-PHA) depending on the carbon source used.
doi:10.1590/S1517-838220110004000017
PMCID: PMC3768727  PMID: 24031764
Burkholderia; Burkholderia cepacia complex; Genomovar-type; mcl-PHA; polyhydroxyalkanoates; polyhydroxybutyrate
13.  Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110 
Journal of Bacteriology  2013;195(14):3145-3155.
Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic redundancy, only phaC1 and phaC2 were expressed at significant rates, while PHA accumulation in stationary-phase cultures was impaired only in the ΔphaC1 mutant. Meanwhile, the ΔphaC2 mutant produced more PHA than the wild type under this condition, and surprisingly, the phaC3 transcript increased in the ΔphaC2 background. A double mutant, the ΔphaC2 ΔphaC3 mutant, consistently accumulated less PHA than the ΔphaC2 mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the ΔphaC1 mutant and increased in the ΔphaC2 mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a ΔphaC1 ΔphaC2 double mutant, to study the B. japonicum PHA requirements for survival, competition for nodulation, and plant growth promotion. All mutants, as well as the wild type, survived for 60 days in a carbon-free medium, regardless of their initial PHA contents. When competing for nodulation against the wild type in a 1:1 proportion, the ΔphaC1 and ΔphaC1 ΔphaC2 mutants occupied only 13 to 15% of the nodules, while the ΔphaC2 mutant occupied 81%, suggesting that the PHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation.
doi:10.1128/JB.02203-12
PMCID: PMC3697631  PMID: 23667236
14.  Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose 
Metabolic engineering  2012;14(6):10.1016/j.ymben.2012.08.003.
Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C12 and C14 fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW – the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.
doi:10.1016/j.ymben.2012.08.003
PMCID: PMC3838867  PMID: 23141473
E. coli; Thioesterase; Polyhydroxyalkanoate; Homopolymer; β-oxidation; Dodecanoic acid
15.  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  1998;64(12):4897-4903.
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. Recombinant Escherichia 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 recombinant E. 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. Recombinant E. 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 recombinant E. 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.
PMCID: PMC90940  PMID: 9835580
16.  Examination of PHB Depolymerases in Ralstonia eutropha: Further Elucidation of the Roles of Enzymes in PHB Homeostasis 
AMB Express  2012;2:26.
Polyhydroxyalkanoates (PHA) are biodegradable polymers that are attractive materials for use in tissue engineering and medical device manufacturing. Ralstonia eutropha is regarded as the model organism for PHA biosynthesis. We examined the effects of PHA depolymerase (PhaZ) expression on PHA homeostasis in R. eutropha strains. In order to analyze the impact of PhaZs on R. eutropha granule architecture, we performed electron microscopy on several phaZ knockout strains and the wild type strain grown under PHA production conditions. Analysis of the acquired micrographs was based on stereology: the ratio of granule area and cell area was determined, along with total granule count per full-size cell image. Cells bearing a phaZ2 knockout mutation alone or in conjunction with a phaZ1 mutation were found to have a high granule volume per cell volume and a higher granule count compared to wild type. A phaZ quadruple knockout strain appeared to have a low granule volume per cell volume and a low granule count per cell. Cells bearing a phaZ3 knockout were found to have a higher granule count than the wild type, whereas granule volume per cell volume was similar. Accordingly, we hypothesize that PhaZs have not only an impact on PHA degradation but also on the 3-dimensional granule architecture. Based on our data, PhaZ2 is postulated to affect granule density. This work increased our knowledge about PHA depolymerases in R. eutropha, including enzymes that had previously been uncharacterized.
doi:10.1186/2191-0855-2-26
PMCID: PMC3430594  PMID: 22537946
Ralstonia eutropha; Polyhydroxyalkanoates; Polyhydroxybutyrate; Biomaterials; Depolymerase; Granules; Carbon utilization; Electron microscopy; Stereology
17.  Application of the BPEC Pathway for Large-Scale Biotechnological Production of Poly(3-Mercaptopropionate) by Recombinant Escherichia coli, Including a Novel In Situ Isolation Method 
Metabolically engineered Escherichia coli JM109 harboring plasmid pBPP1 and expressing the nonnatural BPEC pathway for synthesis of thermoplastic polyhydroxyalkanoates (PHA) and novel polythioesters (PTE) to provide suitable substrates of PHA synthase was investigated with respect to biotechnological production of poly(3-mercaptopropionate) [poly(3MP)]. Fed-batch fermentation processes were established at the 30- and 500-liter scales in stirred tank bioreactors to produce kilogram amounts of poly(3MP). Cultivation was done in a modified M9 mineral salts medium containing glucose or glycerol as the carbon and energy source and with 3-mercaptopropionic acid (3MP) as the precursor substrate for poly(3MP) biosynthesis provided from the late exponential growth phase. Approximately 23 g of cell dry matter (CDM) per liter and poly(3MP) cell contents of up to 45% (wt/wt) were the highest cell densities and polymer contents obtained, respectively. At best, 69.1% (wt/wt) of 3MP was converted into poly(3MP), indicating that 3MP was mostly used for poly(3MP) biosynthesis. Furthermore, a novel in situ process for rapid and convenient isolation of poly(3MP) from the cells in the bioreactor was developed. This was achieved by addition of sodium dodecyl sulfate to the cultivation broth immediately after the fermentation, heating to 90°C for 20 min with intensive stirring, and subsequent washing steps. The purity of such in situ isolated poly(3MP) was more than 98%, as revealed by gas chromatographic and elemental sulfur analyses of the material isolated.
doi:10.1128/AEM.71.2.835-841.2005
PMCID: PMC546776  PMID: 15691938
18.  Tight coupling of polymerization and depolymerization of polyhydroxyalkanoates ensures efficient management of carbon resources in Pseudomonas putida 
Microbial Biotechnology  2013;6(5):551-563.
Summary
Environmental microbes oscillate between feast and famine and need to carefully manage utilization, storage and conversion of reserve products to exploitable sources of carbon and energy. Polyhydroxyalkanoates (PHAs) are storage polymers that serve bacteria as sources of food materials under physiological conditions of carbon demand. In order to obtain insights into the role of PHA depolymerase (PhaZ) and its relationship to a PHA polymerase (PhaC2) in the carbon management activity of Pseudomonas putida strain U, we created a polymerase hyperexpression strain and a depolymerase knockout mutant of this strain, and examined their synthesis of PHA and expression of their PHA genes. This study revealed that hyperexpression of PhaC2 led to the accumulation of higher amounts of PHA (44%wt) than in the wild-type strain (24%wt) after 24 h of cultivation, which then returned to wild-type levels by 48 h, as a result of elevated depolymerization. The phaZ mutant, however, accumulated higher levels of PHA than the parental strain (62%wt), which were maintained for at least 96 h. Transcriptional analysis of the pha cluster by RT-PCR revealed that PHA operon proteins, including depolymerase, are expressed from the beginning of the growth phase. Hyperexpression of the PhaC2 polymerase was accompanied by an increase in the expression of the PhaZ depolymerase and a decrease in expression of another PHA polymerase, PhaC1. This suggests tight regulatory coupling of PHA polymerase and depolymerase activities that act in synergy, and in concert with other PHA proteins, to provide dynamic PHA granule synthesis and remodelling that rapidly and sensitively respond to changes in availability of carbon and the physiological-metabolic needs of the cell, to ensure optimal carbon resource management.
doi:10.1111/1751-7915.12040
PMCID: PMC3918157  PMID: 23445364
19.  Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2▿ 
The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaCCs). PhaCCs showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaCCs expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCs was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaCCs of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCs is a naturally occurring, highly active PHA synthase with superior polymerizing ability.
doi:10.1128/AEM.01997-10
PMCID: PMC3126384  PMID: 21398494
20.  The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach 
Background
Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.
Results
We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system.
Conclusion
This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.
doi:10.1186/1475-2859-11-34
PMCID: PMC3325844  PMID: 22433058
P. putida KT2442; Nutrient limitation; Systems biology; Polyhydroxyalkanoates
21.  Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax mediterranei▿ †  
Journal of Bacteriology  2008;190(12):4173-4180.
The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaECHme) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaEHme and phaCHme genes were constitutively expressed, and both the PhaEHme and PhaCHme proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaECHme genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaECHme genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaCHme was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaEHme/PhaCHme (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaCHme and PhaEHme, accounted for the PHBV synthesis in H. mediterranei.
doi:10.1128/JB.00134-08
PMCID: PMC2446746  PMID: 18408025
22.  Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in Escherichia coli 
Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the β-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.
doi:10.1128/AEM.67.7.3102-3109.2001
PMCID: PMC92987  PMID: 11425728
23.  Effect of carbon and nitrogen sources on simultaneous production of α-amylase and green food packaging polymer by Bacillus sp. CFR 67 
In this paper, effect of different carbon and nitrogen sources, including hydrolysates of rice bran and wheat bran, on simultaneous production of α-amylase (for hydrolysis of starch in food systems) and polyhydroxyalkanoates (PHA, a green biopolymer, which can be used as a packing material for foods) by Bacillus sp. CFR 67 was studied by submerged fermentation. Amongst various carbon sources tested, glucose and sucrose supported production of significantly (P < 0.05) higher amount of α-amylase (66 U/ml) and PHA (444 mg/l), respectively. Of the nitrogen sources tested, ammonium acetate and beef extract led to the production of maximum amount of amylase (36 U/ml) and PHA (592 mg/l), respectively. Supplementation of the production medium with wheat bran hydrolysate (50 ml/l) produced significantly higher amounts of amylase (73 U/ml) and PHA (524 mg/l). Thus this study indicated the potential of agro-residues for the production of value added biomolecules, which can reduce the cost of production of these molecules and enables to reduce the pollution mainly caused by the use of non biodegradable plastics.
doi:10.1007/s13197-012-0639-6
PMCID: PMC3550928  PMID: 24425935
Amylase; Bacillus; Carbon; Nitrogen; Polyhydroxyalkanoates; Rice bran hydrolysate; Submerged fermentation; Wheat bran hydrolysate
24.  Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases 
Applied and Environmental Microbiology  2004;70(11):6789-6799.
PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB−4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.
doi:10.1128/AEM.70.11.6789-6799.2004
PMCID: PMC525123  PMID: 15528546
25.  Structure and Ecological Roles of a Novel Exopolysaccharide from the Arctic Sea Ice Bacterium Pseudoalteromonas sp. Strain SM20310 
The structure and ecological roles of the exopolysaccharides (EPSs) from sea ice microorganisms are poorly studied. Here we show that strain SM20310, with an EPS production of 567 mg liter−1, was screened from 110 Arctic sea ice isolates and identified as a Pseudoalteromonas strain. The EPS secreted by SM20310 was purified, and its structural characteristics were studied. The predominant repeating unit of this EPS is a highly complicated α-mannan with a molecular mass greater than 2 × 106 Da. The backbone of the EPS consists of 2-α-, 6-α-mannosyl residues, in which a considerable part of the 6-α-mannosyl residues are branched at the 2 position with either single t-mannosyl residues or two mannosyl residues. The structure of the described EPS is different from the structures of EPSs secreted by other marine bacteria. Analysis of the ecological roles of the identified EPS showed that the EPS could significantly enhance the high-salinity tolerance of SM20310 and improve the survival of SM20310 after freeze-thaw cycles. These results suggest that the EPS secreted by strain SM20310 enables the strain to adapt to the sea ice environment, which is characterized by low temperature, high salinity, and repeated freeze-thaw cycles. In addition to its functions in strain SM20310, this EPS also significantly improved the tolerance of Escherichia coli to freeze-thaw cycles, suggesting that it may have a universal impact on microorganism cryoprotection.
doi:10.1128/AEM.01801-12
PMCID: PMC3536116  PMID: 23087043

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