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Biopolymers from marine prokaryotes.

Biopolymers from marine prokaryotes, both Bacteria and Archaea, offer a number of novel material properties and commercial opportunities. The characteristics of marine exopolysaccharides and melanins that enhance the survival ability of the organisms producing them can be exploited for a number of products ranging from emulsifiers to adhesives. In the prokaryotes, the polyhydroxyalkanoates form carbon-storage molecules, but their technological application is entirely different, serving as a potential base material for biodegradable plastics. Marine biopolymers are a significant and undeveloped biological resource.

Biofilms↗

Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16.

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.

Aerobiosis↗

Chiral selection on inorganic crystalline surfaces.

From synthetic drugs to biodegradable plastics to the origin of life, the chiral selection of molecules presents both daunting challenges and significant opportunities in materials science. Among the most promising, yet little explored, avenues for chiral molecular discrimination is adsorption on chiral crystalline surfaces - periodic environments that can select, concentrate and possibly even organize molecules into polymers and other macromolecular structures. Here we review experimental and theoretical approaches to chiral selection on inorganic crystalline surfaces - research that is poised to open this new frontier in understanding and exploiting surface-molecule interactions.

Adsorption↗

Roseateles depolymerans gen. nov., sp. nov., a new bacteriochlorophyll a-containing obligate aerobe belonging to the beta-subclass of the Proteobacteria.

Strains 61AT (T = type strain) and 61B2, the first bacteriochlorophyll (BChl) a-containing obligate aerobes to be classified in the beta-subclass of the Proteobacteria, were isolated from river water. The strains were originally isolated as degraders of poly(hexamethylene carbonate) (PHC). The organisms can utilize PHC and some other biodegradable plastics. The strains grow only under aerobic conditions. Good production of BChl a and caroterioid pigments is achieved on PHC agar plates and an equivalent production is observed under oligotrophic conditions on agar medium. Spectrometric results suggest that BChl a is present in light-harvesting complex I and the photochemical reaction centre. The main carotenoids are spirilloxanthin and its precursors. Analysis of the 16S rRNA gene sequence indicated that the phylogenetic positions of the two strains are similar to each other and that their closest relatives are the genera Rubrivivax, ideonella and Leptothrix with similarities of 96.3, 96.2 and 96.1%, respectively. The cells are motile, straight rods and contain poly-beta-hydroxybutyrate granules. Ubiquinone-8 is the predominant quinone. Vitamins are not required for growth. The G + C content of genomic DNA is 66.2-66.3 mol%. Genetic and phenotypic features suggest that the strains represent a new genus in the beta-subclass which is evenly distant from known genera. Consequently, the name Roseateles depolymerans gen. nov., sp. nov. is proposed for the strains; the type strain of Roseateles depolymerans is strain 61AT (= DSM 11813T).

Bacteria, Aerobic↗

Alginate formation in Azotobacter vinelandii UWD during stationary phase and the turnover of poly-beta-hydroxybutyrate.

Azotobacter vinelandii UWD is a mutant of strain UW that is defective in the respiratory oxidation of NADH. This mutation causes an overproduction of polyhydroxyalkanoates (PHAs), as polyester synthesis is used as an alternative electron sink. Since PHAs have potential for use as natural, biodegradable plastics, studies of physiology related to their production are of interest. Alginate production by this strain is limited to < 11 microg (mg cell protein)(-1), which permits high efficiency conversion of carbon source into PHA. However, < or = 400 microg (mg cell protein)(-1) was formed when UWD cells were oxygen-limited and in the stationary phase of growth. Alginate formation was fuelled by PHA turnover, which was coincident with the synthesis of alkyl resorcinols, under conditions of exogenous glucose limitation. However, alginate production was a phenotypic and reversible change. Alginate production was stopped by interruption of algD with Tn5lacZ. LacZ activity in UWD was shown to increase in stationary phase, while LacZ activity in a similarly constructed mutant of strain UW did not. Transcription of algD in strain UWD started from a previously identified RpoD promoter and not from the AlgU (RpoE) promoter. This is because strain UWD has a natural insertion element in algU. Differences between strain UW and UWD may reside in the defective respiratory oxidation of NADH, where the NADH surplus in strain UWD may act as a signal of stationary phase. Indeed, a backcross of UW DNA into UWD generated NADH-oxidase-proficient cells that failed to form alginate in stationary phase. Evidence is also presented to show that the RpoD promoter may be recognized by the stationary phase sigma factor (RpoS), which may mediate alginate production in strain UWD.

Alginates↗

Reduction of starch granule size by expression of an engineered tandem starch-binding domain in potato plants.

Granule size is an important parameter when using starch in industrial applications. An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via the Pro-Thr-rich linker peptide from Xyn10A from Cellulomonas fimi. SBD2 and a single SBD were introduced into the amylose-free potato mutant, amf, using appropriate signal sequences. The accumulation of SBD2 into transgenic starch granules was much higher than that of SBD. In a number of transformants, particularly amfSS3, the starch granules were much smaller than in control plants. The amfSS3 mean granule size was 7.8 microm, compared with 15.2 microm in the control, whereas other starch properties were unaltered. This new starch combines the advantage of the high purity of potato starch with that of the small granule size of other crop species, such as cassava, taro and wheat. This starch may find application in the manufacture of biodegradable plastic films. Both genes were also expressed in Escherichia coli and the affinity for soluble starch of the purified recombinant proteins was determined. SBD2 had an approximately 10-fold higher affinity for starch than SBD, indicating that the two appended SBDs act in synergy when binding to their target polysaccharide ligand.

Journal Article↗

Production of poly-beta-hydroxyalkanoates from soy molasses oligosaccharides by new, rapidly growing Bacillus species.

AIMS: To isolate and characterize bacteria from nature capable of producing poly-beta-hydroxyalkanoates in high yields from soy molasses oligosaccharides. METHODS AND RESULTS: Several strains of bacteria were obtained from enrichment cultures employing raffinose as major carbon source and inoculated with soybean field soil, lake sediment, or lake water. Many of the isolates were Bacillus species and produced polyhydroxyalkanoates (PHAs) to high yield. The raffinose-degrading isolates produced endospores, were highly saccharolytic, and both respired and fermented a variety of mono-, di-, tri- and tetrasaccharides. Strain CL1 produced 90% of cell dry mass as PHA from various sugars, including raffinose, and did so without requiring a nutrient limitation. CONCLUSIONS: Strain CL1 could be the catalyst for an industrial fermentation converting soy molasses and other waste carbohydrates to PHAs. The properties of this organism that make it ideally suited for such a fermentation include (i) its ability to use a wide variety of plant-associated carbohydrates as PHA feedstocks; (ii) its rapid growth; (iii) its ability to grow under anoxic conditions; and (iv) its ability to produce spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of bacteria capable of making biodegradable plastics to high yield from soy molasses oligosaccharides.

Bacillus↗

Synthesis of polyhydroxyalkanoate in the peroxisome of Pichia pastoris.

Polyhydroxyalkanoates (PHAs) are polyesters naturally produced by bacteria that have properties of biodegradable plastics and elastomers. A PHA synthase from Pseudomonas aeruginosa modified at the carboxy-end for peroxisomal targeting was transformed in Pichia pastoris. The PHA synthase was expressed under the control of the promoter of the P. pastoris acyl-CoA oxidase gene. Synthesis of up to 1% medium-chain-length PHA per g dry weight was dependent on both the expression of the PHA synthase and the presence of oleic acid in the medium. PHA accumulated as inclusions within the peroxisomes. P. pastoris could be used as a model system to study how peroxisomal metabolism needs to be modified to increase PHA production in other eukaryotes, such as plants.

Acyltransferases↗

Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions.

Succinate is an interesting chemical for industries producing food and pharmaceutical products, surfactants, detergents and biodegradable plastics. Succinate is produced mainly by a mixed-acid fermentation process using anaerobically growing bacteria. However, succinate excretion is also widespread among fungi. In this article we report results on the intracellular concentration and the excretion of succinate by Penicillium simplicissimum under aerobic and anaerobic conditions. The intracellular concentration of succinate increased slightly with the specific growth rate and strongly if the respiratory chain was inhibited by sodium azide or anaerobic conditions (N(2)). A strong increase of succinate excretion was observed if the respiratory chain was inhibited. It is suggested that succinate synthesis under functional (sodium azide) or environmental (N(2)) anaerobic conditions occurs via the reductive part of the tricarboxylic acid cycle. Succinate is then excreted because the oxidative part of the tricarboxylic acid cycle is inactive. A possible role of succinate synthesis in the regeneration of NAD ('fumarate respiration') is discussed.

Adenosine Triphosphate↗

Amino acid reprogramming and biofilm-specific tricarboxylate transporters in PET-degrading Piscinibacter sakaiensis.

Plastic-degrading bacteria predominantly colonize polymer surfaces as biofilms, yet it remains unclear whether the biofilm phenotype contributes to metabolism beyond retaining extracellular enzymes. Here, we combine population-level RNA-sequencing across three conditions-biofilm cells on polyethylene terephthalate (PET), planktonic cells incubated with PET, and planktonic cells on maltose-with single-cell Raman spectroscopy to characterize the PET response of Piscinibacter sakaiensis (formerly Ideonella sakaiensis). This integrated approach reveals two metabolically distinct response layers. A carbon-source-driven response shared by all PET-exposed cells is dominated by a broad amino acid reprogramming, led by upregulation of branched-chain amino acid transport genes, enhanced serine biosynthesis, and reduced chemotaxis. A biofilm-specific layer selectively induces tripartite tricarboxylate transporter genes from three distinct genomic loci. This transcriptional feature is accompanied by a single-cell phenotype consistent with a protein-rich and saturated membrane. These results suggest that biofilm formation is not limited to enzyme retention but is associated with selective activation of transport systems, consistent with a putative role in capturing PET-derived intermediates at the polymer interface. This two-layer model separates general metabolic adaptation to PET from biofilm-specific functions and provides a framework for understanding how surface-associated bacterial physiology contributes to plastic degradation.IMPORTANCEPolyethylene terephthalate (PET) degradation in natural and engineered environments is largely mediated by surface-attached microbial communities, yet the physiological role of biofilm state during plastic degradation remains poorly understood. Using the model PET degrader Piscinibacter sakaiensis, we show that biofilm-associated cells are not simply retained near the polymer surface but exhibit a distinct metabolic program characterized by selective induction of tripartite tricarboxylate transporters. In contrast, extensive amino acid reprogramming occurs in both biofilm and planktonic PET-exposed cells, indicating that it is driven by carbon source rather than surface attachment. These findings reveal that PET degradation involves two separable physiological layers: a general metabolic response to PET-derived carbon shared across cell phenotypes, and a biofilm-specific transport response potentially linked to substrate capture at the plastic interface. This work advances our understanding of how microbial physiology is organized during plastic biodegradation and identifies transport processes as previously unrecognized components of PET-degrading biofilms.

PET biodegradation↗

Accumulation of Poly (beta-Hydroxybutyrate) by Halobacteria.

Some species of extremely halophilic archaebacteria, Halobacteriaceae, have been shown to accumulate large amounts of poly (beta-hydroxybutyrate) under conditions of nitrogen limitation and abundant carbon source. The production of poly (beta-hydroxybutyrate), at least in large quantities, was restricted to two carbohydrate-utilizing species, Halobacterium mediterranei and H. volcanii. In addition to the nutrients in the media, the salt concentration also influenced poly (beta-hydroxybutyrate) accumulation, which was greater at lower salt concentrations. The possible application of these microorganisms for the production of biodegradable plastics is discussed.

Journal Article↗

Hyperproduction of Poly-beta-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD.

The transformation of Azotobacter vinelandii UW with A. vinelandii 113 DNA resulted in the formation of rifampin-resistant colonies, 13% of which also inherited a previously unrecognized mutation in the respiratory NADH oxidase. These transformants produced colonies with a white-sectored phenotype after prolonged incubation. Cells from these sectors were separated and purified by streaking and were named UWD. The dense white phenotype was due to the production of a large amount of poly-beta-hydroxybutyrate during the exponential growth of strain UWD. The polymer accounted for 65 or 75% of the cell dry weight after 24 h of incubation of cultures containing glucose and either ammonium acetate or N(2), respectively, as the nitrogen source. Under the same conditions, strain UW cells contained 22 to 25% poly-beta-hydroxybutyrate, but O(2)-limited growth was required for these optimal production values. Polymer production was not dependent on O(2) limitation in strain UWD, but the efficiency of conversion of glucose to poly-beta-hydroxybutyrate was enhanced in O(2)-limited cultures. Conversion efficiencies were >0.25 and 0.33 mg of poly-beta-hydroxybutyrate per mg of glucose consumed under vigorous- and low-aeration conditions, respectively, compared with an efficiency of 0.05 achieved by strain UW. Strain UWD, therefore, appeared to from poly-beta-hydroxybutyrate under novel conditions, which may be useful in designing new methods for the industrial production of biodegradable plastics.

Journal Article↗

Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici.

The microbial production of L-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of L-(+) and D-(-) isomers. For most uses of PLA, the L-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a D-(-)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an L-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in L-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native D-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased L-lactate dehydrogenase activity. SZ85 produced L-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for L-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes.

Culture Media↗

Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109.

Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

Whole-genome shotgun optical mapping of Rhodospirillum rubrum.

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.

Contig Mapping↗

Cloning and sequence analysis of poly(tetramethylene succinate) depolymerase from Acidovorax delafieldii strain BS-3.

A gene encoding poly(tetramethylene succinate), PBS, depolymerase, pbsA, has been cloned from Acidovorax delafieldii strain BS-3 chromosomal DNA. The clone expressed in Escherichia coli showed the ability to degrade both PBS and poly[(tetramethylene succinate)-co-adipate] that are kinds of biodegradable plastics. PBS depolymerase was considered to be a kind of lipase, since it also degrades olive oil. It had no apparent hydrophobic-amino-acid-rich region which exists in other known plastic-degrading enzymes. From the result of amino acid homology search, PbsA was found to have some similarities with lipases of Streptomyces sp. and Mollaxella sp. In the motif surrounding the active site Ser residue (Gly-X1-Ser-X2-Gly), PbsA was revealed to have a Trp residue in the X1 position instead of His which is most likely found in other bacterial lipases.

Journal Article↗

Microbial community analysis with a high PHA storage capacity.

Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were affiliated to Azoarcus genus within Betaproteobacteria.

Alkanes↗

Transgenic oil palm: production and projection.

Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020.

Biolistics↗