scholarly journals Lactic Acid Containing Polymers Produced in EngineeredSinorhizobium melilotiandPseudomonas putida

2019 ◽  
Author(s):  
Tam T. Tran ◽  
Trevor C. Charles
Keyword(s):  
Lactic Acid ◽  
Sinorhizobium Meliloti ◽  
Pha Synthase ◽  
Broad Host Range ◽  
Phenotypic Analysis ◽  
Chromatography Analysis ◽  
Broad Host Range Plasmid ◽  
Coa Transferase ◽  
Gas Chromatography Analysis ◽  
Genes Encoding

AbstractThis study demonstrates that novel polymer production can be achieved by introducing pTAM, a broad-host-range plasmid expressing codon-optimized genes encodingClostridium propionicumpropionate CoA transferase (PctCp) and a modifiedPseudomonassp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19), intophaCmutant strains of the native polymer producersSinorhizobium melilotiandPseudomonas putida. Both phenotypic analysis and gas chromatography analysis indicated the synthesis and accumulation of biopolymers inS. melilotiandP. putidastrains. Expression inS. melilotiresulted in the production of PLA homopolymer up to 3.2% dried cell weight (DCW). The quaterpolymer P(3HB-co-LA-co-3HHx-co-3HO) was produced by expression inP. putida. TheP. putida phaCmutant strain produced this type of polymer the most efficiently with polymer content of 42% DCW when cultured in defined media with the addition of sodium octanoate, while the greatest LA fraction (4% mol) was achieved in the same strain cultured in LB with the addition of lactic acid. This is the first report, to our knowledge, of the production of a range of different biopolymers using the same plasmid-based system in different backgrounds. In addition, it is the first time that the novel polymer (P(3HB-co-LA-co-3HHx-co-3HO)), has been reported being produced in bacteria.

scholarly journals Plasmid Donor Affects Host Range of Promiscuous IncP-1β Plasmid pB10 in an Activated-Sludge Microbial Community

2005 ◽  
Vol 71 (9) ◽  
pp. 5309-5317 ◽  
Author(s):  
Leen De Gelder ◽  
Frederik P. J. Vandecasteele ◽  
Celeste J. Brown ◽  
Larry J. Forney ◽  
Eva M. Top
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Host Range ◽  
Sinorhizobium Meliloti ◽  
Ralstonia Eutropha ◽  
Rrna Gene ◽  
Broad Host Range ◽  
Donor Strain ◽  
Broad Host Range Plasmid ◽  
Mating Conditions

ABSTRACT Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI q. The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the α- and γ-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.

scholarly journals Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range PromoterPm

2011 ◽  
Vol 77 (8) ◽  
pp. 2648-2655 ◽  
Author(s):  
Rahmi Lale ◽  
Laila Berg ◽  
Friederike Stüttgen ◽  
Roman Netzer ◽  
Marit Stafsnes ◽  
...  
Keyword(s):  
Host Range ◽  
Untranslated Region ◽  
Micrococcus Luteus ◽  
Background Level ◽  
Broad Host Range ◽  
Continuous Control ◽  
Wild Type ◽  
Content Type ◽  
Broad Host Range Plasmid ◽  
Genes Encoding

ABSTRACTThe induciblePmpromoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in thePmDNA region corresponding to the 5′ untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) inEscherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter,P1(Pantitet), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C50carotenoid sarcinaxanthin in an engineered strain ofE. colithat produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing threeMicrococcus luteusderived genes from the promoterPm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.

scholarly journals “Production of D-lactic acid containing polyhydroxyalkanoate (PHA) polymers in yeast Saccharomyces cerevisiae”

2021 ◽  
Author(s):  
Anna Ylinen ◽  
Hannu Maaheimo ◽  
Adina Anghelescu-Hakala ◽  
Merja Penttilä ◽  
Laura Salusjärvi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Hydroxy Acid ◽  
Raw Materials ◽  
Dry Weight ◽  
Pha Synthase ◽  
Bacterial Strains ◽  
Yeast Saccharomyces Cerevisiae ◽  
Coa Transferase ◽  
Coa Reductase

Abstract Polyhydroxyalkanoates (PHA) provide biodegradable and bio-based alternatives to conventional plastics. Incorporation of 2-hydroxy acid monomers into polymer, in addition to 3-hydroxy acids, offers possibility to tailor the polymer properties. In this study, poly(D-lactic acid) (PDLA) and copolymer P(LA-3HB) were produced and characterized for the first time in the yeast Saccharomyces cerevisiae. Expression of engineered PHA synthase PhaC1437Ps6–19, propionyl-CoA transferase Pct540Cp, acetyl-CoA acetyltransferase PhaA, and acetoacetyl-CoA reductase PhaB1 resulted in accumulation of 3.6% P(LA-3HB) and expression of engineered enzymes PhaC1Pre and PctMe resulted in accumulation of 0.73% PDLA of the cell dry weight. According to NMR, P(LA-3HB) contained D-Lactic acid repeating sequences. For reference, expression of PhaA, PhaB1, and PHA synthase PhaC1 resulted in accumulation 11% poly(hydroxybutyrate) (PHB) of the cell dry weight. Weight average molecular weights of these polymers were comparable to similar polymers produced by bacterial strains, 24.6 kDa, 6.3 kDa, and 1 130 kDa, for P(LA-3HB), PDLA, and PHB, respectively. The results suggest that yeast, as a robust and acid tolerant industrial production organism, could be suitable for production of 2-hydroxy acid containing PHAs from sugars or from 2-hydroxy acid containing raw materials. Moreover, the wide substrate specificity of PHA synthase enzymes employed increases the possibilities for modifying copolymer properties in yeast in the future.

scholarly journals Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1521-1531 ◽  
Author(s):  
Magne Østerås ◽  
Shelley A P O'Brien ◽  
Turlough M Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Root Nodules ◽  
Genetic Regulation ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Type Locus ◽  
Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

Diversity of Root Bacteria from Peanut Cropping Systems

2004 ◽  
Vol 31 (2) ◽  
pp. 86-91 ◽  
Author(s):  
D. T. Gooden ◽  
H. D. Skipper ◽  
J. H. Kim ◽  
K. Xiong
Keyword(s):  
Fatty Acids ◽  
Biological Control ◽  
Gas Chromatography ◽  
Cropping Systems ◽  
Cropping System ◽  
Critical Research ◽  
Chromatography Analysis ◽  
Rotation System ◽  
Gas Chromatography Analysis ◽  
Peanut Production

Abstract Rhizobacteria play an important role in sustainable agriculture via plant growth and biological control of pests in a number of ecosystems. Understanding the interactions of crop rotation and rhizobacteria on peanut production is a critical research need. Development of a database on the rhizobacteria obtained from continuous and rotational fields of peanut was initiated in 1997 and terminated in 2000. Peanut was planted in monoculture for 4 yr. In rotational plots, peanut, cotton, corn, and peanut were planted in sequence. Rhizobacteria were isolated from the roots of crop plants grown in a Norfolk soil near Florence, SC. These isolates were identified by composition of fatty acids from gas chromatography analysis (GC/FAME). Arthrobacter and Bacillus were the major genera from non-rhizosphere soils. At initiation of this study in July 1997, the plots selected for continuous peanut had more diversity in rhizobacteria than those plots selected for rotation. In July 2000, rhizobacteria diversity was greater from peanut roots in the rotation cropping system than continuous peanut. Even though rhizobacteria diversity was greater in the rotation system, higher peanut yields were recorded in the continuous peanut system in 2000. Burkholderia spp. were always isolated from the peanut and other crop rhizospheres at each sampling date.

Cloning of the Nocardia corallina polyhydroxyalkanoate synthase gene and production of poly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate)

1998 ◽  
Vol 44 (7) ◽  
pp. 687-691 ◽  
Author(s):  
Brian Hall ◽  
Jennifer Baldwin ◽  
Ho Gun Rhie ◽  
Douglas Dennis
Keyword(s):  
Fatty Acids ◽  
Ralstonia Eutropha ◽  
Carbon Sources ◽  
Pha Synthase ◽  
Broad Host Range ◽  
Coding Region ◽  
Reading Frame ◽  
Nocardia Corallina ◽  
Odd Chain Fatty Acids ◽  
Chain Fatty Acids

The polyhydroxyalkanoate (PHA) synthase gene (phaCNc) from Nocardia corallina was identified in a lambda library on a 6-kb BamHI fragment. A 2.8-kb XhoII subfragment was found to contain the ntact PHA synthase. This 2.8-kb fragment was subjected to DNA sequencing and was found to contain the coding region for the PHA synthase and a small downstream open reading frame of unknown function. On the basis of DNA sequence, phaCNc is closest in homology to the PHA synthases (phaCPaI and phaCPaII) of Pseudomonas aeruginosa (approximately 41% identity and 55% similarity). The 2.8-kb XhoII fragment containing phaCNc was subcloned into broad host range mobilizable plasmids and transferred into Escherichia coli, Klebsiella aerogenes (both containing a plasmid bearing phaA and phaB from Ralstonia eutropha), and PHA-negative strains of R. eutropha and Pseudomonas putida. The recombinant strains were grown on various carbon sources and the resulting polymers were analyzed. In these strains, the PHA synthase from N. corallina was able to mediate the production of poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) containing high levels of 3-hydroxyhexanoate when grown on hexanoate and larger even-chain fatty acids and poly(3-hydroxyvalerate-co-3-hydroxyheptanoate) containing high levels of 3-hydroxyheptanoate when grown on heptanoate or larger odd-chain fatty acids. Key words: polyhydroxyalkanoates (PHAs), Nocardia corallina, biodegradable, polyester.

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