scholarly journals Employing a Recombinant Strain of Advenella mimigardefordensis for Biotechnical Production of Homopolythioesters from 3,3′-Dithiodipropionic Acid

2012 ◽  
Vol 78 (9) ◽  
pp. 3286-3297 ◽  
Author(s):  
Yongzhen Xia ◽  
Jan Hendrik Wübbeler ◽  
Qingsheng Qi ◽  
Alexander Steinbüchel
Keyword(s):  
Carbon Source ◽  
Dry Weight ◽  
Pha Synthase ◽  
Mineral Salts ◽  
Content Type ◽  
Genes Encoding ◽  
Polyhydroxyalkanoate Synthase ◽  
Encoding Gene ◽  
Different Origins ◽  
Severe Growth

ABSTRACTAdvenella mimigardefordensisstrain DPN7Twas genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3′-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of thebuk-ptboperon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, fromClostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaCAm). These measures yielded the potent PMP production strainA. mimigardefordensisstrain SHX22. The deletion ofmdowas required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-bornebuk-ptboperon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase ofA. mimigardefordensis(phaCAm) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.

scholarly journals From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

2013 ◽  
Vol 79 (12) ◽  
pp. 3582-3589 ◽  
Author(s):  
Daniel Heinrich ◽  
Björn Andreessen ◽  
Mohamed H. Madkour ◽  
Mansour A. Al-Ghamdi ◽  
Ibrahim I. Shabbaj ◽  
...  
Keyword(s):  
Carbon Source ◽  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Biodiesel Production ◽  
Pha Synthase ◽  
Content Type ◽  
Coa Transferase ◽  
Fed Batch Fermentation ◽  
A Cell ◽  
Cultivation Process

ABSTRACTIn 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) ofPseudomonas putidaKT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) ofClostridium propionicumX2, and polyhydroxyalkanoate (PHA) synthase (phaC1) ofRalstonia eutrophaH16 were cloned and expressed in the 1,3-propanediol producerShimwellia blattae. In a two-step cultivation process, recombinantS. blattaecells 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.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe 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 isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed 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 strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof 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. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

2011 ◽  
Vol 77 (9) ◽  
pp. 2847-2854 ◽  
Author(s):  
Charles F. Budde ◽  
Sebastian L. Riedel ◽  
Laura B. Willis ◽  
ChoKyun Rha ◽  
Anthony J. Sinskey
Keyword(s):  
Palm Oil ◽  
Ralstonia Eutropha ◽  
Plasmid Stability ◽  
Dry Weight ◽  
Pha Synthase ◽  
Plant Oil ◽  
Short Chain Length ◽  
Recombinant Gene Expression ◽  
Content Type ◽  
Coa Reductase

ABSTRACTThe polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinantRalstonia eutrophastrains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered twoR. eutrophastrains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacteriumRhodococcus aetherivoransI24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) fromPseudomonas aeruginosawas shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded byphaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so anR. eutrophaplasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase geneproCwas deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

scholarly journals Engineering of a Xylose Metabolic Pathway in Rhodococcus Strains

2012 ◽  
Vol 78 (16) ◽  
pp. 5483-5491 ◽  
Author(s):  
Xiaochao Xiong ◽  
Xi Wang ◽  
Shulin Chen
Keyword(s):  
Carbon Source ◽  
Metabolic Pathway ◽  
Neutral Lipids ◽  
Shuttle Vector ◽  
Dry Weight ◽  
Rhodococcus Opacus ◽  
Xylose Utilization ◽  
Organic Substrates ◽  
Xylose Metabolism ◽  
Content Type

ABSTRACTThe two metabolically versatile actinobacteriaRhodococcus opacusPD630 andR. jostiiRHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway ofd-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes,xylA, encoding xylose isomerase, andxylB, encoding xylulokinase fromStreptomyces lividansTK23, under the control of thetacpromoter with anEscherichia coli-Rhodococcusshuttle vector. The recombinantR. jostiiRHA1 bearingxylAcould grow on xylose as the sole carbon source, and additional expression ofxylBfurther improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strainR. opacusPD630 by expression ofxylAandxylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants ofR. jostiiRHA1 andR. opacusPD630 carryingxylAandxylBrepresented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.

scholarly journals Malic Enzyme and Malolactic Enzyme Pathways Are Functionally Linked but Independently Regulated in Lactobacillus casei BL23

2013 ◽  
Vol 79 (18) ◽  
pp. 5509-5518 ◽  
Author(s):  
José María Landete ◽  
Sergi Ferrer ◽  
Vicente Monedero ◽  
Manuel Zúñiga
Keyword(s):  
Carbon Source ◽  
Gene Cluster ◽  
Malic Enzyme ◽  
Lactobacillus Casei ◽  
Growth Defect ◽  
Two Component System ◽  
Content Type ◽  
Malolactic Enzyme ◽  
Genes Encoding ◽  
Malate Metabolism

ABSTRACTLactobacillus caseiis the only lactic acid bacterium in which two pathways forl-malate degradation have been described: the malolactic enzyme (MLE) and the malic enzyme (ME) pathways. Whereas the ME pathway enablesL. caseito grow onl-malate, MLE does not support growth. Themlegene cluster consists of three genes encoding MLE (mleS), the putativel-malate transporter MleT, and the putative regulator MleR. Themaegene cluster consists of four genes encoding ME (maeE), the putative transporter MaeP, and the two-component system MaeKR. Since both pathways compete for the same substrate, we sought to determine whether they are coordinately regulated and their role inl-malate utilization as a carbon source. Transcriptional analyses revealed that themleandmaegenes are independently regulated and showed that MleR acts as an activator and requires internalization ofl-malate to induce the expression ofmlegenes. Notwithstanding, bothl-malate transporters were required for maximall-malate uptake, although only anmleTmutation caused a growth defect onl-malate, indicating its crucial role inl-malate metabolism. However, inactivation of MLE resulted in higher growth rates and higher final optical densities onl-malate. The limited growth onl-malate of the wild-type strain was correlated to a rapid degradation of the availablel-malate tol-lactate, which cannot be further metabolized. Taken together, our results indicate thatL. caseil-malate metabolism is not optimized for utilization ofl-malate as a carbon source but for deacidification of the medium by conversion ofl-malate intol-lactate via MLE.

Growth of Graphium sp. on natural gas

1969 ◽  
Vol 15 (10) ◽  
pp. 1231-1236 ◽  
Author(s):  
J. E. Zajic ◽  
B. Volesky ◽  
Angela Wellman
Keyword(s):  
Carbon Source ◽  
Natural Gas ◽  
Continuous Culture ◽  
Mixed Culture ◽  
Continuous Cultivation ◽  
Dry Weight ◽  
Mineral Salts ◽  
Ambient Temperatures ◽  
Acid Tolerant ◽  
Enrichment Techniques

A fungus which grows well on a mineral salts solution with natural gas as the carbon source is described and provisionally identified as a Graphium species. Its taxonomic relation to several genera is presented. This organism was isolated from sewage after selection by enrichment techniques and continuous culture. The fermentor was operated at ambient temperatures, 28 °C ± 2, at a volume of 10 liters with a dilution rate of 10 liters/4 days to 10 liters/1.7 days. Coty's mineral salts medium gave the highest tissue yield. When the pH of the incoming mineral salts medium was decreased stepwise from 7.0 to 5.0 the pH of the reactor became self-adjusting, varying from around 2.7 to 3.5, and the dry weight of microbial tissue obtained varied from 65 to 275 mg/h. Also present in the continuous culture was an acid tolerant bacterium, which, when isolated, grew well on natural gas, methanol, and ethanol, and a strain of Trichoderma, which, when isolated, did not use natural gas as a carbon source. In mixed culture the Trichoderma is thought to grow on metabolites produced by either or both the Graphium and the acid-tolerant bacterium during oxidation of natural gas. The nature of the relationship is being investigated. The mixed culture has been under continuous cultivation for 18 months.

scholarly journals Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters

2015 ◽  
Vol 82 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Daiana de Lima-Morales ◽  
Diego Chaves-Moreno ◽  
Melissa L. Wos-Oxley ◽  
Ruy Jáuregui ◽  
Ramiro Vilchez-Vargas ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Contaminated Site ◽  
Degradation Pathways ◽  
Content Type ◽  
Benzene Degradation ◽  
Semialdehyde Dehydrogenase ◽  
Genes Encoding ◽  
Aromatic Degradation ◽  
Pseudomonas Veronii ◽  
Encoding Gene

ABSTRACTPseudomonas veronii1YdBTEX2, a benzene and toluene degrader, andPseudomonas veronii1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR.

scholarly journals Gammaproteobacterial Methanotrophs Dominate Cold Methane Seeps in Floodplains of West Siberian Rivers

2014 ◽  
Vol 80 (19) ◽  
pp. 5944-5954 ◽  
Author(s):  
Igor Y. Oshkin ◽  
Carl-Eric Wegner ◽  
Claudia Lüke ◽  
Mikhail V. Glagolev ◽  
Illiya V. Filippov ◽  
...  
Keyword(s):  
West Siberia ◽  
Dry Weight ◽  
Small Rivers ◽  
Type I ◽  
Content Type ◽  
Cell Counts ◽  
Genes Encoding ◽  
Irtysh Basin ◽  
Methane Fluxes

ABSTRACTA complex system of muddy fluid-discharging and methane (CH4)-releasing seeps was discovered in a valley of the river Mukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH4flux from most (90%) of these gas ebullition sites did not exceed 1.45 g CH4h−1, while some seeps emitted up to 5.54 g CH4h−1. The δ13C value of methane released from these seeps varied between −71.1 and −71.3‰, suggesting its biogenic origin. Although the seeps were characterized by lowin situtemperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH4ml−1day−1) were measured in mud samples. Fluorescencein situhybridization detected 107methanotrophic bacteria (MB) per g of mud (dry weight), which accounted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacterial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing ofpmoAgenes, encoding particulate methane monooxygenase. A total of 53,828pmoAgene sequences of seep-inhabiting methanotrophs were retrieved and analyzed. Nearly all of these sequences affiliated with type I MB, including theMethylobacter-Methylovulum-Methylosomagroup, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuating methane fluxes from these local but strong CH4sources in floodplains of high-latitude rivers have a large proportion of potentially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.

scholarly journals Off-Target Gene Regulation Mediated by Transcriptional Repressors of Antimicrobial Efflux Pump Genes in Neisseria gonorrhoeae

2011 ◽  
Vol 55 (6) ◽  
pp. 2559-2565 ◽  
Author(s):  
Paul J. T. Johnson ◽  
Virginia A. Stringer ◽  
William M. Shafer
Keyword(s):  
Dna Binding ◽  
Neisseria Gonorrhoeae ◽  
Binding Proteins ◽  
Efflux Pump ◽  
Dna Binding Proteins ◽  
Transcriptional Repressors ◽  
Content Type ◽  
Genes Encoding ◽  
Encoding Gene ◽  
Microarray Studies

ABSTRACTDNA-binding proteins that control expression of drug efflux pump genes have been termed “local regulators” as their encoding gene is often located adjacent to the gene(s) that they regulate. However, results from recent studies indicate that they can control genes outside efflux pump-encoding loci, which we term as being “off target.” For example, the MtrR repressor was initially recognized for its ability to repress transcription of themtrCDE-encoded efflux pump operon in the strict human pathogenNeisseria gonorrhoeae, but recent results from genetic and microarray studies have shown that it can control expression of nearly 70 genes scattered throughout the chromosome. One of the off-target MtrR-repressed genes isglnA, which encodes glutamine synthetase. Herein, we confirm the capacity of MtrR to repressglnAexpression and provide evidence that such repression is due to its ability to negatively influence the binding of a second DNA-binding protein (FarR), which activatesglnA. FarR was previously recognized as a transcriptional repressor of thefarAB-encoded efflux pump operon. Thus, two DNA-binding proteins previously characterized as repressors of genes encoding efflux pumps that contribute to gonococcal resistance to antimicrobials can act in an opposing manner to modulate expression of a gene involved in basic metabolism.

scholarly journals Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

2015 ◽  
Vol 81 (24) ◽  
pp. 8294-8306 ◽  
Author(s):  
Heba Khairy ◽  
Jan Hendrik Wübbeler ◽  
Alexander Steinbüchel
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Rhodococcus Erythropolis ◽  
Model Organism ◽  
Hypothetical Protein ◽  
Gene Encoding ◽  
Content Type ◽  
Gene Coding ◽  
Initial Cleavage ◽  
Genes Encoding

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

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