scholarly journals Catabolism of alkylphenols inRhodococcusvia ameta-cleavage pathway associated with genomic islands

2019 ◽  
Author(s):  
David J. Levy-Booth ◽  
Morgan M. Fetherolf ◽  
Gordon Stewart ◽  
Jie Liu ◽  
Lindsay D. Eltis ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Plant Biomass ◽  
Specific Activity ◽  
Industrial Applications ◽  
Genomic Islands ◽  
Phylogenomic Analysis ◽  
Rhodococcus Rhodochrous ◽  
Extradiol Dioxygenase ◽  
Lignin Depolymerization ◽  
Rhodococcus Jostii

AbstractThe bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well elucidated. We isolatedRhodococcus rhodochrousEP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 andRhodococcus jostiiRHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by theaphABCDEFGHIQRSgenes that degrades 4-ethylphenol via themeta-cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by theaphABgenes, which were up-regulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2000 U/mg) but did not detectably transform phenol. Nevertheless, a ΔaphAmutant in RHA1 grew on 4-ethylphenol by compensatory up-regulation of phenol hydroxylase genes (pheA1-3). Deletion ofaphC, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption ofpcaLin the β-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-ethylphenol and 4-propylphenol are catabolized exclusively viameta-cleavage in rhodococci while phenol is subject toortho-cleavage. Putative genomic islands encodingaphgeneswere identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.ImportanceElucidation of bacterial alkylphenol catabolism is important for the development of biotechnologies to upgrade the lignin component of plant biomass. We isolated a new strain,Rhodococcus rhodochrousEP4, on 4-ethylphenol, an alkylphenol that occurs in lignin-derived streams, including reductive catalytic fractionation products of corn stover. We further demonstrated its degradation via ameta-cleavage pathway (Aph) with transcriptomics. A new class of Actinobacterial hydroxylase, AphAB, acts specifically on alkylphenols. Phylogenomic analysis indicated that theaphgenes occur on putative genomic islands in several rhodococcal strains. These genes were identified in the genetically-tractable strainRhodococcus jostiiRHA1. Strains missing this element cannot metabolise 4-ethylphenol and 4-propylphenol. Overall, we advanced the understanding of how aromatic compounds are degraded by environmental bacteria and identified enzymes that can be employed in the transition away from petro-chemicals towards renewable alternatives.

scholarly journals Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Jingen Li ◽  
Shuying Gu ◽  
Zhen Zhao ◽  
Bingchen Chen ◽  
Qian Liu ◽  
...  
Keyword(s):  
Malic Acid ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Cellulase Activity ◽  
Industrial Applications ◽  
Efficient Production ◽  
Myceliophthora Thermophila ◽  
Final Strain ◽  
Cellobiose Metabolism ◽  
Cost Efficient

Abstract Background Lignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered. Results Using the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by β-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular β-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that β-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207. Conclusions This is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

scholarly journals Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bryan Angelo P. Roxas ◽  
Jennifer Lising Roxas ◽  
Rachel Claus-Walker ◽  
Anusha Harishankar ◽  
Asad Mansoor ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Diarrheal Disease ◽  
The United States ◽  
Genomic Islands ◽  
Rapid Expansion ◽  
Phylogenomic Analysis ◽  
Community Settings ◽  
Hamster Model ◽  
Clostridioides Difficile ◽  
Healthcare Associated

AbstractClostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

scholarly journals Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Selfela Restu Adina ◽  
Antonius Suwanto ◽  
Anja Meryandini ◽  
Esti Puspitasari
Keyword(s):  
Fatty Acid ◽  
Pichia Pastoris ◽  
Specific Activity ◽  
Signal Sequence ◽  
Micrococcus Luteus ◽  
Acid Methyl Ester ◽  
Industrial Applications ◽  
Expression Level ◽  
Lipase Gene ◽  
Acidic Lipase

Abstract Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

scholarly journals Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

2017 ◽  
Vol 7 (4) ◽  
pp. 1 ◽  
Author(s):  
Sreedevi Basavaraju ◽  
Chandrasekhar Kathera ◽  
Pramoda Kumari Jasti
Keyword(s):  
Bacillus Cereus ◽  
Ammonium Sulphate ◽  
Alkaline Protease ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Tween 20 ◽  
Original Activity ◽  
Protease Enzyme

The alkaline protease produced by Bacillus cereus UV-15 mutant was purified by precipitation with ammonium sulphate and gel filtration through sephadex G-100. The enzyme has shown to have a molecular weight of 29kDa by SDS polyacrylamide gel electrophoresis. The extracted protease enzyme was purified by 16.64 fold through ammonium sulphate precipitation and chromatography separation in Sephadex G-100. The purified protease had a specific activity of 2915 (U/mg). The zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which coincided with the band obtained with SDS–PAGE. The enzyme was remained active and stable at pH 8-11, with an optimum at pH 10.0. The protease was stable in the temperature ranging from 40°C to 60°C, but gradually decreased at temperature 70°C. The optimum temperature for protease activity was determined at 60°C. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. At 1% concentration of Tween-20 and Tween-80, the enzyme retained 78% and 94% relative activity respectively. Alkaline protease retained 95% activity toward 0.5% concentration of the anionic detergent SDS. The enzyme showed compatibility at 50°C with commercial detergents such as Ariel, Surf excel, Rin, wheel, Tide and Nirma. In the presence of Ariel and Rin the enzyme retained about 72 and 75% of the original activity respectively. The supplementation of the enzyme in detergents could improve the cleansing performance towards the blood stains and suggested to be used as a detergent additive. The enzyme also removed goat hide hairs completely after 15 hr of incubation. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

scholarly journals Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

2017 ◽  
Author(s):  
Chenlu Zhang ◽  
Ligia Acosta-Sampson ◽  
Vivian Yaci Yu ◽  
Jamie H. D. Cate
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Plant Biomass ◽  
Carbon Sources ◽  
Industrial Applications ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cellulosic Biofuel ◽  
Economic Production ◽  
Report Analysis ◽  
Selection Medium

AbstractThe economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

scholarly journals Introducing a Thermo-Alkali-Stable, Metallic Ion-Tolerant Laccase Purified From White Rot Fungus Trametes hirsuta

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Si ◽  
Hongfei Ma ◽  
Yongjia Cao ◽  
Baokai Cui ◽  
Yucheng Dai
Keyword(s):  
Specific Activity ◽  
Endocrine Disrupting Chemicals ◽  
Environmental Pollutants ◽  
Fold Increase ◽  
Industrial Applications ◽  
White Rot ◽  
White Rot Fungus ◽  
Metallic Ions ◽  
Trametes Hirsuta ◽  
Ph Range

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.

Targeted analysis of phenolic compounds by LC-MS v1

2021 ◽  
Author(s):  
Bashar Amer ◽  
Ramu Kakumanu ◽  
yangtian not provided ◽  
Aymerick Eudes ◽  
Edward EK Baidoo
Keyword(s):  
Phenolic Compounds ◽  
Plant Biomass ◽  
Reversed Phase ◽  
Industrial Applications ◽  
Liquid Chromatography Mass Spectrometry ◽  
Reversed Phase Liquid Chromatography ◽  
Sample Matrix ◽  
Targeted Analysis ◽  
Challenges And Opportunities ◽  
Phase Liquid

Cell-wall-bound (CWB) aromatics such as ferulate and p-coumarate play important physiological roles in plant development and response to stresses. Their presence also poses some challenges and opportunities during processing of plant biomass in various agro-industrial applications. To this end, we have developed a robust high-throughput reversed-phase liquid chromatography mass spectrometry method for quantifying CWB phenolic compounds. The method showed excellent linearity (R2 = ≥0.999) and intraday retention time repeatability (≤ 0.31 %RSD) for ferulate and p-coumarate. The limits of detection and quantitation for these analytes were ≤ 39 nM and 130 nM, respectively. Furthermore, there was very little effect of the CWB sample matrix on the retention times of the analytes and analyte percent recoveries from the CWB sample matrix was ≥83.91%.

A novel Swollenin from Talaromyces leycettanus JCM12802 exhibits cellulase activity and enhanced cellulase hydrolysis towards various substrates

2020 ◽  
Author(s):  
Honghai Zhang ◽  
Yuan Wang ◽  
Roman Brunecky ◽  
Bin Yao ◽  
Xiangming Xie ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Functional Diversity ◽  
Specific Activity ◽  
Biomass Conversion ◽  
Hydrolytic Enzymes ◽  
Cellulase Activity ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Carbohydrate Binding ◽  
Cell Wall Polysaccharides

Abstract Background Swollenins are present in some fungal species involved in the biodegradation of cellulosic substrates. They appear to promote a rearrangement in the network of non-covalent interactions between the cell wall polysaccharides, thus making it more accessible for degradation by hydrolytic enzymes. Here, we have reported a detailed characterization of a recombinant swollenin with respect to its disruptive activity on cellulosic substrates and synergistic effect with cellulases. Results In the present study, a novel swollenin gene Tlswo consisting of an open reading frame encoding 503 amino acids was identified from Talaromyces leycettanus JCM12802 and successfully expressed in Trichoderma reesei and Pichia pastoris. Similar to other fungal swollenins, TlSWO contained a N-terminal family 1 carbohydrate binding module (CBM1) followed by a Ser/Thr rich linker connected to expansin-like domain which includes a family 45 endoglucanase-like domain and group-2 grass pollen allergen domain. TlSWO demonstrated disruptive activity on Avicel and displayed a high synergistic effect with cellobiohydrolases, enhancing its hydrolytic performance up to 132%. The activity of TlSWO on various substrates and biomass was also examined. It was shown that TlSWO could release reducing sugars from lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of TlSWO towards the substates above is 9.0 ± 0.100 U/mg, 8.9 ± 0.100U/mg, 2.3 ± 0.002 U/mg and 0.79 ± 0.002 U/mg respectively. Moreover, TlSWO exhibits maximum activity at pH 4.0 and 50 ℃. Conclusion This study reported on a novel swollenin with highly efficient for biomass conversion. It also reveals the functional diversity of swollenin with activity on various substrates. Although the exact mechanism of swollenin catalytic action activity still remains unknown, the functional diversity of TlSWO makes it a good candidate for industrial applications.

scholarly journals A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
James Round ◽  
Raphael Roccor ◽  
Shu-Nan Li ◽  
Lindsay D. Eltis
Keyword(s):  
Specific Activity ◽  
Dry Weight ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
Wild Type ◽  
Considerable Potential ◽  
Commodity Chemicals ◽  
Acyl Coenzyme A ◽  
Rhodococcus Jostii

ABSTRACT Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon- or nitrogen-limited medium. However, the ΔfcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ∼13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C17 versus C16). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.

scholarly journals Macroalgae Derived Fungi Have High Abilities to Degrade Algal Polymers

2019 ◽  
Vol 8 (1) ◽  
pp. 52 ◽  
Author(s):  
Aleksandrina Patyshakuliyeva ◽  
Daniel L. Falkoski ◽  
Ad Wiebenga ◽  
Klaas Timmermans ◽  
Ronald P. de Vries
Keyword(s):  
Algal Biomass ◽  
Marine Fungi ◽  
Plant Biomass ◽  
Brown Seaweed ◽  
Industrial Applications ◽  
Good Growth ◽  
Biomass Degradation ◽  
Degrading Enzymes ◽  
Green Algal ◽  
Poor Understanding

Marine fungi associated with macroalgae are an ecologically important group that have a strong potential for industrial applications. In this study, twenty-two marine fungi isolated from the brown seaweed Fucus sp. were examined for their abilities to produce algal and plant biomass degrading enzymes. Growth of these isolates on brown and green algal biomass revealed a good growth, but no preference for any specific algae. Based on the analysis of enzymatic activities, macroalgae derived fungi were able to produce algae specific and (hemi-)cellulose degrading enzymes both on algal and plant biomass. However, the production of algae specific activities was lower than the production of cellulases and xylanases. These data revealed the presence of different enzymatic approaches for the degradation of algal biomass by macroalgae derived fungi. In addition, the results of the present study indicate our poor understanding of the enzymes involved in algal biomass degradation and the mechanisms of algal carbon source utilization by marine derived fungi.

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