scholarly journals Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification under High-Level Substrate Loading

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Olusola A. Ogunyewo ◽  
Anmoldeep Randhawa ◽  
Mayank Gupta ◽  
Vemula Chandra Kaladhar ◽  
Praveen Kumar Verma ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Minimal Amount ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Lytic Polysaccharide Monooxygenase ◽  
Content Type ◽  
High Level ◽  
Polysaccharide Monooxygenase

ABSTRACT Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identities with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum, PfMig188 (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig88 overexpressing LPMO or CBH1 increased the saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig188 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig188, which accounted for 82% saccharification of PWS under 20% substrate loading. IMPORTANCE The enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for the efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum. Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for the saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high-level substrate loading using a minimal amount of protein.

scholarly journals Insights into structure of Penicillium funiculosum LPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

2020 ◽  
Author(s):  
Olusola A. Ogunyewo ◽  
Anmoldeep Randhawa ◽  
Mayank Gupta ◽  
Vemula Chandra Kaladhar ◽  
Praveen Kumar Verma ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Genetic Manipulation ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Minimal Amount ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Biomass Hydrolysis ◽  
Lytic Polysaccharide Monooxygenase ◽  
High Substrate

AbstractLytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in natural carbon cycle. These enzymes known to possess auxiliary activity are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized thermostable LPMO from Penicillium funiculosum (PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% structural identity to Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Analysis of the molecular interactions during substrate binding revealed that PfLPMO9 demonstrated a higher binding affinity with a ΔG free energy of -46 k kcal/mol when compared with that of TaLPMO9A (−31 kcal/mol). The enzyme was further found to be highly thermostable at elevated temperature with a half-life of ∼88 h at 50 °C. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress this LPMO and Cellobiohydrolase I (CBH1) in catabolite derepressed strain of Penicillium funiculosum, PfMig188, in order to improve its saccharification performance towards acid pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed ∼200% and ∼66% increase in LPMO and Avicelase activities, respectively. While the secretomes of individually overexpressed LPMO and CBH1-strains increased saccharification of PWS by 6% and 13%, respectively, over PfMig188 at same enzyme concentration, the simultaneous overexpression of these two genes led to 20% increase in saccharification efficiency over PfMig188, which accounted for 82% saccharification of PWS at 20% substrate loading.ImportanceEnzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation bio-based industries. While efforts are being intensified at how best to obtain indigenous cellulase for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge confronting its wide availability for efficient utilization of cellulosic materials. This is because it is challenging to get an enzymatic cocktail with balanced activity from a single host. This report provides for the first time the annotation and structural analysis of an uncharacterized thermostable lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact in biomass deconstruction upon overexpression in catabolite derepressed strain of P. funiculosum. Cellobiohydrolase I (CBH1) which is the most important enzyme produced by many cellulolytic fungi for saccharification of crystalline cellulose was further overexpressed simultaneously with the LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities with the corresponding improvement in its saccharification performance at high substrate loading by ∼20% using a minimal amount of protein.

scholarly journals A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

2016 ◽  
Vol 82 (22) ◽  
pp. 6557-6572 ◽  
Author(s):  
Yuka Kojima ◽  
Anikó Várnai ◽  
Takuya Ishida ◽  
Naoki Sunagawa ◽  
Dejan M. Petrovic ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Viscosity ◽  
Brown Rot ◽  
Primary Cell Wall ◽  
Gloeophyllum Trabeum ◽  
Lytic Polysaccharide Monooxygenase ◽  
Content Type ◽  
Brown Rot Fungus ◽  
Sensitive Method ◽  
Polysaccharide Monooxygenase

ABSTRACTFungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such asGloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome ofG. trabeumencodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants ofGtLPMO9A seem to be produced, a single-domain variant,GtLPMO9A-1, and a longer variant,GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinctGtLPMO9A-2 inPichia pastorisand investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed thatGtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs,GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action ofGtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO,NcLPMO9C fromNeurospora crassarevealed thatGtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity ofNcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities ofGtLPMO9A-2. These results provide insight into the LPMO potential ofG. trabeumand provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.IMPORTANCECurrently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme,GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone.GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential ofGtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

scholarly journals Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Narumon Tangthirasunun ◽  
David Navarro ◽  
Sona Garajova ◽  
Didier Chevret ◽  
Laetitia Chan Ho Tong ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulose Degradation ◽  
Degradation Mechanism ◽  
Podospora Anserina ◽  
Cellulosic Biomass ◽  
Striking Difference ◽  
Crystalline Cellulose ◽  
Minor Role ◽  
Wild Type ◽  
Content Type

ABSTRACT Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

scholarly journals Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface

Science ◽  
2011 ◽  
Vol 333 (6047) ◽  
pp. 1279-1282 ◽  
Author(s):  
Kiyohiko Igarashi ◽  
Takayuki Uchihashi ◽  
Anu Koivula ◽  
Masahisa Wada ◽  
Satoshi Kimura ◽  
...  
Keyword(s):  
High Speed ◽  
Cellulose Degradation ◽  
Polymorphic Form ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Cellobiohydrolase I ◽  
Force Microscopy ◽  
Real Time Visualization ◽  
Enzyme Molecules ◽  
Cellulose Surface

A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

scholarly journals Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

2014 ◽  
Vol 80 (7) ◽  
pp. 2062-2070 ◽  
Author(s):  
Chiaki Hori ◽  
Jill Gaskell ◽  
Kiyohiko Igarashi ◽  
Phil Kersten ◽  
Michael Mozuch ◽  
...  
Keyword(s):  
Cellulose Degradation ◽  
Alcohol Oxidase ◽  
White Rot ◽  
Protein Abundance ◽  
Lytic Polysaccharide Monooxygenase ◽  
Content Type ◽  
Ceriporiopsis Subvermispora ◽  
Liquid Chromatography Tandem Mass ◽  
Nano Liquid Chromatography ◽  
Polysaccharide Monooxygenase

ABSTRACTThe white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such asCeriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue,C. subvermisporawas grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis byC. subvermispora.

scholarly journals Chitinase Expression in Listeria monocytogenes Is Influenced by lmo0327, Which Encodes an Internalin-Like Protein

2017 ◽  
Vol 83 (22) ◽  
Author(s):  
Dafni Katerina Paspaliari ◽  
Vicky Gaedt Kastbjerg ◽  
Hanne Ingmer ◽  
Magdalena Popowska ◽  
Marianne Halberg Larsen
Keyword(s):  
Listeria Monocytogenes ◽  
Foodborne Pathogen ◽  
Sole Source ◽  
Chitinase Activity ◽  
Transcriptional Level ◽  
Chitinolytic Activity ◽  
Lytic Polysaccharide Monooxygenase ◽  
Content Type ◽  
New Information ◽  
Polysaccharide Monooxygenase

ABSTRACT The chitinolytic system of Listeria monocytogenes thus far comprises two chitinases, ChiA and ChiB, and a lytic polysaccharide monooxygenase, Lmo2467. The role of the system in the bacterium appears to be pleiotropic, as besides mediating the hydrolysis of chitin, the second most ubiquitous carbohydrate in nature, the chitinases have been deemed important for the colonization of unicellular molds, as well as mammalian hosts. To identify additional components of the chitinolytic system, we screened a transposon mutant library for mutants exhibiting impaired chitin hydrolysis. The screening yielded a mutant with a transposon insertion in a locus corresponding to lmo0327 of the EGD-e strain. lmo0327 encodes a large (1,349 amino acids [aa]) cell wall-associated protein that has been proposed to possess murein hydrolase activity. The single inactivation of lmo0327, as well as of lmo0325 that codes for a putative transcriptional regulator functionally related to lmo0327, led to an almost complete abolishment of chitinolytic activity. The effect could be traced at the transcriptional level, as both chiA and chiB transcripts were dramatically decreased in the lmo0327 mutant. In accordance with that, we could barely detect ChiA and ChiB in the culture supernatants of the mutant strain. Our results provide new information regarding the function of the lmo0325-lmo0327 locus in L. monocytogenes and link it to the expression of chitinolytic activity. IMPORTANCE Many bacteria from terrestrial and marine environments express chitinase activities enabling them to utilize chitin as the sole source of carbon and nitrogen. Interestingly, several bacterial chitinases may also be involved in host pathogenesis. For example, in the important foodborne pathogen Listeria monocytogenes, the chitinases ChiA and ChiB and the lytic polysaccharide monooxygenase Lmo2467 are implicated in chitin assimilation but also act as virulence factors during the infection of mammalian hosts. Therefore, it is important to identify their regulators and induction cues to understand how the different roles of the chitinolytic system are controlled and mediated. Here, we provide evidence for the importance of lmo0327 and lmo0325, encoding a putative internalin/autolysin and a putative transcriptional activator, respectively, in the efficient expression of chitinase activity in L. monocytogenes and thereby provide new information regarding the function of the lmo0325-lmo0327 locus.

scholarly journals Detection and characterization of a novel copper-dependent intermediate in a lytic polysaccharide monooxygenase

2019 ◽  
Author(s):  
Raushan K. Singh ◽  
Bart v. Oort ◽  
Benedikt Möllers ◽  
David A. Russo ◽  
Ranjitha Singh ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Crystalline Cellulose ◽  
Lytic Polysaccharide Monooxygenase ◽  
Paramagnetic Resonance ◽  
Oxidative Reaction ◽  
Polysaccharide Monooxygenases ◽  
Copper Center ◽  
Electron Paramagnetic ◽  
Polysaccharide Monooxygenase

AbstractLytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose and the enzyme has large practical application in the process of refining biomass. The LPMO catalytic mechanism still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t½= 6 – 8 minutes) observed in an LPMO fromThermoascus aurantiacus(TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-Cu(I) reacts with H2O2. UV-vis absorption spectroscopy, electron paramagnetic resonance (EPR), and stopped-flow spectroscopy indicate that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). We propose that the reaction with H2O2first forms a highly reactive short-lived Cu(III)-intermediate which is subsequently transformed into the observed long-lived copper-dependent intermediate. Since sub-equimolar amount of H2O2to LPMO boosts oxidation of phosphoric acid swollen cellulose (PASC) suggests that the long-lived copper-dependent intermediate is part of the catalytic mechanism for LPMOs. The proposed mechanism offers new perspectives in the oxidative reaction mechanism of copper enzymes and hence for the biomass oxidation and the reactivity of copper in biological systems.

Construction of thermostable cellobiohydrolase I from the fungus Talaromyces cellulolyticus by protein engineering

2019 ◽  
Vol 32 (1) ◽  
pp. 33-40
Author(s):  
Makoto Nakabayashi ◽  
Saori Kamachi ◽  
Dominggus Malle ◽  
Toshiaki Yanamoto ◽  
Seiichiro Kishishita ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Structural Information ◽  
Expression System ◽  
Complex Structure ◽  
Hydrolytic Activity ◽  
Cellulosic Biomass ◽  
Crystalline Cellulose ◽  
Cellobiohydrolase I ◽  
Positive Effects ◽  
Talaromyces Cellulolyticus

Abstract Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Anmoldeep Randhawa ◽  
Nandita Pasari ◽  
Tulika Sinha ◽  
Mayank Gupta ◽  
Anju M. Nair ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Drug Tolerance ◽  
Growth Media ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

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