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.

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.

Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering

2019 ◽  
Vol 103 (11) ◽  
pp. 4313-4324 ◽  
Author(s):  
Ying Ding ◽  
Kai-Feng Wang ◽  
Wei-Jian Wang ◽  
Yi-Rong Ma ◽  
Tian-Qiong Shi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Recombination Efficiency ◽  
Eukaryotic Microorganisms

scholarly journals Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Daniel Sommer ◽  
Annika E. Peters ◽  
Tristan Wirtz ◽  
Maren Mai ◽  
Justus Ackermann ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Mouse Embryos ◽  
Transcription Activator

The mycobacterial efflux pump EfpA can induce high drug tolerance to many anti-tuberculosis drugs, including moxifloxacin, in Mycobacterium smegmatis

2021 ◽  
Author(s):  
Deepika Rai ◽  
Sarika Mehra
Keyword(s):  
Mycobacterium Smegmatis ◽  
Efflux Pump ◽  
Drug Tolerance ◽  
Inducible Promoter ◽  
Major Facilitator Superfamily ◽  
Expression Level ◽  
Efflux Pump Inhibitor ◽  
Gene Encoding ◽  
Active Efflux ◽  
Major Facilitator

Active efflux of drugs across the membrane is a major survival strategy of bacteria against many drugs. In this work, we characterize an efflux pump EfpA, from the major facilitator superfamily, that is highly conserved among both slow growing and fast-growing mycobacterium species and has been found to be upregulated in many clinical isolates of Mycobacterium tuberculosis . The gene encoding EfpA from Mycobacterium smegmatis was over-expressed under both constitutive and an inducible promoter. Expression of efpA gene under both the promoters resulted in greater than 32-fold increased drug tolerance of M. smegmatis cells to many first-line (rifampicin, isoniazid and streptomycin) and second-line (amikacin) anti-tuberculosis drugs. Notably, drug tolerance of M. smegmatis cells to moxifloxacin increased by more than 180-fold when efpA was over-expressed. The increase in minimum inhibitory concentration (MIC) correlated with the decreased uptake of drugs including norfloxacin, moxifloxacin and ethidium bromide and the high MIC could be reversed in the presence of an efflux pump inhibitor. A correlation was observed between the MIC of drugs and the efflux pump expression level, suggesting that the latter could be modulated by varying the expression level of the efflux pump. The expression of high levels of efpA did not impact the fitness of the cells when supplemented with glucose.The efpA gene is conserved across both pathogenic and non-pathogenic mycobacteria. The efpA gene from the Mycobacterium bovis BCG/ M. tuberculosis , which is 80% identical to efpA from M. smegmatis , also led to decreased antimicrobial efficacy to many drugs, although the fold-change was lower. When over-expressed in M. bovis BCG, an 8-fold higher drug tolerance to moxifloxacin was observed . This is the first report of an efflux pump from mycobacterium species that leads to higher drug tolerance to moxifloxacin, a promising new drug for the treatment of tuberculosis.

scholarly journals Genetic Manipulation of a Lipolytic Yeast Candida aaseri SH14 Using CRISPR-Cas9 System

2020 ◽  
Vol 8 (4) ◽  
pp. 526 ◽  
Author(s):  
Zool Hilmi Ibrahim ◽  
Jung-Hoon Bae ◽  
Sun-Hee Lee ◽  
Bong Hyun Sung ◽  
Ahmad Hazri Ab Rashid ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Genetic Manipulation ◽  
Plant Oils ◽  
Long Chain Fatty Acids ◽  
Temporal Expression ◽  
Autonomously Replicating Sequence ◽  
Manipulation System ◽  
Episomal Vectors ◽  
Dodecanedioic Acid ◽  
Diploid Cells

A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A β-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of β-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.

scholarly journals CRISPR-nRAGE, a Cas9 nickase-reverse transcriptase assisted versatile genetic engineering toolkit for E. coli

2020 ◽  
Author(s):  
Yaojun Tong ◽  
Tue S. Jørgensen ◽  
Christopher M. Whitford ◽  
Tilmann Weber ◽  
Sang Yup Lee
Keyword(s):  
Reverse Transcriptase ◽  
Genome Engineering ◽  
Genetic Manipulation ◽  
Fragment Size ◽  
Leukemia Virus ◽  
Strand Break ◽  
Engineering System ◽  
E Coli ◽  
Dna Engineering ◽  
Nucleotide Resolution

AbstractIn most prokaryotes, missing and poorly active non-homologous end joining (NHEJ) DNA repair pathways heavily restrict the direct application of CRISPR-Cas for DNA double-strand break (DSB)-based genome engineering without providing editing templates. CRISPR base editors, on the other hand, can be directly used for genome engineering in a number of bacteria, including E. coli, showing advantages over CRISPR-Cas9, since they do not require DSBs. However, as the current CRISPR base editors can only engineer DNA by A to G or C to T/G/A substitutions, they are incapable of mediating deletions, insertions, and combinations of deletions, insertions and substitutions. To address these challenges, we developed a Cas9 nickase (Cas9n)-reverse transcriptase (Moloney Murine Leukemia Virus, M-MLV) mediated, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes, termed CRISPR-nRAGE (CRISPR-Cas9n Reverse transcriptase Assisted Genome Engineering) system. CRISPR-nRAGE can be used to introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli. Notably, small sized-deletion shows better editing efficiency compared to other kinds of DNA engineering. CRISPR-nRAGE has been used to delete and insert DNA fragments up to 97 bp and 33 bp, respectively. Efficiencies, however, drop sharply with the increase of the fragment size. It is not only a useful addition to the genome engineering arsenal for E. coli, but also may be the basis for the development of similar toolkits for other organisms.

scholarly journals Assembly of Radically Recoded E. coli Genome Segments

2016 ◽  
Author(s):  
Julie E. Norville ◽  
Cameron L. Gardner ◽  
Eduardo Aponte ◽  
Conor K. Camplisson ◽  
Alexandra Gonzales ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Next Generation ◽  
Selective Marker ◽  
E Coli ◽  
Cassette Exchange ◽  
Large Potential ◽  
Attachment Sites

AbstractThe large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

scholarly journals Azithromycin resistance through interspecific acquisition of an epistasis dependent efflux pump component and transcriptional regulator in Neisseria gonorrhoeae

2018 ◽  
Author(s):  
Crista B. Wadsworth ◽  
Brian J. Arnold ◽  
Mohamad R. Abdul Sater ◽  
Yonatan H. Grad
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Promoter Region ◽  
Structural Changes ◽  
Population Genomics ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Full Length ◽  
Mechanism Of Resistance ◽  
Promoter Mutations ◽  
Azithromycin Resistance

ABSTRACTMosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with reduced susceptibility to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hotspot of interspecific recombination introducing alleles from N. meningitidis and N. lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrated that resistance is mediated through epistasis between these two loci and that the full length of the mosaic mtrD allele is required. Gene expression profiling revealed the mechanism of resistance in mosaics couples the novel mtrDalleles with promoter mutations enhancing expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple Neisseria has contributed to azithromycin resistance in the gonococcal population.AUTHOR SUMMARYNeisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for over 100 million cases of gonorrhea worldwide each year. The incidence of reduced susceptibility to the macrolide class antibiotic azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance to this drug remains unexplained. Recently, resistance has been shown to be highly associated with mosaic alleles of the multiple transferable resistance (mtr) efflux pump, which have been gained via horizontal gene exchange with other Neisseria. However, if and how these alleles caused resistance was unknown. Here, we demonstrate that resistance has been gained through epistasis between mtrD and the mtr promoter region using evidence from both population genomics and experimental genetic manipulation. Epistasis also acts within the mtrD locus alone, requiring the full length of the gene for phenotypic resistance. Transcriptomic profiling indicates that the mechanism of resistance in mosaics is likely derived from both structural changes to mtrD, coupled with promoter mutations that result in regulatory changes to mtrCDE.

scholarly journals PARP1 Inhibitor Combined With Oxaliplatin Efficiently Suppresses Oxaliplatin Resistance in Gastric Cancer-Derived Organoids via Homologous Recombination and the Base Excision Repair Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Huafu Li ◽  
Chunming Wang ◽  
Linxiang Lan ◽  
Wenhui Wu ◽  
Ian Evans ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Homologous Recombination ◽  
Base Excision Repair ◽  
Genetic Manipulation ◽  
Excision Repair ◽  
Tumor Formation ◽  
Parp Inhibition ◽  
Repair Pathway ◽  
Base Excision Repair Pathway ◽  
Base Excision

Oxaliplatin (OXA) resistance in the treatment of different types of cancer is an important and complex problem. The culture of tumor organoids derived from gastric cancer can help us to provide a deeper understanding of the underlying mechanisms that lead to OXA resistance. In this study, our purpose was to understand the mechanisms that lead to OXA resistance, and to provide survival benefits to patients with OXA through targeted combination therapies. Using sequence analysis of OXA-resistant and non-OXA-resistant organoids, we found that PARP1 is an important gene that mediates OXA resistance. Through the patients’ follow-up data, it was observed that the expression level of PARP1 was significantly correlated with OXA resistance. This was confirmed by genetic manipulation of PARP1 expression in OXA-resistant organoids used in subcutaneous tumor formation. Results further showed that PARP1 mediated OXA resistance by inhibiting the base excision repair pathway. OXA also inhibited homologous recombination by CDK1 activity and importantly made cancers with normal BRCA1 function sensitive to PARP inhibition. As a result, combination of OXA and Olaparib (PARP-1/2/3 inhibitor), inhibited in vivo and in vitro OXA resistant organoid growth and viability.

Sign in / Sign up

Export Citation Format

Share Document