scholarly journals Construction of CRISPR/CAS9 system for industrial Saccharomyces cerevisiae strain and genetic manipulation effect on 2-phenylethanol pathway

2021 ◽  
Author(s):  
Zhiwei Xu ◽  
Zhe Chen ◽  
Lucheng Lin ◽  
Kun Wang ◽  
Jie Sun ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Editing ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Market Demand ◽  
Dna Homology ◽  
Ehrlich Pathway ◽  
Key Factor ◽  
Industrial Strains ◽  
Manipulation Strategy

Abstract Background The market demand for natural 2-phenylethanol (2-PE) continues to increase. Saccharomyces cerevisiae can synthesize 2-PE through the Ehrlich pathway. There are few studies on the improvement of the diploid industrial strains of S. cerevisiae by gene editing technology. There is no report on the comparison of genetic manipulation effect among S.cerevisiae strains with different 2-PE yield background, and the study on knockout of 2-PE downstream product synthesis gene and its effect on the yield of 2-PE have not been found. Results The CRISPR/CAS9 system with high efficiency for diploid S.cerevisiae CWY132 strain for industrial production of 2-PE was constructed. When the length of the homology arm of donor DNA is increased from 60bp to 500bp, the efficiency of gene editing increased from 0–100%. Using CRISPR/CAS9 technology, the branched acetaldehyde dehydrogenase genes ALD2 and ALD3 and the terminal acetyltransferase gene ATF1 in the Ehrlich pathway of S.cerevisiae strains with different 2-PE yields were knocked out. The results showed that in the high-yielding CWY-132 strain, the 2-PE yield decreased from 3.50 g/L to 1.65 g/L when double ALD2 and ALD3 were knocked out, a decrease of 52.8%. When ATF1 was knocked out, the yield of 2-PE decreased to 0.83 g/L, a decrease of 76.2%; In the low-yielding strain PK-2C, the yield of 2-PE increased from 0.21 g/L to 1.20 g/L when ALD2 was knocked out, an increase of 471%. When ATF1 was knocked out, the yield of 2-PE increased to 0.45g/L, an increase of 114%. The results show that the same genetic manipulation strategy for strains with different 2-PE yeilds backgrounds produces significantly different or even opposite effects. In addition, we found that the insufficient supply of NADH in cells can significantly affect the production of 2-PE, and the tolerance of cells to 2-PE is also a key factor that limits the further increase of 2-PE production in high-yielding strain. Conclusions This study shows that the length of the Donor DNA homology arm is a key factor affecting the efficiency of CRISPR/CAS9 gene editing in industrial diploid S. cerevisiae strains. Our result also shows that it is not feasible to increase the 2-PE production in high-yielding strains by blocking the branch pathway in the Ehrlich pathway. Breakthrough in the 2-PE yield of the high-yielding strains requires improved strains’ tolerance to 2-PE and increase the cellular NADH level.

scholarly journals Flavin Adenine Dinucleotide-Dependent Halogenase XanH and Engineering of Multifunctional Fusion Halogenases

2020 ◽  
Vol 86 (18) ◽  
Author(s):  
Lingxin Kong ◽  
Qing Wang ◽  
Zixin Deng ◽  
Delin You
Keyword(s):  
Protein Engineering ◽  
Fusion Protein ◽  
Natural Product ◽  
Flavin Adenine Dinucleotide ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Good Alternative ◽  
Limited Information ◽  
Flavin Reductase ◽  
Free Standing

ABSTRACT Xantholipin (compound 1), a polycyclic xanthone antibiotic, exhibited strong antibacterial activities and showed potent cytotoxicity. The biosynthetic gene cluster of compound 1 has been identified in our previous work, and the construction of xanthone nucleus has been well demonstrated. However, limited information of the halogenation involved in compound 1 biosynthesis is available. In this study, based on the genetic manipulation and biochemical assay, we characterized XanH as an indispensable flavin adenine dinucleotide (FAD)-dependent halogenase (FDH) for the biosynthesis of compound 1. XanH was found to be a bifunctional protein capable of flavin reduction and chlorination and exclusively used the NADH. However, the reduced flavin could not be fully and effectively utilized, and the presence of an extra flavin reductase (FDR) and chemical-reducing agent could promote the halogenation. XanH accepted its natural free-standing substrate with angular fused polycyclic aromatic systems. Meanwhile, it exhibited moderate halogenation activity and possessed high substrate specificity. The requirement of extra FDR for higher halogenation activity is tedious for future engineering. To facilitate efforts in engineering XanH derivative proteins, we constructed the self-sufficient FDR-XanH fusion proteins. The fusion protein E1 with comparable activities to that of XanH could be used as a good alternative for future protein engineering. Taken together, these findings reported here not only improve the understanding of polycyclic xanthones biosynthesis but also expand the substrate scope of FDH and pave the way for future engineering of biocatalysts for new active substance synthesis. IMPORTANCE Halogenation is important in medicinal chemistry and plays an essential role in the biosynthesis of active secondary metabolites. Halogenases have evolved to catalyze reactions with high efficiency and selectivity, and engineering efforts have been made to engage the selective reactivity in natural product biosynthesis. The enzymatic halogenations are an environmentally friendly approach with high regio- and stereoselectivity, which make it a potential complement to organic synthesis. FDHs constitute one of the most extensively elucidated class of halogenases; however, the inventory awaits to be expanded for biotechnology applications and for the generation of halogenated natural product analogues. In this study, XanH was found to reduce flavin and halogenated the freely diffusing natural substrate with an angular fused hexacyclic scaffold, findings which were different from those for the exclusively studied FDHs. Moreover, the FDR-XanH fusion protein E1 with comparable reactivity to that of XanH serves as a successful example of genetic fusions and sets an important stage for future protein engineering.

scholarly journals Rapid target validation in a Cas9-inducible hiPSC derived kidney model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasaman Shamshirgaran ◽  
Anna Jonebring ◽  
Anna Svensson ◽  
Isabelle Leefa ◽  
Mohammad Bohlooly-Y ◽  
...  
Keyword(s):  
High Efficiency ◽  
Disease Modeling ◽  
Genetic Manipulation ◽  
Disease Model ◽  
Genetically Engineered ◽  
Model Systems ◽  
Target Validation ◽  
Direct Differentiation ◽  
Kidney Organoids

AbstractRecent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

scholarly journals Eukaryotic translation factor eIF5A contributes to acetic acid tolerance in Saccharomyces cerevisiae via transcriptional factor Ume6p

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yanfei Cheng ◽  
Hui Zhu ◽  
Zhengda Du ◽  
Xuena Guo ◽  
Chenyao Zhou ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Adaptive Response ◽  
Acid Tolerance ◽  
Peptide Bond ◽  
Transcriptional Factor ◽  
Yeast Cells ◽  
Acetic Acid Tolerance ◽  
Translation Factor ◽  
Industrial Strains

Abstract Background Saccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown. Results In this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance. Conclusion Translation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals A set of novel CRISPR-based integrative vectors for Saccharomyces cerevisiae

2018 ◽  
Vol 3 ◽  
pp. 72
Author(s):  
Peter W Daniels ◽  
Anuradha Mukherjee ◽  
Alastair SH Goldman ◽  
Bin Hu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Manipulation ◽  
Strand Break ◽  
Integration Strategy ◽  
System A ◽  
Target Dna ◽  
Biosynthesis Gene ◽  
Dna Fragment ◽  
Integrative Vectors ◽  
Yeast Genomes

Integrating a desired DNA sequence into yeast genomes is a widely-used genetic manipulation in the budding yeast Saccharomyces cerevisiae. The conventional integration method is to use an integrative plasmid such as pRS or YIplac series as the target DNA carrier. The nature of this method risks multiple integrations of the target DNA and the potential loss of integrated DNA during cell proliferation. In this study, we developed a novel yeast integration strategy based on the widely used CRISPR-Cas9 system and created a set of plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA expression cassettes will induce a double-strand break (DSB) inside a biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated by another plasmid bearing upstream and downstream sequences of the DSB and an integration sequence in between. As a result of this repair the sequence is integrated into genome by replacing the biosynthesis gene, the disruption of which leads to a new auxotrophic genotype. The newly-generated auxotroph can serve as a traceable marker for the integration. In this study, we demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated into the Met15 or Lys2 locus using this system. This novel integration strategy can be applied to various yeasts, including natural yeast isolated from wild environments or different yeast species such as Candida albicans.

Characterization of industrial strains of Saccharomyces cerevisiae exhibiting filamentous growth induced by alcohols and nutrient deprivation

2006 ◽  
Vol 23 (5) ◽  
pp. 697-704 ◽  
Author(s):  
Paula Cristina da Silva ◽  
Jorge Horii ◽  
Viviane Santos Miranda ◽  
Heloísa Gallera Brunetto ◽  
Sandra Regina Ceccato-Antonini
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Filamentous Growth ◽  
Nutrient Deprivation ◽  
Industrial Strains

scholarly journals Gene Transfer to the Desiccation-Tolerant CyanobacteriumChroococcidiopsis

2001 ◽  
Vol 183 (7) ◽  
pp. 2298-2305 ◽  
Author(s):  
Daniela Billi ◽  
E. Imre Friedmann ◽  
Richard F. Helm ◽  
Malcolm Potts
Keyword(s):  
High Efficiency ◽  
Genetic Manipulation ◽  
Recipient Cell ◽  
Conjugative Transfer ◽  
Lower Efficiency ◽  
Genetic Studies ◽  
Variable Regions ◽  
Multiple Cycles ◽  
Experimental Strain

ABSTRACT The coccoid cyanobacterium Chroococcidiopsisdominates microbial communities in the most extreme arid hot and cold deserts. These communities withstand constraints that result from multiple cycles of drying and wetting and/or prolonged desiccation, through mechanisms which remain poorly understood. Here we describe the first system for genetic manipulation ofChroococcidiopsis. Plasmids pDUCA7 and pRL489, based on the pDU1 replicon of Nostoc sp. strain PCC 7524, were transferred to different isolates of Chroococcidiopsisvia conjugation and electroporation. This report provides the first evidence that pDU1 replicons can be maintained in cyanobacteria other than Nostoc and Anabaena. Following conjugation, both plasmids replicated inChroococcidiopsis sp. strains 029, 057, and 123 but not in strains 171 and 584. Both plasmids were electroporated into strains 029 and 123 but not into strains 057, 171, and 584. Expression of P psbA-luxAB on pRL489 was visualized through in vivo luminescence. Efficiencies of conjugative transfer for pDUCA7 and pRL489 into Chroococcidiopsissp. strain 029 were approximately 10−2 and 10−4 transconjugants per recipient cell, respectively. Conjugative transfer occurred with a lower efficiency into strains 057 and 123. Electrotransformation efficiencies of about 10−4electrotransformants per recipient cell were achieved with strains 029 and 123, using either pDUCA7 or pRL489. Extracellular deoxyribonucleases were associated with each of the five strains. Phylogenetic analysis, based upon the V6 to V8 variable regions of 16S rRNA, suggests that desert strains 057, 123, 171, and 029 are distinct from the type species strain Chroococcidiopsis thermalis PCC 7203. The high efficiency of conjugative transfer of Chroococcidiopsis sp. strain 029, from the Negev Desert, Israel, makes this a suitable experimental strain for genetic studies on desiccation tolerance.

scholarly journals Genomic Considerations for the Modification of Saccharomyces cerevisiae for Biofuel and Metabolite Biosynthesis

2020 ◽  
Vol 8 (3) ◽  
pp. 321 ◽  
Author(s):  
James T. Arnone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Native Species ◽  
Genetic Manipulation ◽  
Scale Up ◽  
Biosynthetic Pathways ◽  
Yeast Saccharomyces Cerevisiae ◽  
Position Effects ◽  
Industrial Use ◽  
A Site ◽  
Global Population

The growing global population and developing world has put a strain on non-renewable natural resources, such as fuels. The shift to renewable sources will, thus, help meet demands, often through the modification of existing biosynthetic pathways or the introduction of novel pathways into non-native species. There are several useful biosynthetic pathways endogenous to organisms that are not conducive for the scale-up necessary for industrial use. The use of genetic and synthetic biological approaches to engineer these pathways in non-native organisms can help ameliorate these challenges. The budding yeast Saccharomyces cerevisiae offers several advantages for genetic engineering for this purpose due to its widespread use as a model system studied by many researchers. The focus of this review is to present a primer on understanding genomic considerations prior to genetic modification and manipulation of S. cerevisiae. The choice of a site for genetic manipulation can have broad implications on transcription throughout a region and this review will present the current understanding of position effects on transcription.

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