RESUMO
Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.
Assuntos
Edição de Genes , Saccharomyces cerevisiae , Betaxantinas , Edição de Genes/métodos , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genéticaRESUMO
The success of forward metabolic engineering depends on a thorough understanding of the behaviour of a heterologous metabolic pathway within its host. We have recently described CRI-SPA, a high-throughput gene editing method enabling the delivery of a metabolic pathway to all strains of the Saccharomyces cerevisiae knock-out library. CRI-SPA systematically quantifies the effect of each modified gene present in the library on product synthesis, providing a complete map of host:pathway interactions. In its first version, CRI-SPA relied on the colour of the product betaxanthins to quantify strains synthesis ability. However, only a few compounds produce a visible or fluorescent phenotype limiting the scope of our approach. Here, we adapt CRI-SPA to onboard a biosensor reporting the interactions between host genes and the synthesis of the colourless product cis-cis-muconic acid (CCM). We phenotype >9,000 genotypes, including both gene knock-out and overexpression, by quantifying the fluorescence of yeast colonies growing in high-density agar arrays. We identify novel metabolic targets belonging to a broad range of cellular functions and confirm their positive impact on CCM biosynthesis. In particular, our data suggests a new interplay between CCM biosynthesis and cytosolic redox through their common interaction with the oxidative pentose phosphate pathway. Our genome-wide exploration of host:pathway interaction opens novel strategies for improved production of CCM in yeast cell factories.
Assuntos
Saccharomyces cerevisiae , Ácido Sórbico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Sórbico/análogos & derivados , Ácido Sórbico/metabolismo , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Most microbial species, including model eukaryote Saccharomyces cerevisiae, possess genetic capability to utilize many alternative nutrient sources. Yet, it remains an open question whether these manifest into assimilatory phenotypes. Despite possessing all necessary pathways, S. cerevisiae grows poorly or not at all when glycerol is the sole carbon source. Here we discover, through multiple evolved lineages, genetic determinants underlying glycerol catabolism and the associated fitness trade-offs. Most evolved lineages adapted through mutations in the HOG pathway, but showed hampered osmotolerance. In the other lineages, we find that only three mutations cause the improved phenotype. One of these contributes counter-intuitively by decoupling the TCA cycle from oxidative phosphorylation, and thereby hampers ethanol utilization. Transcriptomics, proteomics and metabolomics analysis of the re-engineered strains affirmed the causality of the three mutations at molecular level. Introduction of these mutations resulted in improved glycerol utilization also in industrial strains. Our findings not only have a direct relevance for improving glycerol-based bioprocesses, but also illustrate how a metabolic pathway can remain unexploited due to fitness trade-offs in other, ecologically important, traits.
Assuntos
Evolução Molecular Direcionada , Glicerol/metabolismo , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.
Assuntos
Cromossomos Fúngicos , Clonagem Molecular/métodos , Mutagênese Insercional , Saccharomyces cerevisiae/genética , Citometria de Fluxo , Expressão Gênica , Ordem dos Genes , Genes Reporter , Recombinação Homóloga , Plasmídeos/genéticaRESUMO
Promoters are crucial elements for engineering microbial production strains used in bioprocesses. For the increasingly popular chassis Komagataella phaffii (formerly Pichia pastoris), a limited number of well-characterized promoters constrain the data-driven engineering of production strains. Here, we present an in silico approach for condition-independent de novo identification of strong native promoters. The method relies on tRNA-codon coadaptation of coding sequences in the K. phaffii genome and is based on two complementary scores: the number of effective codons and the tRNA adaptation index. Genes with high codon bias are expected to be translated efficiently and, thus, also be under control of strong promoters. Using this approach, we identified promising strong promoter candidates and experimentally assessed their activity using fluorescent reporter assays characterizing 50 promoters spanning a 76-fold difference in expression levels in a glucose medium. Overall, we report several promoters that should be added to the molecular toolbox for engineering of K. phaffii and present an approach for identifying promoters in microbial genomes.
Assuntos
Pichia , Saccharomycetales , Pichia/genética , Uso do Códon , Saccharomycetales/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Efficient homologous recombination in baker's yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an Escherichia coli cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput projects depending on DNA construction. With the aim of developing similar tools for filamentous fungi, we first set out to determine the genetic- and sequence-length requirements needed for efficient fusion reactions, and demonstrated that in nonhomologous end-joining deficient strains of Aspergillus nidulans, efficient fusions can be achieved by 25 bp sequence overlaps. Based on these results, we developed a novel fungal in vivo DNA assembly toolbox for simple and flexible genetic engineering of filamentous fungi. Specifically, we have used this method for construction of AMA1-based vectors, complex gene-targeting substrates for gene deletion and gene insertion, and for marker-free CRISPR based gene editing. All reactions were done via single-step transformations involving fusions of up to six different DNA fragments. Moreover, we show that it can be applied in four different species of Aspergilli. We therefore envision that in vivo DNA assembly can be advantageously used for many more purposes and will develop into a popular tool for fungal genetic engineering.
Assuntos
Aspergillus nidulans , Edição de Genes , Edição de Genes/métodos , Marcação de Genes/métodos , Engenharia Genética/métodos , Aspergillus nidulans/genética , DNA Fúngico/genéticaRESUMO
Precise control of the gene copy number in the model yeast Saccharomyces cerevisiae may facilitate elucidation of enzyme functions or, in cell factory design, can be used to optimize production of proteins and metabolites. Currently, available methods can provide high gene-expression levels but fail to achieve accurate gene dosage. Moreover, strains generated using these methods often suffer from genetic instability resulting in loss of gene copies during prolonged cultivation. Here we present a method, CASCADE, which enables construction of strains with defined gene copy number. With our present system, gene(s) of interest can be amplified up to nine copies, but the upper copy limit of the system can be expanded. Importantly, the resulting strains can be stably propagated in selection-free media.
Assuntos
Quebras de DNA de Cadeia Dupla , Amplificação de Genes , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Engenharia Metabólica , Técnicas Microbiológicas , Saccharomyces cerevisiae/metabolismoRESUMO
Halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques do not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism poses further challenges when precise gene targeting is required. In this study, we present a novel plasmid-based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple single-guide RNAs (sgRNAs) that provide the possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed NHEJ-deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support the highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single-stranded DNA oligonucleotides are sufficient for direct repair of DNA breaks induced by sgRNA-Cas9, resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.
RESUMO
Recent sequencing of numerous fungal species revealed large repertoires of putative biotechnologically relevant genes and secondary metabolite gene clusters. However, often the commercial potential of these species is impeded by difficulties to predict host physiological and metabolic compatibility with a given product, and lack of adequate genetic tools. Consequently, most heterologous production is performed in standard hosts where genetic tools and experience are in place. However, these species may not be suitable for all products. To increase chances of successful heterologous production, we have created a flexible platform, DIVERSIFY, for multispecies heterologous gene expression. This reduces the workload to construction of a single gene expression cassette, used to transform all DIVERSIFY strains in order to identify the optimal cell factory host. As proof of principle of the DIVERSIFY concept, we present the first version of our platform, DIVERSIFY 1.0, which we have successfully used for the production of three proteins and a metabolite in four different Aspergilli species, and for the identification of the best producer for each of the products. Moreover, we show that DIVERSIFY 1.0 is compatible with marker-free gene targeting induced by the CRISPR nucleases Cas9 and MAD7.
Assuntos
Fungos/metabolismo , Edição de Genes/métodos , Aspergillus/genética , Aspergillus/metabolismo , Sistemas CRISPR-Cas/genética , Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/metabolismo , Fungos/genética , Glucuronidase/genética , Glucuronidase/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Salicilatos/metabolismoRESUMO
BACKGROUND: CRISPR technology has revolutionized fungal genetic engineering by increasing the speed and complexity of the experiments that can be performed. Moreover, the efficiency of the system often allows genetic engineering to be introduced in non-model species. The efficiency of CRISPR gene editing is due to the formation of specific DNA double-strand breaks made by RNA guided nucleases. In filamentous fungi, only Cas9 has so far been used as the CRISPR nuclease. Since, gene editing with Cas9 is limited by its 5'-NGG-3' protospacer adjacent motif (PAM) sequence, it is important to introduce RNA guided nucleases that depend on other PAM sequences in order to be able to target a larger repertoire of genomic sites. Cpf1 from Lachnospiraceae bacterium employs a PAM sequence composed of 5'-TTTN-3' and therefore serves as an attractive option towards this goal. RESULTS: In this study we showed that Lb_cpf1 codon optimized for Aspergillus nidulans can be used for CRISPR based gene editing in filamentous fungi. We have developed a vector-based setup for Cpf1-mediated CRISPR experiments and showed that it works efficiently at different loci in A. nidulans and in A. niger. Specifically, we used our setup to demonstrate that Cpf1 is able to catalyze oligonucleotide-mediated genomic site-directed mutagenesis and marker-free gene targeting. CONCLUSIONS: In this paper we introduce Cpf1 as a new tool in the fungal CRISPR toolbox. Our experiments demonstrate that Cpf1 can be efficiently used in Aspergilli for gene editing thereby expanding the range of genomic DNA sequences that can be targeted by CRISPR technologies.
RESUMO
Yeast Saccharomyces cerevisiae was engineered for de novo production of six different flavonoids (naringenin, liquiritigenin, kaempferol, resokaempferol, quercetin, and fisetin) directly from glucose, without supplementation of expensive intermediates. This required reconstruction of long biosynthetic pathways, comprising up to eight heterologous genes from plants. The obtained titers of kaempferol 26.57±2.66mgL-1 and quercetin 20.38±2.57mgL-1 exceed the previously reported titers in yeast. This is also the first report of de novo biosynthesis of resokaempferol and fisetin in yeast. The work demonstrates the potential of flavonoid-producing yeast cell factories.
Assuntos
Fermentação , Engenharia Metabólica , Saccharomyces cerevisiae , Vias Biossintéticas , FlavonoidesRESUMO
Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 µ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 µ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme ß-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside.
Assuntos
Amplificação de Genes , Regulação Fúngica da Expressão Gênica , Expressão Gênica , Técnicas de Amplificação de Ácido Nucleico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dosagem de Genes , Genes Reporter , Vetores Genéticos/genética , Glucosídeos/biossíntese , Policetídeos/metabolismo , Recombinação GenéticaRESUMO
The yeast Saccharomyces cerevisiae is a widely used eukaryotic model organism and a key cell factory for production of biofuels and wide range of chemicals. From the broad palette of available yeast strains, the most popular are those derived from laboratory strain S288c and the industrially relevant CEN.PK strain series. Importantly, in recent years these two strains have been subjected to comparative "-omics" analyzes pointing out significant genotypic and phenotypic differences. It is therefore possible that the two strains differ significantly with respect to their potential as cell factories for production of specific compounds. To examine this possibility, we have reconstructed a de novo vanillin-ß-glucoside pathway in an identical manner in S288c and CEN.PK strains. Characterization of the two resulting strains in two standard conditions revealed that the S288c background strain produced up to 10-fold higher amounts of vanillin-ß-glucoside compared to CEN.PK. This study demonstrates that yeast strain background may play a major role in the outcome of newly developed cell factories for production of a given product.