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1.
ACS Synth Biol ; 9(9): 2252-2257, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32841560

RESUMO

Combinatorial metabolic engineering has been widely established for the development of efficient microbial cell factories to produce the products of interest by precisely regulating the expression levels of multiple genes simultaneously. Here, we report a novel multifunctional CRISPR system that enables simultaneous gene activation, repression, and editing (CRISPR-ARE) with a single Cas9-VPR protein for combinatorial metabolic engineering applications in Saccharomyces cerevisiae. Via gRNA engineering, we achieved orthogonal transcriptional regulations and genome editing using the nuclease active Cas9-VPR fusion protein, individually or in a combinatorial manner. After establishing a system for stable expression of multiple gRNAs on the same plasmid, we first demonstrated CRISPR-ARE for simultaneous mCherry activation, mVenus repression, and ADE2 disruption in a fluorescence reporter strain. Subsequently, we adopted CRISPR-ARE for simple and fast combinatorial metabolic engineering, which improved the production of α-santalene for 2.66-fold in a single step. Because of its simplicity and modularity, the developed CRISPR-ARE system could be applied for facile multifunctional metabolic engineering of microbial cell factories, particularly for which only a few CRISPR proteins have been characterized.

2.
Appl Microbiol Biotechnol ; 104(10): 4303-4311, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32221689

RESUMO

High-temperature fermentation using thermophilic microorganisms may provide cost-effective processes for the industrial production of fuels and chemicals, due to decreased hygiene and cooling costs. In the present study, the genetically trackable thermophile Parageobacillus thermoglucosidasius DSM2542T was engineered to produce (2R, 3R)-butanediol (R-BDO), a valuable chemical with broad industrial applications. The R-BDO biosynthetic pathway was optimized by testing different combinations of pathway enzymes, with acetolactate synthase (AlsS) from Bacillus subtilis and acetolactate decarboxylase (AlsD) from Streptococcus thermophilus yielding the highest production in P. thermoglucosidasius DSM2542T. Following fermentation condition optimization, shake flask fermentation at 55 °C resulted in the production of 7.2 g/L R-BDO with ~ 72% theoretical yield. This study details the microbial production of R-BDO at the highest fermentation temperature reported to date and demonstrates that P. thermoglucosidasius DSM2542T is a promising cell factory for the production of fuels and chemicals using high-temperature fermentation.

3.
Chembiochem ; 21(5): 644-649, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-31482654

RESUMO

Pyrazomycin is a rare C-nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that the StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen-nitrogen bond-forming enzyme that catalyzes the linkage of the ϵ-NH2 nitrogen atom of l-N6 -OH-lysine and the α-NH2 nitrogen atom of l-glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.

4.
Nat Commun ; 10(1): 5794, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31857575

RESUMO

Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype-phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Here we report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.


Assuntos
Sistemas CRISPR-Cas/genética , Mapeamento Cromossômico/métodos , Genoma Fúngico/genética , Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Biotecnologia/métodos , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Biblioteca Genômica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Fenótipo , Saccharomyces cerevisiae/genética
5.
Nat Commun ; 10(1): 5150, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723141

RESUMO

Large-scale data acquisition and analysis are often required in the successful implementation of the design, build, test, and learn (DBTL) cycle in biosystems design. However, it has long been hindered by experimental cost, variability, biases, and missed insights from traditional analysis methods. Here, we report the application of an integrated robotic system coupled with machine learning algorithms to fully automate the DBTL process for biosystems design. As proof of concept, we have demonstrated its capacity by optimizing the lycopene biosynthetic pathway. This fully-automated robotic platform, BioAutomata, evaluates less than 1% of possible variants while outperforming random screening by 77%. A paired predictive model and Bayesian algorithm select experiments which are performed by Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB). BioAutomata excels with black-box optimization problems, where experiments are expensive and noisy and the success of the experiment is not dependent on extensive prior knowledge of biological mechanisms.


Assuntos
Algoritmos , Automação , Teorema de Bayes , Vias Biossintéticas , Licopeno/metabolismo , Modelos Teóricos
6.
Appl Microbiol Biotechnol ; 103(21-22): 8911-8922, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31583448

RESUMO

Corynebacterium glutamicum is a versatile workhorse for producing industrially important commodities. The design of an optimal strain often requires the manipulation of metabolic and regulatory genes to different levels, such as overexpression, downregulation, and deletion. Unfortunately, few tools to achieve multiple functions simultaneously have been reported. Here, a dual-functional clustered regularly interspaced short palindromic repeats (CRISPR) (RE-CRISPR) system that combined genome editing and transcriptional repression was designed using a catalytically active Cas12a (a.k.a. Cpf1) in C. glutamicum. Firstly, gene deletion was achieved using Cas12a under a constitutive promoter. Then, via engineering of the guide RNA sequences, transcriptional repression was successfully achieved using a catalytically active Cas12a with crRNAs containing 15 or 16 bp spacer sequences, whose gene repression efficiency was comparable to that of the canonical system (deactivated Cas12a with full-length crRNAs). Finally, RE-CRISPR was developed to achieve genome editing and transcriptional repression simultaneously by transforming a single crRNA plasmid and Cas12a plasmid. The application of RE-CRISPR was demonstrated to increase the production of cysteine and serine for ~ 3.7-fold and 2.5-fold, respectively, in a single step. This study expands the application of CRISPR/Cas12a-based genome engineering and provides a powerful synthetic biology tool for multiplex metabolic engineering of C. glutamicum.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Cisteína/biossíntese , Endodesoxirribonucleases/genética , Edição de Genes/métodos , Engenharia Metabólica/métodos , Serina/biossíntese , Proteínas de Bactérias/metabolismo , Reatores Biológicos/microbiologia , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endodesoxirribonucleases/metabolismo , Deleção de Genes , Genoma Bacteriano/genética , Redes e Vias Metabólicas/genética , RNA Guia/genética
7.
Biotechnol Bioeng ; 116(12): 3312-3323, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31478186

RESUMO

S-Adenosyl-l-methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l-methionine is generally used, the low-cost dl-methionine is more suitable as the substrate for industrial production of SAM. However, d-methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl-methionine, intracellular conversion of d-methionine to l-methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d-amino acid-N-acetyltransferase, d-methionine was accumulated in vivo and no N-acetyl-d-methionine production was observed. Further, codon-optimized d-amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l-phenylalanine dehydrogenase gene (l-PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d-methionine to l-methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d-methionine. Using the recently developed CRISPR tools, the DAAO and l-PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl-methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl-methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl-methionine.

8.
J Ind Microbiol Biotechnol ; 46(12): 1685-1695, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31420796

RESUMO

Glutathione is a bioactive tripeptide composed of glycine, L-cysteine, and L-glutamate, and has been widely used in pharmaceutical, food, and healthy products. The current metabolic studies of glutathione were mainly focused on the native producing strains with precursor amino acid supplementation. In the present work, Corynebacterium glutamicum, a workhorse for industrial production of a series of amino acids, was engineered to produce glutathione. First, the introduction of glutathione synthetase gene gshF from Streptococcus agalactiae fulfilled the ability of glutathione production in C. glutamicum and revealed that L-cysteine was the limiting factor. Then, considering the inherent capability of L-glutamate synthesis and the availability of external addition of low-cost glycine, L-cysteine biosynthesis was enhanced using a varieties of pathway engineering methods, such as disrupting the degradation pathways of L-cysteine and L-serine, and removing the repressor responsible for sulfur metabolism. Finally, the simultaneously introduction of gshF and enhancement of cysteine formation enabled C. glutamicum strain to produce glutathione greatly. Without external addition of L-cysteine and L-glutamate, 756 mg/L glutathione was produced. This is first time to demonstrate the potential of the glutathione non-producing strain C. glutamicum for glutathione production and provide a novel strategy to construct glutathione-producing strains.


Assuntos
Corynebacterium glutamicum/metabolismo , Glutationa/biossíntese , Corynebacterium glutamicum/genética , Cisteína/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Serina/metabolismo
9.
Metab Eng ; 2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31085296

RESUMO

Biotin (Vitamin H or B7) is one of the most important cofactors involved in central metabolism of pro- and eukaryotic cells. Currently, chemical synthesis is the only route for commercial production. This study reports efficient microbial production of biotin in Pseudomonas mutabilis via multi-level metabolic engineering strategies: Level 1, overexpressing rate-limiting enzyme encoding genes involved in biotin synthesis (i.e. promoter and ribosome binding site engineering); Level 2, deregulating biotin biosynthesis (i.e. deletion of the negative regulator and the biotin importer genes); Level 3, enhancing the supply of co-factors (i.e. S-adenosyl-L-methionine and [Fe-S] cluster) for biotin biosynthesis; Level 4, increasing the availability of the precursor pimelate thioester (i.e. introduction of the BioW-BioI pathway from Bacillus subtilis). The combination of these interventions resulted in the establishment of a biotin overproducing strain, with the secretion of biotin increased for more than 460-fold. In combination with bioprocess engineering efforts, biotin was produced at a final titer of 87.17 mg/L in a shake flask and 271.88 mg/L in a fed-batch fermenter with glycerol as the carbon source. This is the highest biotin titer ever reported so far using rationally engineered microbial cell factories.

10.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
11.
ACS Synth Biol ; 8(5): 1047-1054, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31013062

RESUMO

Golden Gate assembly is one of the most widely used DNA assembly methods due to its robustness and modularity. However, despite its popularity, the need for BsaI-free parts, the introduction of scars between junctions, as well as the lack of a comprehensive study on the linkers hinders its more widespread use. Here, we first developed a novel sequencing scheme to test the efficiency and specificity of 96 linkers of 4-bp length and experimentally verified these linkers and their effects on Golden Gate assembly efficiency and specificity. We then used this sequencing data to generate 200 distinct linker sets that can be used by the community to perform efficient Golden Gate assemblies of different sizes and complexity. We also present a single-pot scarless Golden Gate assembly and BsaI removal scheme and its accompanying assembly design software to perform point mutations and Golden Gate assembly. This assembly scheme enables scarless assembly without compromising efficiency by choosing optimized linkers near assembly junctions.


Assuntos
Software , Biologia Sintética/métodos , DNA/química , DNA/metabolismo
12.
Biomed Res Int ; 2018: 3560894, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30363687

RESUMO

Aquaporins (AQPs) are widely applied in biomimetic membranes for water recycling and desalination. In this study, a novel aquaporin was isolated from Photobacterium profundum SS9 (AQP SS9), which showed high water permeability and potential for practical water purification applications. To improve the stability of the AQP SS9 embedded biomimetic membranes, a modified AQP SS9 was obtained by incorporation of an unnatural amino acid (p-propargyloxyphenylalanine, pPpa) (P-AQP SS9) in vitro using a mutated Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and the cell-free expression system. The modified AQP SS9 can covalently link with phospholipids and hence significantly improve the stability of biomimetic membranes. The concentration of Mg2+ and fusion expression with signal peptides were evaluated to enhance the expression level of P-AQP SS9, resulting in a highest yield of 49 mg/L. The modified AQP SS9 was then reconstituted into DOPC liposomes and analyzed by a stopped-flow spectrophotometer. The obtained water permeability coefficient (Pf) of 7.46×10-4 m/s was 5.7 times higher than that of proteoliposomes with the wild-type AQP SS9 (Pf=1.31×10-4 m/s) and 12.1 times higher than that of the DOPC liposomes (Pf=6.15×10-5m/s). This study demonstrates the development of a cell-free system for the expression of membrane proteins with much higher stability and the potential application of the modified aquaporins for water filtration.


Assuntos
Aminoácidos/química , Aquaporinas/química , Sistema Livre de Células/química , Membranas/química , Animais , Biomimética/métodos , Lipossomos/química , Methanocaldococcus/química , Permeabilidade , Sinais Direcionadores de Proteínas , Proteolipídeos/química , Tirosina-tRNA Ligase/química , Água/química , Purificação da Água/métodos
13.
Methods Enzymol ; 608: 265-276, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30173764

RESUMO

Design and construction of an optimal microbial cell factory typically requires overexpression, knockdown, and knockout of multiple gene targets. In this chapter, we describe a combinatorial metabolic engineering strategy utilizing an orthogonal trifunctional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables multiplexed perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. To implement this system, three orthogonal Cas proteins were utilized: dLbCpf1 fused to a transcriptional activator, dSpCas9 fused to a transcriptional repressor, and SaCas9 for gene deletion. Deletion was accomplished by the introduction of a 28bp frame-shift mutation using a homology donor on the guide RNA expression vector. This approach enables the application of metabolic engineering to systematically optimize phenotypes of interest through a combination of gain-, reduction-, and loss-of-function mutations. Finally, we describe the construction of the CRISPR-AID system and its application toward engineering an example phenotype, surface display of recombinant Trichoderma reesei endoglucanase II.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Celulase/genética , Deleção de Genes , RNA Guia/genética , Proteínas Recombinantes/genética , Ativação Transcricional , Trichoderma/enzimologia , Trichoderma/genética
14.
Synth Syst Biotechnol ; 3(2): 90-96, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29900421

RESUMO

Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS) has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors) to speed up design-build-test (DBT) cycles of metabolic engineering and synthetic biology.

15.
Metab Eng ; 48: 279-287, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29933064

RESUMO

Cellular metabolic networks should be carefully balanced using metabolic engineering to produce the desired products at the industrial scale. As the precursor for the biosynthesis of the neurotransmitter serotonin, 5-hydroxytryptophan (5-HTP) is effective in treating a variety of diseases, such as depression, fibromyalgia, obesity, and cerebellar ataxia. Due to the lack of an efficient synthetic method, commercial production of 5-HTP is only achieved by extracting from the seeds of Griffonia Smplicifolia. This study reports efficient microbial production of 5-HTP via metabolically engineered Escherichia coli. Firstly, human tryptophan hydroxylase I (TPH1) gene was functionally expressed. For endogenous supply of the cofactor tetrahydrobiopterin (BH4), human BH4 biosynthesis and regeneration pathway was reconstituted. Whole-cell bioconversion resulted in high-level production of 5-HTP (~1.2 g/L) from 2 g/L L-tryptophan in shake flasks. Further metabolic engineering efforts were employed to achieve 5-HTP biosynthesis from simple carbon sources. The whole biosynthetic pathway was divided into three functional modules, L-tryptophan module, the hydroxylation module, and the BH4 module. By reducing the copy number of L-tryptophan module, replacing TPH1 with a more stable mutant form, and promoter regulation of the BH4 module, 5-HTP was produced at a final titer of 1.3 g/L in the shake flask and 5.1 g/L in a fed-batch fermenter with glycerol as the carbon source, both of which were the highest ever reported for microbial production of 5-HTP.


Assuntos
5-Hidroxitriptofano , Biopterina/análogos & derivados , Escherichia coli , Engenharia Metabólica , Triptofano Hidroxilase , 5-Hidroxitriptofano/biossíntese , 5-Hidroxitriptofano/genética , Biopterina/biossíntese , Biopterina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Triptofano Hidroxilase/biossíntese , Triptofano Hidroxilase/genética
16.
Metab Eng ; 50: 85-108, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29702275

RESUMO

Metabolic engineering aims to develop efficient cell factories by rewiring cellular metabolism. As one of the most commonly used cell factories, Saccharomyces cerevisiae has been extensively engineered to produce a wide variety of products at high levels from various feedstocks. In this review, we summarize the recent development of metabolic engineering approaches to modulate yeast metabolism with representative examples. Particularly, we highlight new tools for biosynthetic pathway optimization (i.e. combinatorial transcriptional engineering and dynamic metabolic flux control) and genome engineering (i.e. clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system based genome engineering and RNA interference assisted genome evolution) to advance metabolic engineering in yeast. We also discuss the challenges and perspectives for high throughput metabolic engineering.


Assuntos
Sistemas CRISPR-Cas , Genoma Fúngico , Engenharia Metabólica/métodos , Interferência de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
17.
Biotechnol J ; 13(9): e1700601, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29436783

RESUMO

Thanks to its ease of use, modularity, and scalability, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been increasingly used in the design and engineering of Saccharomyces cerevisiae, one of the most popular hosts for industrial biotechnology. This review summarizes the recent development of this disruptive technology for metabolic engineering applications, including CRISPR-mediated gene knock-out and knock-in as well as transcriptional activation and interference. More importantly, multi-functional CRISPR systems that combine both gain- and loss-of-function modulations for combinatorial metabolic engineering are highlighted.


Assuntos
Sistemas CRISPR-Cas , Engenharia Metabólica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
18.
Biotechnol Bioeng ; 115(6): 1552-1560, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29460286

RESUMO

Xylose is a major component of lignocellulosic biomass, one of the most abundant feedstocks for biofuel production. Therefore, efficient and rapid conversion of xylose to ethanol is crucial in the viability of lignocellulosic biofuel plants. In this study, RNAi Assisted Genome Evolution (RAGE) was used to improve the xylose utilization rate in SR8, one of the most efficient publicly available xylose utilizing Saccharomyces cerevisiae strains. To identify gene targets for further improvement, we created a genome-scale library consisting of both genetic over-expression and down-regulation mutations in SR8. Followed by screening in media containing xylose as the sole carbon source, yeast mutants with 29% faster xylose utilization, and 45% higher ethanol productivity were obtained relative to the parent strain. Two known and two new effector genes were identified in these mutant strains. Notably, down-regulation of CDC11, an essential gene, resulted in faster xylose utilization, and this gene target cannot be identified in genetic knock-out screens.


Assuntos
Etanol/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Interferência de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Evolução Molecular Direcionada , Testes Genéticos , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento
19.
Biotechnol Bioeng ; 115(6): 1630-1635, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29460422

RESUMO

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Técnicas de Inativação de Genes , Redes e Vias Metabólicas/genética , Poliploidia
20.
Nat Commun ; 8(1): 1688, 2017 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29167442

RESUMO

Designing an optimal microbial cell factory often requires overexpression, knock-down, and knock-out of multiple gene targets. Unfortunately, such rewiring of cellular metabolism is often carried out sequentially and with low throughput. Here, we report a combinatorial metabolic engineering strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. We demonstrate the application of CRISPR-AID not only to increase the production of ß-carotene by 3-fold in a single step, but also to achieve 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner.


Assuntos
Sistemas CRISPR-Cas , Engenharia Metabólica/métodos , Membrana Celular/metabolismo , Celulase/genética , Celulase/metabolismo , Deleção de Genes , Genes Fúngicos , Redes e Vias Metabólicas/genética , Interferência de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Transcricional , beta Caroteno/biossíntese
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