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1.
Appl Microbiol Biotechnol ; 103(11): 4313-4324, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31016357

RESUMO

In recent years, eukaryotic microorganisms have been widely applied to offer many solutions for everyday life and have come to play important roles in agriculture, food, health care, and the fine-chemicals industry. However, the complex genetic background and low homologous recombination efficiency have hampered the implementation of large-scale and high-throughput gene editing in many eukaryotic microorganisms. The low efficiency of homologous recombination (HR) not only makes the modification process labor-intensive but also completely precludes the application of many otherwise very useful genome editing techniques. Thus, increasing the efficiency of HR is clearly an enabling technology for basic research and gene editing in eukaryotic microorganisms. In this review, we summarize the current strategies for enhancing the efficiency of HR in eukaryotic microorganisms (particularly yeasts and filamentous fungi), list some small molecules and candidate genes associated with homologous and non-homologous recombination, and briefly discuss the further development prospects of these strategies.


Assuntos
Fungos/genética , Edição de Genes/métodos , Recombinação Homóloga , Engenharia Metabólica/métodos , Leveduras/genética
2.
Appl Microbiol Biotechnol ; 103(8): 3239-3248, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30877356

RESUMO

Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Microalgas/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica , Marcação de Genes , Engenharia Genética , RNA Guia/genética , Transformação Genética
3.
Bioresour Technol ; 281: 449-456, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30846235

RESUMO

Terpenoids are a large class of natural compounds based on the C5 isoprene unit, with many biological effects such activity against cancer and allergies, while some also have an agreeable aroma. Consequently, they have received extensive attention in the food, pharmaceutical and cosmetic fields. With the identification and analysis of the underlying natural product synthesis pathways, current microbial-based metabolic engineering approaches have yielded new strategies for the production of highly valuable terpenoids. Yarrowia lipolytica is a non-conventional oleaginous yeast that is rapidly emerging as a valuable host for the production of terpenoids due to its own endogenous mevalonate pathway and high oil production capacity. This review aims to summarize the status and strategies of metabolic engineering for the heterologous synthesis of terpenoids in Y. lipolytica in recent years and proposes new methods aiming towards further improvement of terpenoid production.


Assuntos
Engenharia Metabólica/métodos , Terpenos/metabolismo , Yarrowia/metabolismo , Produtos Biológicos/metabolismo
4.
ACS Synth Biol ; 8(2): 445-454, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30616338

RESUMO

The filamentous fungus Fusarium fujikuroi is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research toward making this fungus more efficient and able to produce tailor-made GAs. Here, we established an efficient CRISPR/Cas9-based genome editing tool for F. fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTBNLS) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and 5S rRNA, which were identified in F. fujikuroi, had the highest editing efficiency. The 5S rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What's more, multigene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F. fujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.


Assuntos
Sistemas CRISPR-Cas/genética , Fungos/genética , Fungos/metabolismo , Fusarium/genética , Fusarium/metabolismo , Giberelinas/metabolismo , Edição de Genes/métodos , Genoma Fúngico/genética
5.
Front Microbiol ; 9: 2185, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30298059

RESUMO

The beta-hydroxy acid 3-hydroxypropionic acid (3-HP) is an attractive platform compound that can be used as a precursor for many commercially interesting compounds. In order to reduce the dependence on petroleum and follow sustainable development, 3-HP has been produced biologically from glucose or glycerol. It is reported that 3-HP synthesis pathways can be constructed in microbes such as Escherichia coli, Klebsiella pneumoniae and the yeast Saccharomyces cerevisiae. Among these host strains, yeast is prominent because of its strong acid tolerance which can simplify the fermentation process. Currently, the malonyl-CoA reductase pathway and the ß-alanine pathway have been successfully constructed in yeast. This review presents the current developments in 3-HP production using yeast as an industrial host. By combining genome-scale engineering tools, malonyl-CoA biosensors and optimization of downstream fermentation, the production of 3-HP in yeast has the potential to reach or even exceed the yield of chemical production in the future.

6.
Appl Microbiol Biotechnol ; 102(22): 9541-9548, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30238143

RESUMO

The oleaginous yeast Yarrowia lipolytica is widely used for the production of both bulk and fine chemicals, including organic acids, fatty acid-derived biofuels and chemicals, polyunsaturated fatty acids, single-cell proteins, terpenoids, and other valuable products. Consequently, it is becoming increasingly popular for metabolic engineering applications. Multiple gene manipulation tools including URA blast, Cre/LoxP, and transcription activator-like effector nucleases (TALENs) have been developed for metabolic engineering in Y. lipolytica. However, the low efficiency and time-consuming procedures involved in these methods hamper further research. The emergence of the CRISPR/Cas system offers a potential solution for these problems due to its high efficiency, ease of operation, and time savings, which can significantly accelerate the genomic engineering of Y. lipolytica. In this review, we summarize the research progress on the development of CRISPR/Cas systems for Y. lipolytica, including Cas9 proteins and sgRNA expression strategies, as well as gene knock-out/knock-in and repression/activation applications. Finally, the most promising and tantalizing future prospects in this area are highlighted.


Assuntos
Engenharia Metabólica/métodos , Yarrowia/genética , Yarrowia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/tendências
7.
Appl Microbiol Biotechnol ; 101(20): 7435-7443, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28887634

RESUMO

In recent years, a variety of genetic tools have been developed and applied to various filamentous fungi, which are widely applied in agriculture and the food industry. However, the low efficiency of gene targeting has for many years hampered studies on functional genomics in this important group of microorganisms. The emergence of CRISPR/Cas9 genome-editing technology has sparked a revolution in genetic research due to its high efficiency, versatility, and easy operation and opened the door for the discovery and exploitation of many new natural products. Although the application of the CRISPR/Cas9 system in filamentous fungi is still in its infancy compared to its common use in E. coli, yeasts, and mammals, the deep development of this system will certainly drive the exploitation of fungal diversity. In this review, we summarize the research progress on CRISPR/Cas9 systems in filamentous fungi and finally highlight further prospects in this area.


Assuntos
Sistemas CRISPR-Cas , Fungos/genética , Edição de Genes/métodos , Genoma Fúngico , Microbiologia Industrial/métodos , Agricultura/métodos , Indústria Alimentícia/métodos
8.
Front Microbiol ; 8: 793, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28507542

RESUMO

Microbial oils, which are mainly extracted from yeasts, molds, and algae, have been of considerable interest as food additives and biofuel resources due to their high lipid content. While these oleaginous microorganisms generally produce only small amounts of lipids under optimal growth conditions, their lipid accumulation machinery can be induced by environmental stresses, such as nutrient limitation and an inhospitable physical environmental. As common second messengers of many stress factors, reactive oxygen species (ROS) may act as a regulator of cellular responses to extracellular environmental signaling. Furthermore, increasing evidence indicates that ROS may act as a mediator of lipid accumulation, which is associated with dramatic changes in the transcriptome, proteome, and metabolome. However, the specific mechanisms of ROS involvement in the crosstalk between extracellular stress signaling and intracellular lipid synthesis require further investigation. Here, we summarize current knowledge on stress-induced lipid biosynthesis and the putative role of ROS in the control of lipid accumulation in oleaginous microorganisms. Understanding such links may provide guidance for the development of stress-based strategies to enhance microbial lipid production.

9.
Bioengineered ; 8(2): 124-128, 2017 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-27459344

RESUMO

Plant hormones are a class of organic substances which are synthesized during the plant metabolism. They have obvious physiological effect on plant growth at very low concentrations. Generally, plant hormones are mainly divided into 5 categories: auxins, cytokinins, ethylene, gibberellins (GAs) and abscisic acid (ABA). With the deepening of research, some novel plant hormones such as brassinosteroid and salicylates have been found and identified. The plant hormone products are mainly obtained through plant extraction, chemical synthesis as well as microbial fermentation. However, the extremely low yield in plants and relatively complex chemical structure limit the development of the former 2 approaches. Therefore, more attention has been paid into the microbial fermentative production. In this commentary, the developments and technological achievements of the 2 important plant hormones (GAs and ABA) have been discussed. The discovery, producing strains, fermentation technologies, and their accumulation mechanisms are first introduced. Furthermore, progresses in the industrial mass scale production are discussed. Finally, guidelines for future studies for GAs and ABA production are proposed in light of the current progress, challenges and trends in the field. With the widespread use of plant hormones in agriculture, we believe that the microbial production of plant hormones will have a bright future.


Assuntos
Fungos/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Ácido Abscísico/metabolismo , Biotecnologia/tendências , Etilenos/metabolismo , Giberelinas/metabolismo , Ácidos Indolacéticos/metabolismo
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