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1.
Nucleic Acids Res ; 51(15): 8283-8292, 2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37486765

RESUMO

As an enabling technique of synthetic biology, the scale of DNA assembly largely determines the scale of genetic manipulation. However, large DNA assembly technologies are generally cumbersome and inefficient. Here, we developed a YLC (yeast life cycle)-assembly method that enables in vivo iterative assembly of large DNA by nesting cell-cell transfer of assembled DNA in the cycle of yeast mating and sporulation. Using this method, we successfully assembled a hundred-kilobase (kb)-sized endogenous yeast DNA and a megabase (Mb)-sized exogenous DNA. For each round, over 104 positive colonies per 107 cells could be obtained, with an accuracy ranging from 67% to 100%. Compared with other Mb-sized DNA assembly methods, this method exhibits a higher success rate with an easy-to-operate workflow that avoid in vitro operations of large DNA. YLC-assembly lowers the technical difficulty of Mb-sized DNA assembly and could be a valuable tool for large-scale genome engineering and synthetic genomics.


Assuntos
Técnicas Genéticas , Saccharomyces cerevisiae , Biologia Sintética , Estágios do Ciclo de Vida , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Biologia Sintética/métodos
2.
PLoS Genet ; 18(12): e1010518, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36459502

RESUMO

Cancer progression is associated with the evolutionary accumulation of genetic mutations that are biologically significant. Mutations of the androgen receptor (AR) are associated with the development of prostate cancer (PCa) by responding to non-androgenic hormones, and the lack of annotations in their responsiveness to hormone ligands remains a daunting challenge. Here, we have used a yeast reporter system to quickly evaluate the responsiveness of all fifty clinical AR mutations to a variety of steroidal ligands including dihydrotestosterone (DHT), 17ß-estradiol (E2), progesterone (PROG), and cyproterone acetate (CPA). Based on an AR-driven reporter that synthesizes histidine, a basic amino acid required for yeast survival and propagation, the yeast reporter system enabling clonal selection was further empowered by combining with a random DNA mutagenesis library to simulate the natural evolution of AR gene under the selective pressures of steroidal ligands. In a time-frame of 1-2 weeks, 19 AR mutants were identified, in which 11 AR mutants were validated for activation by tested steroidal compounds. The high efficiency of our artificial evolution strategy was further evidenced by a sequential selection that enabled the discovery of multipoint AR mutations and evolution directions under the pressure of steroidal ligands. In summary, our designer yeast is a portable reporter module that can be readily adapted to streamline high-throughput AR-compound screening, used as a PCa clinical reference, and combined with additional bioassay systems to further extend its potential.


Assuntos
Receptores Androgênicos , Saccharomyces cerevisiae , Masculino , Humanos , Saccharomyces cerevisiae/genética , Receptores Androgênicos/genética , Mutação , Mutagênese , Seleção Genética
3.
Crit Rev Biotechnol ; : 1-21, 2024 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-38246753

RESUMO

Stress tolerance is a vital attribute for all living beings to cope with environmental adversities. IrrE (also named PprI) from Deinococcus radiodurans enhances resistance to extreme radiation stress by functioning as a global regulator, mediating the transcription of genes involved in deoxyribonucleic acid (DNA) damage response (DDR). The expression of IrrE augmented the resilience of various species to heat, radiation, oxidation, osmotic stresses and inhibitors, encompassing bacterial, fungal, plant, and mammalian cells. Moreover, IrrE was employed in a global regulator engineering strategy to broaden its applications in stress tolerance. The regulatory impacts of heterologously expressed IrrE have been investigated at the molecular and systems level, including the regulation of genes, proteins, modules, or pathways involved in DNA repair, detoxification proteins, protective molecules, native regulators and other aspects. In this review, we discuss the regulatory role and mechanism of IrrE in the antiradiation response of D. radiodurans. Furthermore, the applications and regulatory effects of heterologous expression of IrrE to enhance abiotic stress tolerance are summarized in particular.

4.
J Nat Prod ; 87(1): 28-37, 2024 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-38204395

RESUMO

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.


Assuntos
Bacillus subtilis , Consórcios Microbianos , Bacillus subtilis/química , Lipopeptídeos/química , Antifúngicos/química
5.
Microb Cell Fact ; 21(1): 79, 2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35527251

RESUMO

BACKGROUND: Mutational technology has been used to achieve genome-wide variations in laboratory and industrial microorganisms. Genetic polymorphisms of natural genome evolution include nucleotide variations and structural variations, which inspired us to suggest that both types of genotypic variations are potentially useful in improving the performance of chassis cells for industrial applications. However, highly efficient approaches that simultaneously generate structural and nucleotide variations are still lacking. RESULTS: The aim of this study was to develop a method of increasing biosynthesis of astaxanthin in yeast by Combining Nucleotide variations And Structure variations (CNAS), which were generated by combinations of Atmospheric and room temperature plasma (ARTP) and Synthetic Chromosome Recombination and Modification by LoxP-Mediated Evolution (SCRaMbLE) system. CNAS was applied to increase the biosynthesis of astaxanthin in yeast and resulted in improvements of 2.2- and 7.0-fold in the yield of astaxanthin. Furthermore, this method was shown to be able to generate structures (deletion, duplication, and inversion) as well as nucleotide variations (SNPs and InDels) simultaneously. Additionally, genetic analysis of the genotypic variations of an astaxanthin improved strain revealed that the deletion of YJR116W and the C2481G mutation of YOL084W enhanced yield of astaxanthin, suggesting a genotype-to-phenotype relationship. CONCLUSIONS: This study demonstrated that the CNAS strategy could generate both structure variations and nucleotide variations, allowing the enhancement of astaxanthin yield by different genotypes in yeast. Overall, this study provided a valuable tool for generating genomic variation diversity that has desirable phenotypes as well as for knowing the relationship between genotypes and phenotypes in evolutionary processes.


Assuntos
Nucleotídeos , Saccharomyces cerevisiae , Fenótipo , Saccharomyces cerevisiae/genética , Xantofilas
6.
Microb Cell Fact ; 21(1): 152, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35918699

RESUMO

Scopoletin is a typical example of coumarins, which can be produced in plants. Scopoletin acts as a precursor for pharmaceutical and health care products, and also possesses promising biological properties, including antibacterial, anti-tubercular, anti-hypertensive, anti-inflammatory, anti-diabetic, and anti-hyperuricemic activity. Despite the potential benefits, the production of scopoletin using traditional extraction processes from plants is unsatisfactory. In recent years, synthetic biology has developed rapidly and enabled the effective construction of microbial cell factories for production of high value-added chemicals. Herein, this review summarizes the progress of scopoletin biosynthesis in artificial microbial cell factories. The two main pathways of scopoletin biosynthesis are summarized firstly. Then, synthetic microbial cell factories are reviewed as an attractive improvement strategy for biosynthesis. Emerging techniques in synthetic biology and metabolic engineering are introduced as innovative tools for the efficient synthesis of scopoletin. This review showcases the potential of biosynthesis of scopoletin in artificial microbial cell factories.


Assuntos
Engenharia Metabólica , Escopoletina , Engenharia Metabólica/métodos , Plantas , Escopoletina/metabolismo , Biologia Sintética
7.
Chem Soc Rev ; 50(22): 12788-12807, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34651628

RESUMO

Directed genome evolution simulates the process of natural evolution at the genomic level in the laboratory to generate desired phenotypes. Here we review the applications of recent technological advances in genome writing and editing to directed genome evolution, with a focus on structural rearrangement techniques. We highlight how these techniques can be used to generate diverse genotypes, and to accelerate the evolution of phenotypic traits. We also discuss the perspectives of directed genome evolution.


Assuntos
Evolução Molecular , Genômica , Evolução Molecular Direcionada , Fenótipo
8.
Metab Eng ; 66: 51-59, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33857581

RESUMO

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a "push-pull-restrain" strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Monoterpenos Acíclicos , Geraniltranstransferase , Engenharia Metabólica , Monoterpenos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
Metab Eng ; 61: 160-170, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32553944

RESUMO

Yeast productivity in lignocellulosic ethanol fermentation is clearly impeded by stress. Enhancing the robustness of xylose-fermenting yeast is important for improving lignocellulosic ethanol production. In this study, the glutathione biosynthesis pathway and acetic acid degradation pathway were strengthened to enhance yeast tolerance to stress due to elevated reactive oxygen species (ROS) and acetic acid. Dynamic feedback regulation of the anti-stress genetic circuits was achieved using stress-driven promoters discovered from the transcriptome to maintain low intracellular ROS, relieve the metabolic burden, and ultimately improve the robustness and ethanol production of yeast. The cell growth, xylose utilization and ethanol production of the engineered strain were enhanced under both stress and nonstress conditions. The engineered strain showed 49.5% and 17.5% higher ethanol productivity in laboratory media and industrial lignocellulosic media, respectively, at 36 °C compared with the parent strain. This study provides novel insights on the rational design and construction of feedback genetic circuits for dynamically improving yeast robustness.


Assuntos
Etanol/metabolismo , Lignina/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
10.
FEMS Yeast Res ; 20(2)2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32188997

RESUMO

Genomic structural variations (SVs) promote the evolution of Saccharomyces cerevisiae, and play an important role in phenotypic diversities. Yeast genomic structures can be remodeled by design and bottom-up synthesis. The synthesis of yeast genome creates novel copy number variations (CNVs) and SVs and develops new strategies to discover gene functions. Further, an inducible evolution system SCRaMbLE, consisted of 3,932 loxPsym sites, was incorporated on synthetic yeast genome. SCRaMbLE enables genomic rearrangements at will and rapidly generates chromosomal number variations, and massive SVs under customized conditions. The impacts of genetic variations on phenotypes can be revealed by genome analysis and chromosome restructuring. Yeast genome synthesis and SCRaMbLE provide a new research paradigm to explore the genotypic mechanisms of phenotype diversities, and can be used to improve biological traits and optimize industrial chassis.


Assuntos
Evolução Molecular Direcionada/métodos , Genoma Fúngico , Variação Estrutural do Genoma , Genótipo , Saccharomyces cerevisiae/genética , Fenótipo , Saccharomyces cerevisiae/classificação
11.
Microb Cell Fact ; 19(1): 103, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398013

RESUMO

BACKGROUND: Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway. RESULTS: To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologous ß-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11-8.51 folds and 3.0-9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg g-1 DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains. CONCLUSIONS: The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.


Assuntos
Vias Biossintéticas , Engenharia Metabólica/métodos , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Escherichia coli/metabolismo , Oxigenases de Função Mista/genética , Oxigenases/genética , Saccharomyces cerevisiae/genética , Xantofilas/metabolismo
12.
Microb Cell Fact ; 19(1): 38, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070349

RESUMO

BACKGROUND: Synthetic biology requires toolbox of promoters to finely tune gene expression levels for building up efficient cell factories. Yeast promoters owned variable core promoter regions between the TATA-box and transcriptional starting site (TSS) at the length mostly around 20-80 bases. This region allowed flexible design of artificial promoter but potentially demand special base motifs to maintain or enhance the promoter's strength. RESULTS: Here, we designed and screened the base motifs and tested the activities of yeast artificial core promoters. Different 30 bases of artificial sequences led to variable expression levels of CrtY enzyme which determined the lycopene-carotene compositions, represented in the colony-color spectrum of red-orange-yellow. The upstream sequences of two strong promoter PEXP1 and PGPD and two starting strains with distinguishable lycopene production levels were utilized to characterize the promoter sequences. Different partition designs of T-rich or G/C-rich base motifs led to distinguishable colony-color distributions. Finally, we screened a champion promoter with a highest 5.5-fold enhancement of lycopene-carotene transformation. Another selected promoter generated a highest beta-carotene production as 7.4 mg/g DCW. CONCLUSIONS: This work offered an approach to redesign promoter with artificial sequences. We concluded that the core promoter region could be designated as 30 bases and different base motifs would enhance or weaken the promoter's strength. Generally, more T-rich elements, higher %T and lower G/C percentage were beneficial to enhance the strength of artificial core promoter.


Assuntos
Proteínas Fúngicas/metabolismo , Genes Fúngicos , Liases Intramoleculares/metabolismo , Regiões Promotoras Genéticas , Yarrowia/genética , Proteínas Fúngicas/genética , Engenharia Genética , Liases Intramoleculares/genética , Licopeno/metabolismo , Biologia Sintética , Transcrição Gênica , Yarrowia/metabolismo , beta Caroteno/biossíntese
13.
J Ind Microbiol Biotechnol ; 47(6-7): 551-562, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32495197

RESUMO

Polymyxins are used as the last-line therapy against multidrug-resistant bacteria. However, their further clinical development needs to solve problems related to the presence of heterogeneous analogs, but there is still no platform or methods that can regulate the biosynthesis of polymyxin analogs. In this study, we present an approach to swap domains in the polymyxin gene cluster to regulate the production of different analogs. Following adenylation domain swapping, the proportion of polymyxin B1 increased from 41.36 to 52.90%, while that of B1-1 decreased from 18.25 to 3.09%. The ratio of polymyxin B1 and B3 following starter condensation domain swapping changed from 41.36 and 16.99 to 55.03 and 6.39%, respectively. The two domain-swapping strains produced 62.96% of polymyxin B1, 6.70% of B3 and 3.32% of B1-1. This study also revealed the presence of overflow fluxes between acetoin, 2,3-butanediol and polymyxin. To our best knowledge, this is the first report of engineering the polymyxin synthetase gene cluster in situ to regulate the relative proportions of polymyxin analogs. This research paves a way for regulating lipopeptide analogs and will facilitate the development of novel lipopeptide derivatives.


Assuntos
Farmacorresistência Bacteriana Múltipla , Paenibacillus polymyxa/enzimologia , Peptídeo Sintases/química , Peptídeo Sintases/genética , Polimixinas/análogos & derivados , Ágar , Antibacterianos , Meios de Cultura , Fermentação , Lipopeptídeos , Engenharia Metabólica , Paenibacillus polymyxa/genética , Polimixinas/biossíntese , Polimixinas/química , Tensoativos/química
14.
Appl Environ Microbiol ; 85(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683746

RESUMO

Saccharomyces cerevisiae is an established cell factory for production of terpenoid pharmaceuticals and chemicals. Numerous studies have demonstrated that deletion or overexpression of off-pathway genes in yeast can improve terpenoid production. The deletion of YPL062W in S. cerevisiae, in particular, has benefitted carotenoid production by channeling carbon toward carotenoid precursors acetyl coenzyme A (acetyl-CoA) and mevalonate. The genetic function of YPL062W and the molecular mechanisms for these benefits are unknown. In this study, we systematically examined this gene deletion to uncover the gene function and its molecular mechanism. RNA sequencing (RNA-seq) analysis uncovered that YPL062W deletion upregulated the pyruvate dehydrogenase bypass, the mevalonate pathway, heterologous expression of galactose (GAL) promoter-regulated genes, energy metabolism, and membrane composition synthesis. Bioinformatics analysis and serial promoter deletion assay revealed that YPL062W functions as a core promoter for ALD6 and that the expression level of ALD6 is negatively correlated to terpenoid productivity. We demonstrate that ΔYPL062W increases the production of all major terpenoid classes (C10, C15, C20, C30, and C40). Our study not only elucidated the biological function of YPL062W but also provided a detailed methodology for understanding the mechanistic aspects of strain improvement.IMPORTANCE Although computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms of YPL062W deletion in S. cerevisiae, which is shown to improve the production of all terpenoid classes. This study redefines the genetic function of YPL062W, demonstrates a strong correlation between YPL062W and terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.


Assuntos
Deleção de Genes , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Metabolismo Secundário/genética , Terpenos/metabolismo , Acetilcoenzima A/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Fermentação , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Ácido Mevalônico/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNA , Biologia de Sistemas
15.
J Exp Bot ; 70(18): 4819-4834, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31056664

RESUMO

Crocus sativus is generally considered the source of saffron spice which is rich in apo-carotenoid compounds such as crocins, crocetin, picrocrocin, and safranal, which possess effective pharmacological activities. However, little is known about the exact genes involved in apo-carotenoid biosynthesis in saffron and the potential mechanism of specific accumulation in the stigma. In this study, we integrated stigmas at different developmental stages to perform in-depth transcriptome and dynamic metabolomic analyses to discover the potential key catalytic steps involved in apo-carotenoid biosynthesis in saffron. A total of 61 202 unigenes were obtained, and 28 regulators and 32 putative carotenogenic genes were captured after the co-expression network analysis. Moreover, 15 candidate genes were predicted to be closely related to safranal and crocin production, in which one aldehyde dehydrogenase (CsALDH3) was validated to oxidize crocetin dialdehyde into crocetin and a crocetin-producing yeast strain was created. In addition, a new branch pathway that catalyses the conversion of geranyl-geranyl pyrophosphate to copalol and ent-kaurene by the class II diterpene synthase CsCPS1 and three class I diterpene synthases CsEKL1/2/3 were investigated for the first time. Such gene to apo-carotenoid landscapes illuminate the synthetic charactersistics and regulators of apo-carotenoid biosynthesis, laying the foundation for a deep understanding of the biosynthesis mechanism and metabolic engineering of apo-carotenoids in plants or microbes.


Assuntos
Carotenoides/metabolismo , Crocus/metabolismo , Metaboloma , Saccharomyces cerevisiae/metabolismo , Crocus/enzimologia , Flores/química , Perfilação da Expressão Gênica , Genes de Plantas , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Saccharomyces cerevisiae/genética , Vitamina A/análogos & derivados
16.
Microb Cell Fact ; 18(1): 52, 2019 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-30857530

RESUMO

BACKGROUND: Strains with increased alkali tolerance have a broad application in industrial, especially for bioremediation, biodegradation, biocontrol and production of bio-based chemicals. A novel synthetic chromosome recombination and modification by LoxP-mediated evolution (SCRaMbLE) system has been introduced in the synthetic yeast genome (Sc 2.0), which enables generation of a yeast library with massive structural variations and potentially drives phenotypic evolution. The structural variations including deletion, inversion and duplication have been detected within synthetic yeast chromosomes. RESULTS: Haploid yeast strains harboring either one (synV) or two (synV and synX) synthetic chromosomes were subjected to SCRaMbLE. Seven of evolved strains with increased alkali tolerance at pH 8.0 were generated through multiple independent SCRaMbLE experiments. Various of structural variations were detected in evolved yeast strains by PCRTag analysis and whole genome sequencing including two complex structural variations. One possessed an inversion of 20,743 base pairs within which YEL060C (PRB1) was deleted simultaneously, while another contained a duplication region of 9091 base pairs in length with a deletion aside. Moreover, a common deletion region with length of 11,448 base pairs was mapped in four of the alkali-tolerant strains. We further validated that the deletion of YER161C (SPT2) within the deleted region could increase alkali tolerance in Saccharomyces cerevisiae. CONCLUSIONS: SCRaMbLE system provides a simple and efficient way to generate evolved yeast strains with enhanced alkali tolerance. Deletion of YER161C (SPT2) mapped by SCRaMbLE can improve alkali tolerance in S. cerevisiae. This study enriches our understanding of alkali tolerance in yeast and provides a standard workflow for the application of SCRaMbLE system to generate various phenotypes that may be interesting for industry and extend understanding of phenotype-genotype relationship.


Assuntos
Álcalis/metabolismo , Cromossomos Artificiais de Levedura/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Fenótipo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Biologia Sintética
17.
Biotechnol Lett ; 41(8-9): 951-961, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31278569

RESUMO

OBJECTIVES: A three-species consortium for one-step fermentation of 2-keto-L-gulonic acid (2-KGA) was constructed to better strengthen the cell-cell communication. And the programmed cell death module based on the LuxI/LuxR quorum-sensing (QS) system was established in Gluconobacter oxydans to reduce the competition that between G. oxydans and Ketogulonicigenium vulgare. RESULTS: By constructing and optimizing the core region of the promoter, which directly regulated the expression of lethal ccdB genes in QS system, IR3C achieved the best lethal effect. The consortium of IR3C- K. vulgare-Bacillus megaterium (abbreviated as 3C) achieved the highest 2-KGA titer (68.80 ± 4.18 g/l), and the molar conversion rate was 80.7% within 36 h in 5 l fermenter. Metabolomic analysis on intracellular small molecules of consortia 3C and 1C showed that most amino acids (such as glycine, leucine, methionine and proline) and TCA cycle intermediates (such as succinic acid, fumaric acid and malic acid) were significantly affected. These results further validated that the programmed cell death module based on the LuxI/LuxR QS system in G. oxydans could also faciliate better growth and higher production of consortium 3C for one-step fermentation. CONCLUSIONS: We successfully constructed a novel three-species consortia for one-step vitamin C fermentation by strengthening the cell-cell communication. This will be very useful for probing the rational design principles of more complex multi-microbial consortia.


Assuntos
Ácido Ascórbico/metabolismo , Bacillus megaterium/metabolismo , Fermentação , Gluconobacter oxydans/metabolismo , Consórcios Microbianos , Rhodobacteraceae/metabolismo , Açúcares Ácidos/metabolismo , Bacillus megaterium/crescimento & desenvolvimento , Comunicação Celular , Gluconobacter oxydans/crescimento & desenvolvimento , Interações Microbianas , Rhodobacteraceae/crescimento & desenvolvimento , Vitaminas/metabolismo
18.
J Ind Microbiol Biotechnol ; 46(1): 21-31, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30368638

RESUMO

Microbial consortia, with the merits of strong stability, robustness, and multi-function, played critical roles in human health, bioenergy, and food manufacture, etc. On the basis of 'build a consortium to understand it', a novel microbial consortium consisted of Gluconobacter oxydans, Ketogulonicigenium vulgare and Bacillus endophyticus was reconstructed to produce 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. With this synthetic consortium, 73.7 g/L 2-KGA was obtained within 30 h, which is comparable to the conventional industrial method. A combined time-series proteomic and metabolomic analysis of the fermentation process was conducted to further investigate the cell-cell interaction. The results suggested that the existence of B. endophyticus and G. oxydans together promoted the growth of K. vulgare by supplying additional nutrients, and promoted the 2-KGA production by supplying more substrate. Meanwhile, the growth of B. endophyticus and G. oxydans was compromised from the competition of the nutrients by K. vulgare, enabling the efficient production of 2-KGA. This study provides valuable guidance for further study of synthetic microbial consortia.


Assuntos
Ácido Ascórbico/metabolismo , Metabolômica , Consórcios Microbianos , Proteômica , Açúcares Ácidos/metabolismo , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Fermentação , Gluconobacter oxydans/metabolismo , Microbiologia Industrial , Rhodobacteraceae/metabolismo
19.
Metab Eng ; 47: 243-253, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29596994

RESUMO

Synthetic microbial coculture to express heterologous biosynthetic pathway for de novo production of medicinal ingredients is an emerging strategy for metabolic engineering and synthetic biology. Here, taking efficient production of salidroside as an example of glycosides, we design and construct a syntrophic Escherichia coli-E. coli coculture composed of the aglycone (AG) strain and the glycoside (GD) strain, which convergently accommodate biosynthetic pathways of tyrosol and salidroside, respectively. To accomplish this the phenylalanine-deficient AG strain was engineered to utilize xylose preferentially and to overproduce precursor tyrosol, while the tyrosine-deficient GD strain was constructed to consume glucose exclusively and to enhance another precursor UDP-glucose availability for synthesis of salidroside. The AG and GD strains in the synthetic consortium are obligatory cooperators through crossfeeding of tyrosine and phenylalanine and compatible in glucose and xylose mixture. Through balancing the metabolic pathway strength, we show that the syntrophic coculture was robust and stable, and produced 6.03 g/L of salidroside. It was the de novo production of salidroside for the first time in E. coli coculture system, which would be applicable for production of other important glycosides and natural products.


Assuntos
Glucosídeos , Engenharia Metabólica , Fenóis , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Glucosídeos/biossíntese , Glucosídeos/genética , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Uridina Difosfato Glucose/genética , Uridina Difosfato Glucose/metabolismo , Xilose/genética , Xilose/metabolismo
20.
Microb Cell Fact ; 17(1): 62, 2018 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-29678175

RESUMO

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest. RESULTS: In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized. CONCLUSION: Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.


Assuntos
Sistemas CRISPR-Cas/genética , Expressão Gênica/genética , RNA Guia de Cinetoplastídeos/genética , Yarrowia/genética , Yarrowia/metabolismo
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