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1.
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.

2.
J Agric Food Chem ; 69(39): 11626-11636, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34554747

RESUMO

Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD) is the critical enzyme to synthesize crocetin. So, in this study, we used "hybrid-tunnel" engineering to obtain variants of Crocus sativus-derived CsCCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the ß-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency (kcat/Km) toward zeaxanthin than the wild-type in vitro. This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications.


Assuntos
Crocus , Dioxigenases , Carotenoides , Vitamina A/análogos & derivados , Zeaxantinas
3.
ACS Synth Biol ; 10(9): 2222-2230, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34420293

RESUMO

DNA inversion is a type of site-specific recombination system that plays an important role in the generation of genetic diversity and phenotypic adaptation by programmed rearrangements in bacteria. However, no such inversion system exhibiting a strong directionality bias has been identified or developed in eukaryotes yet. Here, using directed evolution of Rci recombinase, a tyrosine recombinase from a bacterial DNA inversion system, we identified a mutant Rci8 with a ratio of inversion/deletion up to ∼4320 in yeast. Based on Rci8 recombinase and sfxa101 sites, we have established a DNA inversion system in yeast and mammalian cells, enabling specificity for DNA inversions between inverted sites over deletions between directly repeated sites. Our results validated that the reversible DNA inversion system can act as an on/off transcriptional switch. Moreover, we demonstrate that the inversion system can also work on linear chromosomes. The eukaryotic DNA inversion system would provide a new tool for fields of genetic circuits, cellular barcoding, and synthetic genomes.

4.
Chem Sci ; 12(15): 5381-5389, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34168782

RESUMO

Synthetic genomics aims to de novo synthesize a functional genome redesigned from natural sequences with custom features. Designed genomes provide new toolkits for better understanding organisms, evolution and the construction of cellular factories. Currently maintaining the fitness of cells with synthetic genomes is particularly challenging as defective designs and unanticipated assembly errors frequently occur. Mapping and correcting bugs that arise during the synthetic process are imperative for the successful construction of a synthetic genome that can sustain a desired cellular function. Here, we review recently developed methods used to map and fix various bugs which arise during yeast genome synthesis with the hope of providing guidance for putting the synthetic yeast chromosome to work.

5.
Front Microbiol ; 12: 663973, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093477

RESUMO

7-Dehydrocholesterol (7-DHC) is the direct precursor to manufacture vitamin D3. Our previous study has achieved 7-DHC synthesis in Saccharomyces cerevisiae based on the endogenous post-squalene pathway. However, the distribution of post-squalene enzymes between the endoplasmic reticulum (ER) and lipid bodies (LD) might raise difficulties for ERG proteins to catalyze and deliver sterol intermediates, resulting in unbalanced metabolic flow and low product yield. Herein, we intended to rearrange the subcellular location of post-squalene enzymes to alleviate metabolic bottleneck and boost 7-DHC production. After identifying the location of DHCR24 (C-24 reductase, the only heterologous protein for 7-DHC biosynthesis) on ER, all the ER-located enzymes were grouped into four modules: ERG1/11/24, ERG25/26/27, ERG2/3, and DHCR24. These modules attempted to be overexpressed either on ER or on LDs. As a result, expression of LD-targeted DHCR24 and ER-located ERG1/11/24 could promote the conversion efficiency among the sterol intermediates to 7-DHC, while locating module ERG2/3 into LDs improved the whole metabolic flux of the post-squalene pathway. Coexpressing LD-targeted ERG2/3 and DHCR24 (generating strain SyBE_Sc01250035) improved 7-DHC production from 187.7 to 308.2 mg/L at shake-flask level. Further expressing ER-targeted module ERG1/11/24 in strain SyBE_Sc01250035 dramatically reduced squalene accumulation from 620.2 mg/L to the lowest level (by 93.8%) as well as improved 7-DHC production to the highest level (to 342.2 mg/L). Then targeting module ERG25/26/27 to LDs further increased 7-DHC titer to 360.6 mg/L, which is the highest shake-flask level production for 7-DHC ever reported. Our study not only proposes and further proves the concept of pathway compartmentalized reconstitution to regulate metabolic flux but also provides a promising chassis to produce other steroidal compounds through the post-squalene pathway.

6.
Front Microbiol ; 12: 631462, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33664720

RESUMO

In Saccharomyces cerevisiae, conventional 2µ-plasmid based plasmid (pC2µ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2µ plasmid (pE2µ) brings the issues concerning plasmid stability and copy number of pC2µ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2µ and built the pE2µ multi-copy system by insertion of the target DNA element and elimination of the original pE2µ plasmid. The resulting plasmid pE2µRAF1 and pE2µREP2 demonstrated higher copy number and slower loss rate than a pC2µ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2µRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2µ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2µ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2µRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2µ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2µ due to the more stable pE2µ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.

7.
Biotechnol Biofuels ; 13(1): 193, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33292418

RESUMO

BACKGROUND: Stress tolerance is one of the important desired microbial traits for industrial bioprocesses, and global regulatory protein engineering is an efficient approach to improve strain tolerance. In our study, IrrE, a global regulatory protein from the prokaryotic organism Deinococcus radiodurans, was engineered to confer yeast improved tolerance to the inhibitors in lignocellulose hydrolysates or high temperatures. RESULTS: Three IrrE mutations were developed through directed evolution, and the expression of these mutants could improve the yeast fermentation rate by threefold or more in the presence of multiple inhibitors. Subsequently, the tolerance to multiple inhibitors of single-site mutants based on the mutations from the variants were then evaluated, and 11 mutants, including L65P, I103T, E119V, L160F, P162S, M169V, V204A, R244G, Base 824 Deletion, V299A, and A300V were identified to be critical for the improved representative inhibitors, i.e., furfural, acetic acid and phenol (FAP) tolerance. Further studies indicated that IrrE caused genome-wide transcriptional perturbation in yeast, and the mutant I24 led to the rapid growth of Saccharomyces cerevisiae by primarily regulating the transcription level of transcription activators/factors, protecting the intracellular environment and enhancing the antioxidant capacity under inhibitor environments, which reflected IrrE plasticity. Meanwhile, we observed that the expression of the wild-type or mutant IrrE could also protect Saccharomyces cerevisiae from the damage caused by thermal stress. The recombinant yeast strains were able to grow with glucose at 42 â„ƒ. CONCLUSIONS: IrrE from Deinococcus radiodurans can be engineered as a tolerance-enhancer for Saccharomyces cerevisiae. Systematic research on the regulatory model and mechanism of a prokaryotic global regulatory factor IrrE to increase yeast tolerance provided valuable insights for the improvements in microbial tolerance to complex industrial stress conditions.

8.
Curr Opin Biotechnol ; 66: 165-170, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32818746

RESUMO

Genome structural variations enable microbes to evolve quickly under environmental stress. Recent efforts in synthetic biology have shown the ability of yeast chromosomal engineering to generate a larger scale of genome structural variations, which require a high efficiency of DNA rearrangement technology. In this review, we summarize the recent development of the SCRaMbLE system, an evolutionary approach, and the CRISPR/Cas9 technology to generate yeast genome rearrangement. Both technologies exhibit the powerful applications of chromosomal engineering to accelerate phenotypic evolution. We highlight several studies where yeast genome rearrangement has successfully improved industrially-relevant phenotypes, including the production of novel medicine, nutrition supplements, anti-tumor molecules, and the tolerance of environmental stress and drug resistance.


Assuntos
Genoma Fúngico , Saccharomyces cerevisiae , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Genoma Fúngico/genética , Fenótipo , Saccharomyces cerevisiae/genética , Biologia Sintética
9.
Biotechnol Biofuels ; 13: 133, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760447

RESUMO

Background: Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunity-enhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small molecular compounds, triterpenes are generally secreted into the extracellular space. The effect of increasing triterpene efflux on the synthesis capacity remains unknown. Results: In this study, we developed a strategy to enhance triterpene efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme Δ9-fatty acid desaturase (OLE1) and disturbing phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). By this strategy combined with two-phase fermentation, the highest lupeol production reported to date was achieved, where the titer in the organic phase reached 381.67 mg/L and the total production was 411.72 mg/L in shake flasks, exhibiting a 33.20-fold improvement over the initial strain. Lipid manipulation led to a twofold increase in the unsaturated fatty acid (UFA) content, up to 61-73%, and an exceptionally elongated cell morphology, which might have been caused by enhanced membrane phospholipid biosynthesis flux. Both phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity for triterpenoid synthesis, which was proven by the 5.11-fold higher ratio of extra/intracellular lupeol concentrations, 2.79-fold higher biomass accumulation and 2.56-fold higher lupeol productivity per unit OD in the modified strains. This strategy was also highly efficient for the biosynthesis of other triterpenes and sesquiterpenes, including α-amyrin, ß-amyrin, longifolene, longipinene and longicyclene. Conclusions: In conclusion, we successfully created a high-yield lupeol-producing strain via lipid manipulation. We demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased UFA content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.

10.
Nat Commun ; 11(1): 4344, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859906

RESUMO

Self-propagating drive systems are capable of causing non-Mendelian inheritance. Here, we report a drive system in yeast referred to as a chromosome drive that eliminates the target chromosome via CRISPR-Cas9, enabling the transmission of the desired chromosome. Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast. Chromosome drive enables entire chromosome elimination and biased inheritance on a chromosomal scale, facilitating genomic engineering and chromosome-scale genetic mapping, and extending applications of self-propagating drives.


Assuntos
Sistemas CRISPR-Cas , Cromossomos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Saccharomyces cerevisiae/genética , Centrômero , Indóis , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Termotolerância/genética , Sequenciamento Completo do Genoma
11.
Aging (Albany NY) ; 12(14): 15021-15036, 2020 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-32712598

RESUMO

Aberrant activation of the cholesterol biosynthesis supports tumor cell growth. In recent years, significant progress has been made by targeting rate-limiting enzymes in cholesterol biosynthesis pathways to prevent carcinogenesis. However, precise mechanisms behind cholesterol degradation in cancer cells have not been comprehensively investigated. Here, we report that codon optimization of the orthologous cholesterol 7-desaturase, NVD-BM from Bombyx mori, significantly slowed melanoma cell proliferation and migration, and inhibited cancer cell engraftment in nude mice, by converting cholesterol to toxic 7-dehydrocholesterol. Based on these observations, we established a synthetic genetic circuit to induce melanoma cell regression by sensing tumor specific signals in melanoma cells. The dual-input signals, RELA proto-oncogene (RELA) and signal transducer and activator of transcription 1 (STAT1), activated NVD-BM expression and repressed melanoma cell proliferation and migration. Mechanically, we observed that NVD-BM decreased Akt1-ser473 phosphorylation and inhibited cytoplasmic RELA translocation. Taken together, NVD-BM was identified as a tumor suppressor in malignant melanoma, and we established a dual-input biosensor to promote cancer cell regression, via Akt1/NF-κB signaling. Our results demonstrate the potential therapeutic effects of cholesterol 7-desaturase in melanoma metabolism, and provides insights for genetic circuits targeting 7-dehydrocholesterol accumulation in tumors.


Assuntos
Desidrocolesteróis/metabolismo , Melanoma , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Técnicas Biossensoriais/métodos , Bombyx , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ácidos Graxos Dessaturases/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Oxirredutases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
12.
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
13.
ACS Synth Biol ; 9(7): 1753-1762, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32579850

RESUMO

Carotenoids, a variety of natural products, have significant pharmaceutical and commercial potential. Phytoene dehydrogenase (CrtI) is the rate-limit enzyme for carotenoid synthesis, whose catalysis specificity results in various carotenoids. However, the structural characteristics of CrtI for controlling the catalysis specificity on dehydrogenation steps are still unclear, which limited the development of CrtI function. Here we confirmed two mutation sites H136 and H453 in the mutant library of CrtI from Blakeslea trispora, which markedly regulated catalytic specificity. Interestingly, the sequence alignment features at H136 and H453 were consistent with the phylogenetic analysis of CrtI families. Subsequently, the functions of saturated mutants at H136 and H453 were clustered by principal component analysis (PCA) and k-means. According to the clustering results, diversiform mutants with specific dehydrogenation function provided important application value for carotenoid product customization. Meanwhile, this study also enriched the theory of enzyme evolution and guided the functional development of enzymes.


Assuntos
Biocatálise , Carotenoides/síntese química , Proteínas Fúngicas/química , Mucorales/enzimologia , Mucorales/genética , Oxirredutases/química , Sequência de Aminoácidos , Aminoácidos/genética , Cianobactérias/enzimologia , Escherichia coli/genética , Evolução Molecular , Mutação , Filogenia , Plantas/enzimologia , Plasmídeos/genética , Análise de Componente Principal , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Especificidade por Substrato
14.
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
15.
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
16.
Artigo em Inglês | MEDLINE | ID: mdl-32258005

RESUMO

Direct bioproduction of DHAA (dihydroartemisinic acid) rather than AA (artemisinic acid), as suggested by previous work would decrease the cost of semi-biosynthesis artemisinin by eliminating the step of initial hydrogenation of AA. The major challenge in microbial production of DHAA is how to efficiently manipulate consecutive key enzymes ADH1 (artemisinic alcohol dehydrogenase), DBR2 [artemisinic aldehyde Δ11(13) reductase] and ALDH1 (aldehyde dehydrogenase) to redirect metabolic flux and elevate the ratio of DHAA to AA (artemisinic acid). Herein, DHAA biosynthesis was achieved in Saccharomyces cerevisiae by introducing a series of heterologous enzymes: ADS (amorpha-4,11-diene synthase), CYP71AV1 (amorphadiene oxidase), ADH1, DBR2 and ALDH1, obtaining initial DHAA/AA ratio at 2.53. The flux toward DHAA was enhanced by pairing fusion proteins DBR2-ADH1 and DBR2-ALDH1, leading to 1.75-fold increase in DHAA/AA ratio (to 6.97). Moreover, to promote the substrate preference of ALDH1 to dihydroartemisinic aldehyde (the intermediate for DHAA synthesis) over artemisinic aldehyde (the intermediate for AA synthesis), two rational engineering strategies, including downsizing the active pocket and enhancing the stability of enzyme/cofactor complex, were proposed to engineer ALDH1. It was found that the mutant H194R, which showed better stability of the enzyme/NAD+ complex, obtained the highest DHAA to AA ratio at 3.73 among all the mutations. Then the mutant H194R was incorporated into above rebuilt fusion proteins, resulting in the highest ratio of DHAA to AA (10.05). Subsequently, the highest DHAA reported titer of 1.70 g/L (DHAA/AA ratio of 9.84) was achieved through 5 L bioreactor fermentation. The study highlights the synergy of metabolic engineering and protein engineering in metabolic flux redirection to get the most efficient product to the chemical process, and simplified downstream conversion process.

17.
ACS Synth Biol ; 9(5): 1181-1189, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32268063

RESUMO

Genome-scale gene knockout is an important approach to the study of global genetic interactions. SCRaMbLEing of synthetic yeast chromosomes provides an efficient way to generate random deletion mutants. Here, we demonstrate the use of SCRaMbLE to explore synthetic lethal interactions. First, all essential genes of yeast chromosome III (chrIII) were clustered in a centromeric plasmid. We found that three types of reorganized clustered chrIII essential genes had similar transcriptional levels. Further, SCRaMbLEing of synthetic chromosome III (synIII) with supplementary clustered essential genes enables deletion of large chromosomal regions. Investigation of 141 SCRaMbLEd strains revealed varied deletion frequencies of synIII chromosomal regions. Among the no deletion detected regions, a hidden synthetic lethal interaction was revealed in the region of synIII 82-88 kb. This study shows that SCRaMbLE with clustered essential genes enhances streamlining of synthetic yeast chromosome and provides a novel strategy to uncover complex genetic interactions.


Assuntos
Cromossomos Fúngicos , Engenharia Genética/métodos , Saccharomyces cerevisiae/genética , Genes Essenciais , Plasmídeos/genética , Plasmídeos/metabolismo , Deleção de Sequência
18.
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
19.
Sci Total Environ ; 710: 136329, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-31918182

RESUMO

The contamination of the aquatic environments by tetracycline antibiotics (TCs) is an increasingly pressing issue. Here, we used the addition of exogenous surfactants and in situ biosynthesis of biosurfactants to remove tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and their mixtures using the co-culture of probiotic Bacillus clausii T and Bacillus amyloliquefaciens HM618 producing surfactin. The addition of exogenous biosurfactants to remove TCs was superior to nonionic surfactants. The maximal bio-removal efficiencies for OTC and CTC among mixed antibiotics under the co-culture of B. clausii T and B. amyloliquefaciens HM618 were 76.6% and 88.9%, respectively, which were both better than the efficiency of the pure culture of B. clausii T. TCs were removed mainly through biotransformation rather than absorption and hydrolysis. The removal efficiency was in the order CTC > OTC > TC. The co-culture of B. clausii T and B. amyloliquefaciens HM618 alleviated the cytotoxicity of OTC and CTC. The toxicity of the biotransformation products was lower than that of the parent compounds. Demethylation, hydroxylation, and dehydration are likely the major mechanisms of TC biotransformation. These results illustrate the potential of using surfactants in the bioremediation of tetracycline antibiotics, and provide new avenues for further exploration of the bioremediation of antibiotics pollution.


Assuntos
Bacillus amyloliquefaciens , Bacillus clausii , Probióticos , Antibacterianos , Tetraciclina
20.
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
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