共查询到20条相似文献，搜索用时 15 毫秒
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Nurzhan Kuanyshev Christopher V. Rao Bruce Dien Yong‐Su Jin 《Biotechnology and bioengineering》2021,118(1):372-382
Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid‐tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN‐ARS‐based CRISPR‐Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site‐specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR‐Cas9 system in Z. bailii, which can be applied for a fundamental study of the species. 相似文献
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Li-Yu Sung Meng-Ying Wu Mei-Wei Lin Mu-Nung Hsu Vu Anh Truong Chih-Che Shen Yi Tu Kuen-Yuan Hwang An-Pang Tu Yu-Han Chang Yu-Chen Hu 《Biotechnology and bioengineering》2019,116(5):1066-1079
CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering. 相似文献
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Mathew M Jessop‐Fabre Tadas Jakočiūnas Vratislav Stovicek Zongjie Dai Michael K Jensen Jay D Keasling Irina Borodina 《Biotechnology journal》2016,11(8):1110-1117
Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available. 相似文献
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丁二酸(又称琥珀酸)是一种重要的C4平台化合物,可作为1,4-丁二醇、四氢呋喃以及生物可降解塑料聚丁二酸丁二醇酯(polybutylene succinate, PBS)的生产原料。与传统的以顺酐为原料的石化基路线相比,采用微生物发酵法生产丁二酸不仅具有更高的经济可持续性,同时也展现出更佳的环境友好性。酵母具有良好的耐酸性,能够实现丁二酸的低pH发酵,从而大幅降低产物提取成本。因此,通过代谢工程改造构建高产丁二酸酵母菌株受到越来越多的关注。本文系统介绍了丁二酸的应用价值及其市场规模,总结了微生物中参与丁二酸合成的途径及其关键酶,详细阐述了利用酵母细胞工厂合成丁二酸的最新研究进展,同时还展示了酵母工程菌株以甘油、乙酸、木质纤维素水解液等非粮原料为底物进行丁二酸合成的现状,最后对基于酵母细胞工厂的低pH丁二酸生物制造进行了展望。 相似文献
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The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA–tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast. 相似文献
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Céline Morineau Yannick Bellec Frédérique Tellier Lionel Gissot Zsolt Kelemen Fabien Nogué Jean‐Denis Faure 《Plant biotechnology journal》2017,15(6):729-739
In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta‐12‐desaturase (FAD2) genes was achieved by CRISPR‐Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss‐of‐function mutants revealed the importance of delta‐12‐desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding. 相似文献
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The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing. 相似文献
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通过分析大肠杆菌的碳源代谢途径, 利用基因敲除手段, 以Escherichia coli MG1655为出发菌株, 成功构建了琥珀酸好氧发酵生产工程菌E. coli QZ1111 (MG1655?ptsG?poxB?pta?iclR?sdhA)。检测结果表明该菌株能以葡萄糖为碳源, 在好氧发酵且不表达任何异源基因的条件下大量积累琥珀酸。摇瓶试验证明, 琥珀酸发酵产量达到26.4 g/L, 乙酸盐作为唯一检测到的副产物产量为2.3 g/L。二者浓度比达到11.5:1。 相似文献
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Cas9 and Cas12a are multidomain CRISPR‐associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best‐characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool. This article is categorized under:
- RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
- Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
- RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
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Soon Ho Hong 《Biotechnology and Bioprocess Engineering》2007,12(2):73-79
Succinic acid is a cellular metabolite belonging to the C4-dicarboxylic acid family, and the fermentative production of succinic
acid via the use of recombinant microorganisms has recently become the focus of an increasing amount of attention. Considering
the difficulty inherent to the direct application of natural succinic acid producers to the industrial process, a variety
of systems biology studies have been conducted regarding the development of enhanced succinic acid production systems. This
review shows how the metabolic processes of microorganisms, includingEscherichia coli andMannheimia succiniciproducens, have been optimized in order to achieve enhanced succinic acid production. First, their metabolic networks were constructed
on the basis of complete genome sequences, after which their metabolic characteristics were estimated viain silico computer modeling. Metabolic engineering strategies were designed in accordance with the results ofin silico modeling and metabolically engineered versions of bothE. coli andM. succiniciproducens have been constructed. The succinic acid productivity and yield obtained using metabolically engineered bacteria was significantly
higher than that obtained using wild-type bacteria. 相似文献
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A Cas9/sgRNA RNA-guided endonuclease expression system including a codon-optimized Streptococcus pyogenes A20 Cas9 recombinant protein expression vector and a spacer-guide chimeric RNA expression vector using the porcine U6 promoter was constructed for application in pigs. Only the Flag2-NLS1-Cas9-NLS2 recombinant protein in complex with sgRNA was translocated into the nucleus; the Flag2-NLS1-Cas9-NLS2 protein alone was excluded from the nucleus. Up to 13% of porcine PK1 cells targeted in vitro were observed, regardless of transfection efficiency. 相似文献
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Lu-Feng Hu Yu-Xuan Li Jia-Zhen Wang Yu-Ting Zhao Yangming Wang 《Wiley interdisciplinary reviews. RNA》2023,14(1):e1731
The clustered regularly interspaced short palindromic repeats (CRISPR) system is a product of million years of evolution by microbes to fight against invading genetic materials. Around 10 years ago, scientists started to repurpose the CRISPR as genetic tools by molecular engineering approaches. The guide RNA provides a versatile and unique platform for the innovation to improve and expand the application of CRISPR-Cas9 system. In this review, we will first introduce the basic sequence and structure of guide RNA and its role during the function of CRISPR-Cas9. We will then summarize recent progress on the development of various guide RNA engineering strategies. These strategies have been dedicated to improve the performance of CRISPR-Cas9, to achieve precise spatiotemporal control of CRISPR-Cas9, and to broaden the application of CRISPR-Cas9. Finally, we will briefly discuss the uniqueness and advantage of guide RNA-engineering based systems versus those with engineered Cas9 proteins and speculate potential future directions in guide RNA engineering. This article is categorized under:
- RNA Methods > RNA Analyses In Vitro and In Silico
- RNA Methods > RNA Nanotechnology
- Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
- RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes
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Mengyan Bai Juehui Yuan Huaqin Kuang Pingping Gong Suning Li Zhihui Zhang Bo Liu Jiafeng Sun Maoxiang Yang Lan Yang Dong Wang Shikui Song Yuefeng Guan 《Plant biotechnology journal》2020,18(3):721-731
The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR‐Cas9 as a mutant screening tool. Here, we report a pooled CRISPR‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1‐1/1‐2/1‐3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops. 相似文献
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Joan Miquel Bernab‐Orts Ivn Casas‐Rodrigo Eugenio G. Minguet Viola Landolfi Victor Garcia‐Carpintero Silvia Gianoglio Marta Vzquez‐Vilar Antonio Granell Diego Orzaez 《Plant biotechnology journal》2019,17(10):1971-1984
The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of ?10 to ?2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. 相似文献