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Programming cells by multiplex genome engineering and accelerated evolution
Open accessAbstract
The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
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“(c) epistasis is a major factor for adaptive optimization of gene regulation [89,157].”
From Adaptive laboratory evolution—Principles and applications for biotechnology (2013) · cites this paper as [157]
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“Each MAGE project involves six steps: (1) determining genome editing goals; (2) identifying target loci for mutagenesis; (3) designing mutagenic MAGE oligos; (4) predicting the number of MAGE cycles to accomplish genome editing goals; (5) performing MAGE cycling; and (6) screening or selecting for the desired genotype…”
BackgroundSelf-citationUtrilla, J., O’Brien, E. J., Chen, K., McCloskey, D., Cheung, J., Wang, H. H., Armenta‐Medina, D., Feist, A. M., & Palsson, B. Ø. (2016) · Cell Systems · doi:10.1016/j.cels.2016.04.003 · Cited by 114
“To assess whether other amino-acid substitutions in the RNAP ALE-selected loci affect growth phenotypes, we used multiplex automated genome engineering (MAGE) (Wang et al., 2009) to generate variants in all possible 546 and 672 positions.”
MethodSelf-citationAn Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism
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Biotechnological advances for improving natural pigment production: a state-of-the-art review
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Metabolic Engineering of Microorganisms for the Production of Natural Compounds
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Automating the design-build-test-learn cycle towards next-generation bacterial cell factories
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Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli
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“Mutations acquired during adaptive evolution were identified by sequencing and introduced into the unadapted strain AXcp through genome editing, as described (Ryu et al., 2014; Wang et al., 2009).”
MethodDNA synthesis, assembly and applications in synthetic biology
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Advanced strategies and tools to facilitate and streamline microbial adaptive laboratory evolution
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“Pioneering efforts to combinatorially mutagenize the chromosome of Escherichia coli have driven home the message that it is essential to comprehensively explore this potential diversity to identify unexpected synergistic effects (40, 41).”
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“Using oligo-mediated recombineering (15), all 321 instances of the TAG stop codon in E.”
MethodA dynamic metabolite valve for the control of central carbon metabolism
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Metabolic flux analysis and pharmaceutical production
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Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis
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“PCC 7002 will facilitate strain construction in this host and possibly enable the adoption of high throughput genetic engineering techniques [47].”
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“Nevertheless, some improvements still need to be considered to carry on: (1) MAGE is limited to integrate large genes (>1 kb) since it depends on ssDNAs; (2) it is still a challenge to widely use this methodology in other non-model bacteria.”
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“Published by E F-1, release factor-1 (prfA); l-tRNA synthetase; WNK4, hoserine; MAGE, multiplex itute, Yale University, West s) acs), jesse.rinehart@yale.edu (EF-Sep) [2].”
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“We seeded 14 independent populations for serial batch evolution of each of the following four strains: (i) “Parent” (ECNR2, which is the nonrecoded parent strain of C321.”
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“Further improvements to MAGE and TRMR, along with methods to track mutations and link them to phenotypes, have also been reported, including co-selection MAGE (CoS-MAGE) [124], microarray oligonucleotide-MAGE (MO-MAGE) [18], CRISPR-enabled trackable genome engineering (CREATE) [77], CRISPR-optimized MAGE (CRMAGE) [104…”
MethodWang, Y., Cheng, H., Liu, Y., Liu, Y., Xiao, W., Zhang, K., Ni, X., Gao, N., Fan, L., Zhang, Z., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-21003-y · Cited by 108
Analytics for Metabolic Engineering
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“New technologies have advanced the Build component via low cost gene synthesis, rapid genome modifications [e.g., CAS9/CRISPR (Jinek et al., 2012)], and facile methods to rationally generate strain diversity [i.e., multiplex automated genome engineering (MAGE) (Wang et al., 2009) and trackable multiplex recombineering…”
MethodPolishing the craft of genetic diversity creation in directed evolution
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Combinatorial pathway optimization for streamlined metabolic engineering
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“This approach can now potentially be applied to the combinatorial diversification of targeted regions, similar to combina-D torial recombineering-based platforms such as MAGE (8), YOGE (9) or iCREATE (10).”
InfluentialA versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing
Tong, Y., Jørgensen, T. S., Whitford, C. M., Weber, T., & Lee, S. Y. (2021) · Nature Communications · doi:10.1038/s41467-021-25541-3 · Cited by 104
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“…approaches, multiplex automated genome engineering (mage) allows targeted, multiplexed editing, and evolution of microbial genomes using short oligonucleotides that are used as templates for allelic replacements mediated by the bacteriophage λ-Red ssDNA-binding protein β (Wang et al., 2009).”
BackgroundMethodTools and Principles for Microbial Gene Circuit Engineering
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Synthetic Evolution of Metabolic Productivity Using Biosensors
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“Instead of relying on random mutations, multiplexed automated genome engineering (MAGE) [33] was used to target specific metabolic genes for mutation.”
BackgroundMethodGenome engineering for improved recombinant protein expression in Escherichia coli
Mahalik, S., Sharma, A. K., & Mukherjee, K. J. (2014) · Microbial Cell Factories · doi:10.1186/s12934-014-0177-1 · Cited by 109
“CsrA, is a posttranscriptional global regulator that regulates mRNA stability and translation, which in turn is regulated by two sRNAs csrB and csrC [58-60].”
BackgroundInfluentialKim, S. K., Seong, W., Han, G. H., Lee, D., & Lee, S. (2017) · Microbial Cell Factories · doi:10.1186/s12934-017-0802-x · Cited by 103
“However, MAGE exhibits limited applicability to diverse microbial hosts, because it requires a certain strain deficient in the DNAmismatch-repair system, and the frequency of desired variants harboring multiple mutations is much lower than that of single-mutation variants [7, 8].”
BackgroundMethodPham, H. L., Wong, A., Chua, N., Teo, W. S., Yew, W. S., & Chang, M. W. (2017) · Nature Communications · doi:10.1038/s41467-017-00511-w · Cited by 103
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“Advances in next-generation sequencing-based assays have enabled quantitative measurements of mRNA levels and ribosome occupancies across an organism’s transcriptome (37,45).”
BackgroundDevelopments in the Tools and Methodologies of Synthetic Biology
Kelwick, R. J. R., MacDonald, J. T., Webb, A. J., & Freemont, P. S. (2014) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2014.00060 · Cited by 106
“Indeed, MAGE has been used to optimize the DXP pathway in E. coli, such that isolated variants that are capable of a fivefold increase in lycopene production were engineered in just 3 days (Wang et al., 2009).”
InfluentialYang, S. P., Sleight, S. C., & Sauro, H. M. (2012) · Nucleic Acids Research · doi:10.1093/nar/gks972 · Cited by 102
High-level diterpene production by transient expression in Nicotiana benthamiana
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“coli that the first step of the pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), constitutes a bottleneck which can be overcome by overexpression [39,40].”
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“sively parallel directed bacterial evolution (Wang et al. 2009) and sequencing (Loman et al.”
BackgroundScalable amplification of strand subsets from chip-synthesized oligonucleotide libraries
Schmidt, T. L., Beliveau, B. J., Uca, Y. O., Theilmann, M. R., Cruz, F. D., Wu, C.-T., & Shih, W. M. (2015) · Nature Communications · doi:10.1038/ncomms9634 · Cited by 98
“Examples for such application include DNA origami 1–3 ; single-stranded tile 4 or brick structures 5 ; multiplexed PCR and targeted sequencing 6 ; gene and genome synthesis 7–10 ; multiplexed genome engineering 11,12 ; and fluorescence in situ hybridization (FISH) 13–15 .”
CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli
Liang, L., Liu, R., Garst, A. D., Lee, T., Nogué, V. S. I., Beckham, G. T., & Gill, R. T. (2017) · Metabolic Engineering · doi:10.1016/j.ymben.2017.02.009 · Cited by 99
Metabolic engineering for enhancing microbial biosynthesis of advanced biofuels
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Protein mistranslation protects bacteria against oxidative stress
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“coli strains with various translational error rates using a genome editing approach (40).”
Methodλ Recombination and Recombineering
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Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelae
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Directed evolution: an evolving and enabling synthetic biology tool
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“coli genome [52 ].”
Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination
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Self-citationDirected Evolution of Microbial Communities
Sánchez, Á., Vila, J. C. C., Chang, C., Díaz‐Colunga, J., Estrela, S., & Rebolleda‐Gómez, M. (2021) · Annual Review of Biophysics · doi:10.1146/annurev-biophys-101220-072829 · Cited by 97
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“coli genes (dxs, dxr, idi, ispA) documented to increase lycopene [13, 34, 35] were selected and a combinatorial approach of metabolic engineering was applied to identify the bottleneck of the cyanobacterial MEP pathway toward FPP (Fig.”
Using natural products for drug discovery: the impact of the genomics era
Zhang, M. M., Qiao, Y., Ang, E. L., & Zhao, H. (2017) · Expert Opinion on Drug Discovery · doi:10.1080/17460441.2017.1303478 · Cited by 98
Multiplexed in Vivo His-Tagging of Enzyme Pathways for in Vitro Single-Pot Multienzyme Catalysis
Wang, H. H., Huang, P.-Y., Xu, G. J., Haas, W., Marblestone, A. H., Li, J., Gygi, S. P., Forster, A. C., Jewett, M. C., & Church, G. M. (2012) · ACS Synthetic Biology · doi:10.1021/sb3000029 · Cited by 91
Self-citationImproved sgRNA design in bacteria via genome-wide activity profiling
Guo, J., Wang, T., Guan, C., Liu, B., Luo, C. Y., Xie, Z., Zhang, C., & Xing, X. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky572 · Cited by 99
Santos, C. N. S., Regitsky, D. D., & Yoshikuni, Y. (2013) · Nature Communications · doi:10.1038/ncomms3503 · Cited by 93
A survey of enabling technologies in synthetic biology
Kahl, L. J., & Endy, D. (2013) · Journal of Biological Engineering · doi:10.1186/1754-1611-7-13 · Cited by 93
“More recently established tools such as PCR [10,11], fluorescent reporter molecules [90], and directed evolution [91] were also included.”
MethodEnhanced multiplex genome engineering through co-operative oligonucleotide co-selection
Carr, P. A., Wang, H. H., Sterling, B., Isaacs, F. J., Lajoie, M. J., Xu, G. J., Church, G. M., & Jacobson, J. M. (2012) · Nucleic Acids Research · doi:10.1093/nar/gks455 · Cited by 93
“We have found that protection of the oligo with at minimum two phosphorothioate linkages at the 50 terminus improves MAGE efficiency by a factor of 2 (10).”
BackgroundMethodInfluentialSelf-citationSimon, A. J., d’Oelsnitz, S., & Ellington, A. D. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0157-4 · Cited by 90
Recent advances in genetic tools for engineering probiotic lactic acid bacteria
Mugwanda, K., Hamese, S., van Zyl, W. F., Prinsloo, E., Plessis, M. D., Dicks, L. M. T., & Raj, D. B. T. G. (2023) · Bioscience Reports · doi:10.1042/bsr20211299 · Cited by 93
GPCR-Based Chemical Biosensors for Medium-Chain Fatty Acids
Mukherjee, K., Bhattacharyya, S., & Peralta‐Yahya, P. (2015) · ACS Synthetic Biology · doi:10.1021/sb500365m · Cited by 89
Wang, C., Zada, B., Wei, G., & Kim, S.-W. (2017) · Bioresource Technology · doi:10.1016/j.biortech.2017.05.168 · Cited by 92
The emerging age of cell‐free synthetic biology
Smith, M. T., Wilding, K. M., Hunt, J. M., Bennett, A. M., & Bundy, B. C. (2014) · FEBS Letters · doi:10.1016/j.febslet.2014.05.062 · Cited by 91
“Examples of these tools include MAGE and Cas9-CRISPR technologies for rapid directed genome editing [3-5], the development of diverse well-characterized BioBricks (gene parts/circuits) [1], and the synthesis of entirely synthetic viable genomes [10].”
BackgroundMethodPrecise genome editing across kingdoms of life using retron-derived DNA
Lopez, S. C., Crawford, K. D., Lear, S. K., Bhattarai-Kline, S., & Shipman, S. L. (2021) · Nature Chemical Biology · doi:10.1038/s41589-021-00927-y · Cited by 91
Automated in vivo enzyme engineering accelerates biocatalyst optimization
Orsi, E., von Borzyskowski, L. S., Noack, S., Nikel, P. I., & Lindner, S. N. (2024) · Nature Communications · doi:10.1038/s41467-024-46574-4 · Cited by 95
“Multiplex automated genome engineering (MAGE) as well as CRISPR-Cas technologies 91 or zinc fi nger nucleases 92 mediated tools are examples of in vivo directed mutagenesis based on mediated allelic replacement 93 .”
BackgroundMethodPathway engineering strategies for production of beneficial carotenoids in microbial hosts
Ye, V. M., & Bhatia, S. K. (2012) · Biotechnology Letters · doi:10.1007/s10529-012-0921-8 · Cited by 91
Mosberg, J. A., Gregg, C. J., Lajoie, M. J., Wang, H. H., & Church, G. M. (2012) · PLoS ONE · doi:10.1371/journal.pone.0044638 · Cited by 90
“Strain Creation EcNR2 (Escherichia coli MG1655 DmutS::cat D(ybhBbioAB)::[lcI857 N(cro-ea59)::tetR-bla]) [18] was used as the basis for all strains created in this work.”
BackgroundMethodInfluentialSelf-citationFine-tuning gene networks using simple sequence repeats
Egbert, R. G., & Klavins, E. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1205693109 · Cited by 90
Informing Biological Design by Integration of Systems and Synthetic Biology
Smolke, C. D., & Silver, P. A. (2011) · Cell · doi:10.1016/j.cell.2011.02.020 · Cited by 90
“…the integration of a number of pathway construction and optimization tools, including genomic discovery and engineering (Bayer et al., 2009; Ro et al., 2006; Wang et al., 2009), in vivo screens for enzyme activity (Pfleger et al., 2006), and enzyme localization strategies (Dueber et al., 2009).”
A navigation guide of synthetic biology tools for Pseudomonas putida
Martín‐Pascual, M., Batianis, C., Bruinsma, L., Asin‐Garcia, E., Morales, L. G., Weusthuis, R. A., van Kranenburg, R., & dos Santos, V. A. P. M. (2021) · Biotechnology Advances · doi:10.1016/j.biotechadv.2021.107732 · Cited by 92
Kumar, A., Mosa, K. A., Ji, L., Kage, U., Dhokane, D., Karre, S., Madalageri, D. M., & Pathania, N. (2017) · Critical Reviews in Food Science and Nutrition · doi:10.1080/10408398.2017.1285752 · Cited by 90
“Various types of genetic modifications in genome engineering include gene deletion, overexpression, and precise regulation, which are vital in improving pathway efficiency and product yield (Esvelt and Wang, 2013; Wang et al., 2009; Woodruff and Gill, 2011).”
BackgroundDirected evolution of multiple genomic loci allows the prediction of antibiotic resistance
Nyerges, Á., Csörgő, B., Draskovits, G., Kintses, B., Szili, P. É., Ferenc, G., Révész, T., Ari, E. E., Nagy, I., Bálint, B., et al. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1801646115 · Cited by 89
“Similarly to prior approaches (26, 28), DIvERGE proceeds via cell growth (1), oligo delivery and incorporation (2), andmutagenesis (3), leading to a highly elevated genetic diversity of the mutagenized population (4).”
BackgroundMethodResultInfluentialChallenges and tackles in metabolic engineering for microbial production of carotenoids
Wang, C., Zhao, S., Shao, X., Park, J.-B., Jeong, S.-H., Park, H.-J., Kwak, W.-J., Wei, G., & Kim, S.-W. (2019) · Microbial Cell Factories · doi:10.1186/s12934-019-1105-1 · Cited by 89
Volke, D. C., Martino, R. A., Kozaeva, E., Smania, A. M., & Nikel, P. I. (2022) · Nature Communications · doi:10.1038/s41467-022-30780-z · Cited by 89
In situ genotyping of a pooled strain library after characterizing complex phenotypes
Lawson, M. J., Camsund, D., Larsson, J., Baltekin, Ö., Fange, D., & Elf, J. (2017) · Molecular Systems Biology · doi:10.15252/msb.20177951 · Cited by 82
“…indicate assigned genotype (10: lacI knockdown; 01: lacY knockdown; 11: no knockdown). ª 2017 The Authors Molecular Systems Biology 13: 947 | 2017 chromosomal libraries with variants of promoters, ribosome binding sites (RBS), or coding sequences (Wang et al, 2009; Keren et al, 2016) can be made.”
BackgroundStrategy for directing combinatorial genome engineering in Escherichia coli
Sandoval, N. R., Kim, J. Y. H., Glebes, T. Y., Reeder, P. J., Aucoin, H. R., Warner, J. R., & Gill, R. T. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1206299109 · Cited by 87
“(5) to generate several different combinatorial RBS libraries specific to acetate, hydrolysate, or low pH tolerance.”
BackgroundMethodInfluentialPan, S., & Reed, J. L. (2017) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2017.12.012 · Cited by 86
Time-resolved imaging-based CRISPRi screening
Camsund, D., Lawson, M. J., Larsson, J., Jones, D., Zikrin, S., Fange, D., & Elf, J. (2019) · Nature Methods · doi:10.1038/s41592-019-0629-y · Cited by 79
Engineering biosynthetic enzymes for industrial natural product synthesis
Galanie, S. S., Entwistle, D. A., & Lalonde, J. J. (2020) · Natural Product Reports · doi:10.1039/c9np00071b · Cited by 91
Metabolism of the fast-growing bacterium Vibrio natriegens elucidated by 13C metabolic flux analysis
Long, C. P., Gonzalez, J. E., Cipolla, R. M., & Antoniewicz, M. R. (2017) · Metabolic Engineering · doi:10.1016/j.ymben.2017.10.008 · Cited by 83
“A wealth of knowledge has been generated over the past century for this model microbe, and many molecular tools have been developed for genetic engineering (Datsenko and Wanner, 2000; Gibson et al., 2009; Li et al., 2015; Wang et al., 2009).”
BackgroundÁlvarez, B., Mencía, M., de Lorenzo, V., & Fernández, L. Á. (2020) · Nature Communications · doi:10.1038/s41467-020-20230-z · Cited by 89
Metadata
- Authors
- Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M.
- Affiliations
- Harvard University (US)
- MIT Media Lab (US)
- Georgia Institute of Technology (US)
- Corresponding author
- Wang, Harris H.
- Isaacs, Farren J.
- ORCID
- Venue
- Nature
- Published
- 2009-07-26
- Volume
- 460
- Issue
- 7257
- Pages
- 894-898
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4590770
- OpenAlex
- W2144779133
- Cited by
- 1,529
- References
- 32
- Citations by year
- 2026: 56 · 2025: 60 · 2024: 70 · 2023: 58 · 2022: 97 · 2021: 94 · 2020: 88 · 2019: 96 · 2018: 119 · 2017: 113 · 2016: 121 · 2015: 141 · 2014: 114 · 2013: 94 · 2012: 81
- FWCI
- 27.54
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- National Institutes of Health (DP5OD009172)
- National Defense Science and Engineering Graduate
- Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Keywords
- accelerated evolution, directed evolution, genome editing, lycopene production, isoprenoid biosynthesis, Escherichia coli, synthetic DNA, metabolic engineering
- MeSH terms
- Lycopene, Alleles, Biotechnology, Carotenoids, Chromosomes, Bacterial, DNA, Escherichia coli, Pentosephosphates, Genetic Variation, Genome, Bacterial, Directed Molecular Evolution
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