High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

Elmore, Joshua R.; Dexter, Gara N.; Baldino, Henri; Huenemann, Jay D.; Francis, Ryan; Peabody, George L.; Martinez-Baird, Jessica; Riley, Lauren A.; Simmons, Tuesday; Coleman-Derr, Devin; Guss, Adam M.; Egbert, Robert G.

doi:10.1126/sciadv.ade1285

High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

Journal Article · Thu Mar 09 23:00:00 EST 2023 · Science Advances

DOI:https://doi.org/10.1126/sciadv.ade1285· OSTI ID:1962851

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Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Science Division
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Univ. of Tennessee, Knoxville, TN (United States)
Univ. of California, Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States); US Dept. of Agriculture (USDA), Albany, CA (United States). Agricultural Research Service (ARS)

Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase–assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker–free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-76RL01830; AC05-00OR22725

OSTI ID:: 1962851

Alternate ID(s):: OSTI ID: 1962852
OSTI ID: 1997679

Report Number(s):: PNNL-SA--181397

Journal Information:: Science Advances, Journal Name: Science Advances Journal Issue: 10 Vol. 9; ISSN 2375-2548

Publisher:: AAASCopyright Statement

Country of Publication:: United States

Language:: English

References (80)

Genome engineering and gene expression control for bacterial strain development Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup Biotechnology Journal, Vol. 10, Issue 1 https://doi.org/10.1002/biot.201400057	journal	August 2014
Complete Nucleotide Sequence of Birmingham IncPα Plasmids Pansegrau, Werner; Lanka, Erich; Barth, Peter T. Journal of Molecular Biology, Vol. 239, Issue 5 https://doi.org/10.1006/jmbi.1994.1404	journal	June 1994
Nitrogen Fixation Through Genetic Engineering: A Future Systemic Approach of Nitrogen Fixation Bahuguna, Vivekanand; Bhatt, Gaurav; Maikhuri, Richa Microbial Metatranscriptomics Belowground https://doi.org/10.1007/978-981-15-9758-9_5	book	January 2021
Effect of the bacterial growth rate on replication control of plasmid pBR322 in Escherichia coli Lin-Chao, S.; Bremer, H. Molecular and General Genetics MGG, Vol. 203, Issue 1 https://doi.org/10.1007/BF00330395	journal	April 1986
A diversity of serine phage integrases mediate site-specific recombination in mammalian cells Keravala, Annahita; Groth, Amy C.; Jarrahian, Sohail Molecular Genetics and Genomics, Vol. 276, Issue 2 https://doi.org/10.1007/s00438-006-0129-5	journal	May 2006
Genetic tools for reliable gene expression and recombineering in Pseudomonas putida Cook, Taylor B.; Rand, Jacqueline M.; Nurani, Wasti Journal of Industrial Microbiology & Biotechnology, Vol. 45, Issue 7 https://doi.org/10.1007/s10295-017-2001-5	journal	January 2018
Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis Xu, Ning; Wei, Liang; Liu, Jun World Journal of Microbiology and Biotechnology, Vol. 35, Issue 2 https://doi.org/10.1007/s11274-019-2606-0	journal	January 2019
Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Bierman, M.; Logan, R.; O'Brien, K. Gene, Vol. 116, Issue 1 https://doi.org/10.1016/0378-1119(92)90627-2	journal	July 1992
Replication and copy number control of the broad-host-range plasmid RSF1010 Frey, Joachim; Bagdasarian, Mira M.; Bagdasarian, Michael Gene, Vol. 113, Issue 1 https://doi.org/10.1016/0378-1119(92)90675-F	journal	April 1992
Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria Quandt, Jürgen; Hynes, Michael F. Gene, Vol. 127, Issue 1 https://doi.org/10.1016/0378-1119(93)90611-6	journal	May 1993
Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum Schäfer, Andreas; Tauch, Andreas; Jäger, Wolfgang Gene, Vol. 145, Issue 1, p. 69-73 https://doi.org/10.1016/0378-1119(94)90324-7	journal	July 1994
Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes Kovach, Michael E.; Elzer, Philip H.; Steven Hill, D. Gene, Vol. 166, Issue 1 https://doi.org/10.1016/0378-1119(95)00584-1	journal	December 1995
Enzymatic ω-Oxidation McKenna, Eva J.; Coon, Minor J. Journal of Biological Chemistry, Vol. 245, Issue 15 https://doi.org/10.1016/S0021-9258(18)62932-1	journal	August 1970
Synthetic biology approaches for chromosomal integration of genes and pathways in industrial microbial systems Li, Lei; Liu, Xiaocao; Wei, Keke Biotechnology Advances, Vol. 37, Issue 5 https://doi.org/10.1016/j.biotechadv.2019.04.002	journal	September 2019
Investigating the interactions between physical and biological heterogeneities in bioreactors using compartment, population balance and metabolic models Pigou, Maxime; Morchain, Jérôme Chemical Engineering Science, Vol. 126 https://doi.org/10.1016/j.ces.2014.11.035	journal	April 2015
Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida Troeschel, Sonja Christina; Thies, Stephan; Link, Olga Journal of Biotechnology, Vol. 161, Issue 2 https://doi.org/10.1016/j.jbiotec.2012.02.020	journal	October 2012
Investigation of plasmid-induced growth defect in Pseudomonas putida Mi, Jia; Sydow, Anne; Schempp, Florence Journal of Biotechnology, Vol. 231 https://doi.org/10.1016/j.jbiotec.2016.06.001	journal	August 2016
Phage Integrases: Biology and Applications Groth, Amy C.; Calos, Michele P. Journal of Molecular Biology, Vol. 335, Issue 3 https://doi.org/10.1016/j.jmb.2003.09.082	journal	January 2004
New Applications for Phage Integrases Fogg, Paul C. M.; Colloms, Sean; Rosser, Susan Journal of Molecular Biology, Vol. 426, Issue 15 https://doi.org/10.1016/j.jmb.2014.05.014	journal	July 2014
Innovative Chemicals and Materials from Bacterial Aromatic Catabolic Pathways Johnson, Christopher W.; Salvachúa, Davinia; Rorrer, Nicholas A. Joule, Vol. 3, Issue 6 https://doi.org/10.1016/j.joule.2019.05.011	journal	June 2019
Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440 Elmore, Joshua R.; Furches, Anna; Wolff, Gara N. Metabolic Engineering Communications, Vol. 5 https://doi.org/10.1016/j.meteno.2017.04.001	journal	December 2017
Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon Silo-Suh, Laura A.; Elmore, Brett; Ohman, Dennis E. Journal of Microbiological Methods, Vol. 78, Issue 3 https://doi.org/10.1016/j.mimet.2009.07.002	journal	September 2009
Transcription control engineering and applications in synthetic biology Engstrom, Michael D.; Pfleger, Brian F. Synthetic and Systems Biotechnology, Vol. 2, Issue 3 https://doi.org/10.1016/j.synbio.2017.09.003	journal	September 2017
aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes Li, Lei; Wei, Keke; Liu, Xiaocao Metabolic Engineering, Vol. 52 https://doi.org/10.1016/j.ymben.2018.12.001	journal	March 2019
Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose, xylose, arabinose, p-coumaric acid, and acetic acid Elmore, Joshua R.; Dexter, Gara N.; Salvachúa, Davinia Metabolic Engineering, Vol. 62 https://doi.org/10.1016/j.ymben.2020.08.001	journal	November 2020
Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440 Werner, Allison Z.; Clare, Rita; Mand, Thomas D. Metabolic Engineering, Vol. 67 https://doi.org/10.1016/j.ymben.2021.07.005	journal	September 2021
Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass Kim, Jiwon; Hwang, Sungmin; Lee, Sun-Mi Metabolic Engineering, Vol. 71 https://doi.org/10.1016/j.ymben.2021.10.002	journal	May 2022
Serine recombinases as tools for genome engineering Brown, William R. A.; Lee, Nicholas C. O.; Xu, Zhengyao Methods, Vol. 53, Issue 4 https://doi.org/10.1016/j.ymeth.2010.12.031	journal	April 2011
Noise in a minimal regulatory network: plasmid copy number control Paulsson, Johan; Ehrenberg, Måns Quarterly Reviews of Biophysics, Vol. 34, Issue 1 https://doi.org/10.1017/S0033583501003663	journal	February 2001
CRAGE-Duet Facilitates Modular Assembly of Biological Systems for Studying Plant–Microbe Interactions Wang, Bing; Zhao, Zhiying; Jabusch, Lauren K. ACS Synthetic Biology, Vol. 9, Issue 9 https://doi.org/10.1021/acssynbio.0c00280	journal	July 2020
Expression of Alternative Nitrogenases in Rhodopseudomonas palustris Is Enhanced Using an Optimized Genetic Toolset for Rapid, Markerless Modifications du Toit, Jan-Pierre; Lea-Smith, David J.; Git, Anna ACS Synthetic Biology, Vol. 10, Issue 9 https://doi.org/10.1021/acssynbio.0c00496	journal	August 2021
Serine Integrases: Advancing Synthetic Biology Merrick, Christine A.; Zhao, Jia; Rosser, Susan J. ACS Synthetic Biology, Vol. 7, Issue 2 https://doi.org/10.1021/acssynbio.7b00308	journal	January 2018
Shifts in the host range of a promiscuous plasmid through parallel evolution of its replication initiation protein Sota, Masahiro; Yano, Hirokazu; M. Hughes, Julie The ISME Journal, Vol. 4, Issue 12 https://doi.org/10.1038/ismej.2010.72	journal	June 2010
Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria Isabella, Vincent M.; Ha, Binh N.; Castillo, Mary Joan Nature Biotechnology, Vol. 36, Issue 9 https://doi.org/10.1038/nbt.4222	journal	October 2018
Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris Larimer, Frank W; Chain, Patrick; Hauser, Loren Nature Biotechnology, Vol. 22, Issue 1, p. 55-61 https://doi.org/10.1038/nbt923	journal	December 2003
A replication clock for Mycobacterium tuberculosis Gill, Wendy P.; Harik, Nada S.; Whiddon, Molly R. Nature Medicine, Vol. 15, Issue 2 https://doi.org/10.1038/nm.1915	journal	February 2009
A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum Shaner, Nathan C.; Lambert, Gerard G.; Chammas, Andrew Nature Methods, Vol. 10, Issue 5 https://doi.org/10.1038/nmeth.2413	journal	March 2013
Characterization of 582 natural and synthetic terminators and quantification of their design constraints Chen, Ying-Ja; Liu, Peng; Nielsen, Alec A. K. Nature Methods, Vol. 10, Issue 7 https://doi.org/10.1038/nmeth.2515	journal	June 2013
Permanent genetic memory with >1-byte capacity Yang, Lei; Nielsen, Alec A. K.; Fernandez-Rodriguez, Jesus Nature Methods, Vol. 11, Issue 12 https://doi.org/10.1038/nmeth.3147	journal	October 2014
Metagenomic mining of regulatory elements enables programmable species-selective gene expression Johns, Nathan I.; Gomes, Antonio L. C.; Yim, Sung Sun Nature Methods, Vol. 15, Issue 5 https://doi.org/10.1038/nmeth.4633	journal	March 2018
Integrative analysis of fitness and metabolic effects of plasmids in Pseudomonas aeruginosa PAO1 San Millan, Alvaro; Toll-Riera, Macarena; Qi, Qin The ISME Journal, Vol. 12, Issue 12 https://doi.org/10.1038/s41396-018-0224-8	journal	August 2018
Fungal community assembly in drought-stressed sorghum shows stochasticity, selection, and universal ecological dynamics Gao, Cheng; Montoya, Liliam; Xu, Ling Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-019-13913-9	journal	January 2020
Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria Lammens, Eveline-Marie; Nikel, Pablo Ivan; Lavigne, Rob Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-19124-x	journal	October 2020
Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion Elmore, Joshua R.; Dexter, Gara N.; Salvachúa, Davinia Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-22556-8	journal	April 2021
Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria Brophy, Jennifer A. N.; Triassi, Alexander J.; Adams, Bryn L. Nature Microbiology, Vol. 3, Issue 9 https://doi.org/10.1038/s41564-018-0216-5	journal	August 2018
CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria Wang, Gaoyan; Zhao, Zhiying; Ke, Jing Nature Microbiology, Vol. 4, Issue 12 https://doi.org/10.1038/s41564-019-0573-8	journal	October 2019
CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering Vo, Phuc Leo H.; Ronda, Carlotta; Klompe, Sanne E. Nature Biotechnology, Vol. 39, Issue 4 https://doi.org/10.1038/s41587-020-00745-y	journal	November 2020
Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome Durrant, Matthew G.; Fanton, Alison; Tycko, Josh Nature Biotechnology https://doi.org/10.1038/s41587-022-01494-w	journal	October 2022
ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways Jones, J. Andrew; Vernacchio, Victoria R.; Lachance, Daniel M. Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep11301	journal	June 2015
Thermochemical wastewater valorization via enhanced microbial toxicity tolerance Jayakody, Lahiru N.; Johnson, Christopher W.; Whitham, Jason M. Energy & Environmental Science, Vol. 11, Issue 6 https://doi.org/10.1039/C8EE00460A	journal	January 2018
Far-red fluorescent tags for protein imaging in living tissues Shcherbo, Dmitry; Murphy, Christopher S.; Ermakova, Galina V. Biochemical Journal, Vol. 418, Issue 3 https://doi.org/10.1042/BJ20081949	journal	February 2009
Tuning genetic control through promoter engineering Alper, H.; Fischer, C.; Nevoigt, E. Proceedings of the National Academy of Sciences, Vol. 102, Issue 36, p. 12678-12683 https://doi.org/10.1073/pnas.0504604102	journal	August 2005
Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria Xu, Ling; Naylor, Dan; Dong, Zhaobin Proceedings of the National Academy of Sciences, Vol. 115, Issue 18 https://doi.org/10.1073/pnas.1717308115	journal	April 2018
Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination Colloms, Sean D.; Merrick, Christine A.; Olorunniji, Femi J. Nucleic Acids Research, Vol. 42, Issue 4 https://doi.org/10.1093/nar/gkt1101	journal	November 2013
Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer. Bailey, M. J.; Lilley, A. K.; Thompson, I. P. Molecular Ecology, Vol. 4, Issue 6 https://doi.org/10.1111/j.1365-294X.1995.tb00276.x	journal	December 1995
A phage protein that binds φC31 integrase to switch its directionality: Excision by φC31 integrase Khaleel, Thanafez; Younger, Ellen; McEwan, Andrew R. Molecular Microbiology, Vol. 80, Issue 6 https://doi.org/10.1111/j.1365-2958.2011.07696.x	journal	May 2011
Site-specific integration ofStreptomycesÎ¦C31 integrase-based vectors in the chromosome ofRhodococcus equi Hong, Yang; Hondalus, Mary K. FEMS Microbiology Letters, Vol. 287, Issue 1 https://doi.org/10.1111/j.1574-6968.2008.01298.x	journal	October 2008
Optimized gene expression from bacterial chromosome by high-throughput integration and screening Saleski, Tatyana E.; Chung, Meng Ting; Carruthers, David N. Science Advances, Vol. 7, Issue 7 https://doi.org/10.1126/sciadv.abe1767	journal	February 2021
Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System Jiang, Yu; Chen, Biao; Duan, Chunlan Applied and Environmental Microbiology, Vol. 81, Issue 7, p. 2506-2514 https://doi.org/10.1128/AEM.04023-14	journal	January 2015
Integration Site for Streptomyces Phage φBT1 and Development of Site-Specific Integrating Vectors Gregory, Matthew A.; Till, Rob; Smith, Margaret C. M. Journal of Bacteriology, Vol. 185, Issue 17 https://doi.org/10.1128/JB.185.17.5320-5323.2003	journal	September 2003
A Novel Transformation of Polychlorinated Biphenyls by Rhodococcus sp. Strain RHA1. Seto, M.; Kimbara, K.; Shimura, M. Applied and environmental microbiology, Vol. 61, Issue 9 https://doi.org/10.1128/aem.61.9.3353-3358.1995	journal	January 1995
Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers. Durland, R. H.; Toukdarian, A.; Fang, F. Journal of Bacteriology, Vol. 172, Issue 7 https://doi.org/10.1128/jb.172.7.3859-3867.1990	journal	January 1990
Structural organization of pBC1, a cryptic plasmid from Bacillus coagulans De Rossi, E.; Milano, A.; Brigidi, P. Journal of Bacteriology, Vol. 174, Issue 2 https://doi.org/10.1128/jb.174.2.638-642.1992	journal	January 1992
Bacterial Longevity Requires Protein Synthesis and a Stringent Response Yin, Liang; Ma, Hongyu; Nakayasu, Ernesto S. mBio, Vol. 10, Issue 5 https://doi.org/10.1128/mBio.02189-19	journal	October 2019
Genomic Characterization and Copy Number Variation of Bacillus anthracis Plasmids pXO1 and pXO2 in a Historical Collection of 412 Strains Pena-Gonzalez, Angela; Rodriguez-R, Luis M.; Marston, Chung K. mSystems, Vol. 3, Issue 4 https://doi.org/10.1128/mSystems.00065-18	journal	August 2018
New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species Guss, Adam M.; Rother, Michael; Zhang, Jun Kai Archaea, Vol. 2, Issue 3 https://doi.org/10.1155/2008/534081	journal	January 2008
The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis Thomson, James G.; Chan, Ronald; Smith, Jamison BMC Biotechnology, Vol. 12, Issue 1 https://doi.org/10.1186/1472-6750-12-9	journal	March 2012
Process inhomogeneity leads to rapid side product turnover in cultivation of Corynebacterium glutamicum Käß, Friedrich; Junne, Stefan; Neubauer, Peter Microbial Cell Factories, Vol. 13, Issue 1 https://doi.org/10.1186/1475-2859-13-6	journal	January 2014
BglBrick vectors and datasheets: A synthetic biology platform for gene expression Lee, Taek; Krupa, Rachel A.; Zhang, Fuzhong Journal of Biological Engineering, Vol. 5, Issue 1 https://doi.org/10.1186/1754-1611-5-12	journal	January 2011
Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae Xu, Zhengyao; Brown, William R. A. BMC Biotechnology, Vol. 16, Issue 1 https://doi.org/10.1186/s12896-016-0241-5	journal	February 2016
Engineered Pseudomonas putida KT2440 co-utilizes galactose and glucose Peabody, George L.; Elmore, Joshua R.; Martinez-Baird, Jessica Biotechnology for Biofuels, Vol. 12, Issue 1 https://doi.org/10.1186/s13068-019-1627-0	journal	December 2019
Approaches to genetic tool development for rapid domestication of non-model microorganisms Riley, Lauren A.; Guss, Adam M. Biotechnology for Biofuels, Vol. 14, Issue 1 https://doi.org/10.1186/s13068-020-01872-z	journal	January 2021
Control of Phage Bxb1 Excision by a Novel Recombination Directionality Factor Ghosh, Pallavi; Wasil, Laura R.; Hatfull, Graham F. PLoS Biology, Vol. 4, Issue 6 https://doi.org/10.1371/journal.pbio.0040186	journal	May 2006
A One Pot, One Step, Precision Cloning Method with High Throughput Capability Engler, Carola; Kandzia, Romy; Marillonnet, Sylvestre PLoS ONE, Vol. 3, Issue 11, Article No. e3647 https://doi.org/10.1371/journal.pone.0003647	journal	November 2008
Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems Yim, Sung Sun; Johns, Nathan I.; Park, Jimin Molecular Systems Biology, Vol. 15, Issue 8 https://doi.org/10.15252/msb.20198875	journal	August 2019
Adaptive Plasmid Evolution Results in Host-Range Expansion of a Broad-Host-Range Plasmid De Gelder, Leen; Williams, Julia J.; Ponciano, José M. Genetics, Vol. 178, Issue 4 https://doi.org/10.1534/genetics.107.084475	journal	April 2008
Charging State Analysis of Transfer RNA from an α-proteobacterium Yin, Liang; Harwood, Caroline BIO-PROTOCOL, Vol. 10, Issue 23 https://doi.org/10.21769/BioProtoc.3834	journal	January 2020
Molecular Biology in the Improvement of Biological Nitrogen Fixation by Rhizobia and Extending the Scope to Cereals Goyal, Ravinder K.; Schmidt, Maria Augusta; Hynes, Michael F. Microorganisms, Vol. 9, Issue 1 https://doi.org/10.3390/microorganisms9010125	journal	January 2021
Genome Editing in Bacteria: CRISPR-Cas and Beyond Arroyo-Olarte, Ruben D.; Bravo Rodríguez, Ricardo; Morales-Ríos, Edgar Microorganisms, Vol. 9, Issue 4 https://doi.org/10.3390/microorganisms9040844	journal	April 2021
Consolidated Bioprocessing: Synthetic Biology Routes to Fuels and Fine Chemicals Banner, Alec; Toogood, Helen S.; Scrutton, Nigel S. Microorganisms, Vol. 9, Issue 5 https://doi.org/10.3390/microorganisms9051079	journal	May 2021

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