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Engineering complex biological systems in bacteria through recombinase-assisted genome engineering

Nature Protocols volume 9, pages 1320–1336 (2014)Cite this article

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Abstract

Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. This engineering strategy was previously applied to the development of an Escherichia coli–based platform, which enabled the use of brown macroalgae as a feedstock for the production of biofuels and renewable chemicals. In this approach, functional genetic modules are first designed in silico and constructed on a bacterial artificial chromosome (BAC) by using a recombineering-based inchworm extension technique. Stable integration into the recipient chromosome is then mediated through the use of recombinase-assisted genome engineering (RAGE). The flexibility, simplicity and speed of this method enable a comprehensive optimization of several different parameters, including module configuration, strain background, integration locus, gene copy number and intermodule compatibility. This paradigm therefore has the potential to markedly expedite most strain-engineering endeavors. Once a biological system has been designed and constructed on a BAC, its implementation and optimization in a recipient host can be carried out in as little as 1 week.

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Figure 1: Strain-engineering design principle based on the use of RAGE for the optimal implementation of complex biological systems.
Figure 2: Module construction and recombineering into BACs.
Figure 3: Two methods for inchworm extension of BACs through recombineering.
Figure 4: RAGE is a genome engineering strategy that facilitates the stable integration of complex biological systems into the bacterial chromosome by recombinase-mediated cassette exchange.

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Author information

Author notes

  1. Christine Nicole S Santos

    Present address: Present address: Manus Biosynthesis, Inc., Cambridge, Massachusetts, USA.,

Authors and Affiliations

  1. Bio Architecture Lab, Inc., Berkeley, California, USA

    Christine Nicole S Santos & Yasuo Yoshikuni

  2. BALChile S.A., Santiago, Chile

    Yasuo Yoshikuni

  3. BAL Biofuels S.A., Santiago, Chile

    Yasuo Yoshikuni

Authors
  1. Christine Nicole S Santos
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All authors contributed equally to the preparation of this manuscript.

Corresponding author

Correspondence to Yasuo Yoshikuni.

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Integrated supplementary information

Supplementary Figure 1 Construction of pALG3 plasmid for alginate degradation and metabolism.

1: pALG1 was first constructed by screening a fosmid library of the Vibrio splendidus 12B01 genome. We found several other genetic components that might be responsible for the degradation and metabolism of alginate downstream of the segment found in pALG1 in its genome. 2-4: Two genes downstream of the genomic segment found in pALG1 (V12B01_24254 and V12B01_24259) were cloned in the pKD13-based plasmid. The integration cassette was prepared by PCR (2) and integrated into pALG1 using recombineering techniques (3). The kanamycin selection marker was excised with a FLP recombinase (4) to yield pALG1.1. 5-7: Three other genes downstream of the genomic segment found in pALG1.1 (V12B01_24264, V12B01_24269, and V12B01_24274) were cloned in the pKD13-based plasmid. The integration cassette was prepared by PCR (5) and integrated into pALG1.1 using recombineering techniques (6). The kanamycin selection marker was excised with a FLP recombinase (7) to yield pALG1.2. 8 and 9: Two other genetic components were found downstream of the genomic segment in pALG1.2 (V12B01_24309 and V12B01_24324) and cloned in the pKm-based plasmid. The integration cassette was prepared by PCR (8), and the cassette was integrated into pALG1.2 using recombineering techniques (9) to yield pALG3.

Supplementary Figure 2 Construction of pALG7 plasmid for alginate and cellobiose degradation and metabolism.

1: We found several other genetic components that might be responsible for the degradation and metabolism of alginate downstream of the genomic segment in pALG1 and in the genomes of other microbes such as Agrobacterium tumefaciens C58 and Pseudoalteromonas sp. SM0524. We also found genes that may be responsible for cellobiose degradation and metabolism in Saccharophagus degradans 2-40. These genetic components were integrated into the module. 2-3: Three genetic components were found downstream of the genomic segment in pALG1 (V12B01_24309, V12B01_24324, and V12B01_24269) and cloned in the pKm-based plasmid (pKm_V12B01_24309_24324_24269). The integration cassette was prepared by PCR (2) and integrated into pALG1 using recombineering techniques (3) to yield pALG2. 4-5: An operon comprising genetic components Atu_3019-3026 (suspected to be responsible for alginate metabolism in Agrobacterium tumefaciens C58) was cloned in the pCm-based plasmid (pCm_Atu2019-3026). The integration cassette was prepared using PCR (4) and integrated into pALG2 using recombineering techniques (5) to yield pALG3.0. 6-8: Two genes downstream of the genomic segment found in pALG1 (V12B01_24254 and V12B01_24259) were cloned in the pKD13-based plasmid (pKD_V12B01_24254-24259). The integration cassette was prepared by PCR (6) and integrated into pALG3.0 using recombineering techniques (7). The kanamycin selection marker was excised via FLP recombinase (8) to yield pALG3.5. 9-10: Genetic components that were suspected to be responsible for cellobiose metabolism in Saccharophagus degradans 2-40 were cloned in the pKm-based plasmid (pKm_Sdes). The integration cassette was prepared with PCR (9) and integrated into pALG3.5 using recombineering techniques (10) to yield pALG4.0. 11-12: A gene encoding a bifunctional alginate lyase derived from Pseudoalteromonas sp. SM0524 was engineered to be secretable in E. coli and was cloned in pBeloBAC11-based plasmid (pCm and pBeloBAC11 share the chloramphenicol resistance gene). This integration cassette was prepared by PCR (11) and integrated into pALG4.0 using recombineering techniques (12) to yield pALG7.2. 13-15; three other genes downstream of the genomic segment found in pALG1 (V12B01_24264, V12B01_24269, and V12B01_24274) were cloned in the pKD13-based plasmid (pKD_V12B01_24264-24274). The integration cassette was prepared by PCR (13). This cassette was integrated into pAL7.2 using recombineering techniques (14). The kanamycin selection marker was excised via FLP recombinase (15) to yield pALG7.8.

Supplementary information

Supplementary Figure 1

Construction of pALG3 plasmid for alginate degradation and metabolism. (PDF 341 kb)

Supplementary Figure 2

Construction of pALG7 plasmid for alginate and cellobiose degradation and metabolism. (PDF 363 kb)

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Santos, C., Yoshikuni, Y. Engineering complex biological systems in bacteria through recombinase-assisted genome engineering. Nat Protoc 9, 1320–1336 (2014). https://doi.org/10.1038/nprot.2014.084

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