Improved betulinic acid biosynthesis using synthetic yeast chromosome recombination and semi-automated rapid LC-MS screening.
Biotechnology
Chromatography, Liquid
Chromosomes, Fungal
Directed Molecular Evolution
Gene Library
Genes, Synthetic
Genetic Association Studies
Genetic Engineering
Genetic Testing
Genome, Fungal
Mass Spectrometry
Pentacyclic Triterpenes
Recombination, Genetic
Saccharomyces cerevisiae
/ genetics
Synthetic Biology
Triterpenes
/ metabolism
Betulinic Acid
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
13 02 2020
13 02 2020
Historique:
received:
21
08
2019
accepted:
24
01
2020
entrez:
15
2
2020
pubmed:
15
2
2020
medline:
28
4
2020
Statut:
epublish
Résumé
Synthetic biology, genome engineering and directed evolution offer innumerable tools to expedite engineering of strains for optimising biosynthetic pathways. One of the most radical is SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeast chromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours. SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited by screening capabilities. To address this bottleneck, we combine automated sample preparation, an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidly isolate and characterise the best performing strains. Here, we use SCRaMbLE to optimise yeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automated workflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidly isolate improved strains from a variant library makes this a valuable tool for biotechnology.
Identifiants
pubmed: 32054834
doi: 10.1038/s41467-020-14708-z
pii: 10.1038/s41467-020-14708-z
pmc: PMC7018806
doi:
Substances chimiques
Pentacyclic Triterpenes
0
Triterpenes
0
Betulinic Acid
4G6A18707N
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
868Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M025632/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/L027852/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/R002614/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/P504579/1
Pays : United Kingdom
Références
Nielsen, J. & Keasling, J. D. Engineering cellular metabolism. Cell 164, 1185–1197 (2016).
doi: 10.1016/j.cell.2016.02.004
Liu, W. & Jiang, R. Combinatorial and high-throughput screening approaches for strain engineering. Appl. Microbiol. Biotechnol. 99, 2093–2104 (2015).
doi: 10.1007/s00253-015-6400-0
Jones, J. A. & Koffas, M. A. G. Optimizing metabolic pathways for the improved production of natural products. Methods Enzymol. 575, 179–193 (2016).
doi: 10.1016/bs.mie.2016.02.010
Dietrich, J. A., McKee, A. E. & Keasling, J. D. High-throughput metabolic engineering: advances in small-molecule screening and selection. Annu. Rev. Biochem. 79, 563–590 (2010).
doi: 10.1146/annurev-biochem-062608-095938
Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth. Biol. 4, 975–986 (2015).
doi: 10.1021/sb500366v
Mitchell, L. A. et al. Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic Acids Res. 43, 6620–6630 (2015).
doi: 10.1093/nar/gkv466
Andreou, A. I. & Nakayama, N. Mobius assembly: a versatile golden-gate framework towards universal DNA assembly. PLoS ONE 13, 1–18 (2018).
doi: 10.1371/journal.pone.0189892
Fonseca, J. P. et al. A toolkit for rapid modular construction of biological circuits in mammalian cells. ACS Synth. Biol. 8, 2593–2606 (2019).
doi: 10.1021/acssynbio.9b00322
Storch, M. et al. BASIC: a new biopart assembly standard for idempotent cloning provides accurate, single-tier DNA assembly for synthetic biology. ACS Synth. Biol. 4, 781–787 (2015).
doi: 10.1021/sb500356d
Taylor, G. M., Mordaka, P. M. & Heap, J. T. Start-stop assembly: a functionally scarless DNA assembly system optimized for metabolic engineering. Nucleic Acids Res. 47, e17 (2019).
doi: 10.1093/nar/gky1182
Richardson, S. M. et al. Design of a synthetic yeast genome. Science 355, 1040–1044 (2017).
doi: 10.1126/science.aaf4557
Annaluru, N. et al. Total synthesis of a functional designer eukaryotic chromosome. Science 344, 55–59 (2014).
doi: 10.1126/science.1249252
Dymond, J. S. et al. Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471–476 (2011).
doi: 10.1038/nature10403
Dymond, J. & Boeke, J. The Saccharomyces cerevisiae SCRaMbLE system and genome minimization. Bioeng. Bugs 3, 168–171 (2012).
pubmed: 22572789
pmcid: 3370935
Ma, L. et al. SCRaMbLE generates evolved yeasts with increased alkali tolerance. Microb. Cell Fact. 18, 52 (2019).
doi: 10.1186/s12934-019-1102-4
Liu, W. et al. Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods. Nat. Commun. 9, 1–12 (2018).
doi: 10.1038/s41467-017-02088-w
Luo, Z. et al. Identifying and characterizing SCRaMbLEd synthetic yeast using ReSCuES. Nat. Commun. 9, 1–10 (2018).
doi: 10.1038/s41467-017-02088-w
Blount, B. A. et al. Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome. Nat. Commun. 9, 1932 (2018).
doi: 10.1038/s41467-018-03143-w
Wang, J. et al. Ring synthetic chromosome V SCRaMbLE. Nat. Commun. 9, 3783 (2018).
doi: 10.1038/s41467-018-06216-y
Jia, B. et al. Precise control of SCRaMbLE in synthetic haploid and diploid yeast. Nat. Commun. 9, 1933 (2018).
doi: 10.1038/s41467-018-03084-4
Wu, Y. et al. In vitro DNA SCRaMbLE. Nat. Commun. 9, 1935 (2018).
doi: 10.1038/s41467-018-03743-6
Petzold, C. J., Chan, L. J. G., Nhan, M. & Adams, P. D. Analytics for metabolic engineering. Front. Bioeng. Biotechnol. 3, 1–11 (2015).
doi: 10.3389/fbioe.2015.00135
Hollywood, K. A., Schmidt, K., Takano, E. & Breitling, R. Metabolomics tools for the synthetic biology of natural products. Curr. Opin. Biotechnol. 54, 114–120 (2018).
doi: 10.1016/j.copbio.2018.02.015
Couchman, L. et al. Ultra-fast LC–MS/MS in therapeutic drug monitoring: quantification of clozapine and norclozapine in human plasma. Drug Test. Anal. 10, 323–329 (2018).
doi: 10.1002/dta.2223
Xie, Z.-X. et al. “Perfect” designer chromosome V and behavior of a ring derivative. Science 355, eaaf4704 (2017).
doi: 10.1126/science.aaf4704
Cheng, X. et al. Quantitative analysis of betulinic acid in mouse, rat and dog plasma using electrospray liquid chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2089–2092 (2003).
doi: 10.1002/rcm.1155
Kosyakov, D. S., Ul’yanovskii, N. V. & Falev, D. I. Determination of triterpenoids from birch bark by liquid chromatography-tandem mass spectrometry. J. Anal. Chem. 69, 1264–1269 (2014).
doi: 10.1134/S1061934814130061
Shen, Y. et al. SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes. Genome Res. 26, 36–49 (2016).
doi: 10.1101/gr.193433.115
Strauss, S. K. et al. Evolthon: a community endeavor to evolve lab evolution. PLoS Biol. 17, 1–17 (2019).
Auxillos, J. Y. et al. Multiplex genome engineering for optimizing bioproduction in Saccharomyces cerevisiae. Biochemistry 58, 1492–1500 (2019).
doi: 10.1021/acs.biochem.8b01086
Kowalski, L. R. Z., Kondo, K. & Inouye, M. Cold-shock induction of a family of TIP1-related proteins associated with the membrane in Saccharomyces cerevisiae. Mol. Microbiol. 15, 341–353 (1995).
doi: 10.1111/j.1365-2958.1995.tb02248.x
Werner, E. D., Brodsky, J. L. & McCracken, A. A. Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate. Proc. Natl. Acad. Sci. USA 93, 13797–13801 (1996).
doi: 10.1073/pnas.93.24.13797
Kempa, E. E., Hollywood, K. A., Smith, C. A. & Barran, P. E. High throughput screening of complex biological samples with mass spectrometry-from bulk measurements to single cell analysis. Analyst 144, 872–891 (2019).
doi: 10.1039/C8AN01448E
Bolt, F. et al. Automated high-throughput identification and characterization of clinically important bacteria and fungi using rapid evaporative ionization mass spectrometry. Anal. Chem. 88, 9419–9426 (2016).
doi: 10.1021/acs.analchem.6b01016
Zhang, L., Smart, S. & Sandrin, T. R. Biomarker- and similarity coefficient-based approaches to bacterial mixture characterization using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sci. Rep. 5, 1–10 (2015).
Hillson, N. et al. Building a global alliance of biofoundries. Nat. Commun. 10, 1–4 (2019).
doi: 10.1038/s41467-018-07882-8
Milne, I. et al. Using tablet for visual exploration of second-generation sequencing data. Brief. Bioinformatics 14, 193–202 (2013).
doi: 10.1093/bib/bbs012