Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry

Glen-Oliver F Gowers, Simon J S Cameron, Alvaro Perdones-Montero, David Bell, Soo Mei Chee, Marcelo Kern, David Tew, Tom Ellis, Zoltan Takats

Research output: Contribution to journalArticle

Abstract

Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) - a form of ambient laser desorption ionisation mass spectrometry - to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies in under 4 hours, while simultaneously generating recoverable glycerol stocks of each colony in real-time. This showcases LA-REIMS as a pre-screening tool to complement downstream quantification methods such as LCMS. Through pre-screening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.

Original languageEnglish
JournalACS Synthetic Biology
Early online date17 Oct 2019
DOIs
Publication statusEarly online date - 17 Oct 2019

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Libraries
Mass Spectrometry
Lasers
Yeasts
Synthetic Biology
Technology
Mass Screening
Workflow
Biosynthetic Pathways
Molecular Cloning
Glycerol
Agar
Phenotype
DNA

Cite this

Gowers, Glen-Oliver F ; Cameron, Simon J S ; Perdones-Montero, Alvaro ; Bell, David ; Chee, Soo Mei ; Kern, Marcelo ; Tew, David ; Ellis, Tom ; Takats, Zoltan. / Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry. In: ACS Synthetic Biology. 2019.
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Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry. / Gowers, Glen-Oliver F; Cameron, Simon J S; Perdones-Montero, Alvaro; Bell, David; Chee, Soo Mei; Kern, Marcelo; Tew, David; Ellis, Tom; Takats, Zoltan.

In: ACS Synthetic Biology, 17.10.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry

AU - Gowers, Glen-Oliver F

AU - Cameron, Simon J S

AU - Perdones-Montero, Alvaro

AU - Bell, David

AU - Chee, Soo Mei

AU - Kern, Marcelo

AU - Tew, David

AU - Ellis, Tom

AU - Takats, Zoltan

PY - 2019/10/17

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AB - Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) - a form of ambient laser desorption ionisation mass spectrometry - to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies in under 4 hours, while simultaneously generating recoverable glycerol stocks of each colony in real-time. This showcases LA-REIMS as a pre-screening tool to complement downstream quantification methods such as LCMS. Through pre-screening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.

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JO - ACS Synthetic Biology

JF - ACS Synthetic Biology

SN - 2161-5063

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