Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

Abstract

Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet–MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)–MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10–50 nL) by tuning the sheath flow within the CE–MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet–MS and LC–MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription–translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

“…Droplet microfluidics enables experiments to be performed at nanoliter to femtoliter scale, increasing throughput and decreasing unit costs of chemical and biological experimentation . A decade of research in the field has demonstrated the utility of droplet systems for a range of applications, including single cell gene expression profiling, small molecule screening, and diagnostics .…”

“…FADS has found use in ultrahigh throughput screening for directed evolution . Screening large libraries is often the rate‐limiting step in biocatalyst development, where thousands of enzyme variants must be tested for catalytic activity, and plate based screens are time and resource intensive. FADS has enabled screening of libraries containing millions of variants in a few hours.…”

Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research

“…These droplets can be directly interfaced to an ESI source for MS-based screening at a rate of < 1 s ( Fig. 1b) [24,29]. To achieve robust screening, it is necessary to perform systematic optimization of many parameters including flow rate, emitter configuration, and droplet-stabilizing surfactants [29].…”

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

“…Droplet microfluidics enables experiments to be performed at nanoliter to femtoliter scale, increasing throughput and decreasing unit costs of chemical and biological experimentation . A decade of research in the field has demonstrated the utility of droplet systems for a range of applications, including single cell gene expression profiling, small molecule screening, and diagnostics .…”

“…MS offers nearly universal label‐free detection with high sensitivity and selectivity, as well as the flexibility for multiplexing. Recent work with ESI and matrix‐assisted laser desorption/ionization (MALDI) MS has shown that MS can be used for droplet analysis; however, sample destruction by ESI has been an obstacle to coupling it to microfluidic droplet sorting.…”