Basecamp Research: Predictive enzyme development through nature and AI

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Growth amplification in ultrahigh-throughput microdroplet screening increases sensitivity of clonal enzyme assays and minimizes phenotypic variation.

Microfluidic ultrahigh-throughput screening of enzyme activities provides information on libraries with millions of variants in a day. Each individual library member is represented by a recombinant single cell, compartmentalised in an emulsion droplet, in which an activity assay is carried out. Key to the success of this approach is the precision and sensitivity of the assay. Assay quality is most profoundly challenged when initially weak, promiscuous activities are to be enhanced in early rounds of directed evolution or when entirely novel catalysts are to be identified from metagenomic sources. Implementation of measures to widen the dynamic range of clonal assays would increase the chances of finding and generating new biocatalysts. Here, we demonstrate that the assay sensitivity and DNA recovery can be improved by orders of magnitude by growth of initially singly compartmentalised cells in microdroplets. Homogeneous cell growth is achieved by continuous oxygenation and recombinant protein expression is regulated by diffusion of an inducer from the oil phase. Reaction conditions are adjusted by directed droplet coalescence to enable full control of buffer composition and kinetic incubation time, creating level playing field conditions for library selections. The clonal amplification multiplies the product readout because more enzyme is produced per compartment. At the same time, phenotypic variation is reduced by measuring monoclonal populations rather than single cells and recovery efficiency is increased. Consequently, this workflow increases the efficiency of lysate-based microfluidic enzyme assays and will make it easier for protein engineers to identify or evolve new enzymes for applications in synthetic and chemical biology.EPSRC, H2020, ER

TreasureDrop – enzyme engineering for applied biocatalysis using microfluidics

Enzymes have established as a new class of catalysts in the field of modern synthetic chemistry. Engineering is arguable the most promising approach to generate desired catalytic activities and its success directly correlates with the library size that can be screened. One of the most powerful technologies enabling the quick and cost-effective testing of millions of enzyme variants is the recently introduced microfluidic droplet-based screening. Interestingly, even though numerous publications highlight its potential, an unambiguous evidence of its ability to provide synthetically relevant biocatalysts still needs to be furnished. We present the engineering of an alcohol dehydrogenase for the challenging enantioselective reduction of a prochiral ketone targeting an important key building block for biologically active compounds. The final aim is not only to obtain an improved variant which allows to perform the selected biotransformation efficiently, but also a comparison of varying evolution paths. Please click Additional Files below to see the full abstract

UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution

Funder: EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011264; Grant(s): 722610Abstract: The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein’s amino acids (‘intra-gene epistasis’). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets

Ultrahigh-Throughput Detection of Enzymatic Alcohol Dehydrogenase Activity in Microfluidic Droplets with a Direct Fluorogenic Assay.

The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for >6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an >800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.ERC, H2020 Marie-Curi

Expanding the Boundary of Water-Tolerant Frustrated Lewis Pair Hydrogenation: Enhanced Back Strain in the Lewis Acid Enables the Reductive Amination of Carbonyls

The development of a boron/nitrogen-centered frustrated Lewis pair (FLP) with remarkably high water tolerance is presented. As systematic steric tuning of the boron-based Lewis acid (LA) component revealed, the enhanced back-strain makes water binding increasingly reversible in the presence of relatively strong base. This advance allows the limits of FLP's hydrogenation to be expanded, as demonstrated by the FLP reductive amination of carbonyls. This metal-free catalytic variant displays a notably broad chemo-selectivity and generality

Made in Bangalore: How social enterprise is transforming business-as-usual

Social enterprise has emerged as an innovative and creative alternative model for providing solutions to the continuing problems of poverty, along with inadequate social services, such as health and education. This study seeks to understand the potential of social enterprise in contributing to development, so that it can be leveraged to maximum effect. This research project, conducted between October 2010 and March 2011, focused upon the current leaders, sculptors and thinkers of the social enterprise landscape of Bangalore, a hub for creative enterprise grappling with rapid urbanisation and its associated opportunities and issues. The enterprises considered are promoting poverty reduction and sustainability through a 'fruit salad' of approaches and models. We look at how they achieve success, how they determine a workable business model, what impact their enterprises have, and what has helped or hindered them along the way

UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution

Funder: EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011264; Grant(s): 722610Abstract: The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein’s amino acids (‘intra-gene epistasis’). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets

Technical Considerations for Scale-Up of Imine-Reductase-Catalyzed Reductive Amination:A Case Study

Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein a number of guidelines and considerations toward the development and scale-up of IRED catalyzed reactions have been determined based on the reductive amination of cyclohexanone (<b>1</b>) with cyclopropylamine (<b>2</b>). A Design of Experiments (DoE) strategy has been followed to study the different reaction parameters, facilitating resource-efficient and informative screening. Enzyme stability was identified to be the limiting factor. By moving from batch to fed-batch, it was possible to double the concentration of the substrate and turnover number (TON). Kinetic studies revealed that IRED-33 was the best enzyme for the reaction with respect to both activity and stability. Under the optimal reaction conditions, it was possible to react <b>1</b> and <b>2</b> at 750 mM concentration and reach 100% conversion to the desired amine (>90% isolated yield) in the space of 8 h. Hence, excellent volumetric productivity of 12.9 g L^–1 h^–1 and TON above 48 000 were achieved

Intensified biocatalytic production of enantiomerically pure halophenylalanines from acrylic acids using ammonium carbamate as the ammonia source

An industrial-scale method employing a phenylalanine ammonia lyase enzyme.</p