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Biotechnology is increasingly relying on high-throughput experiments in which large populations of cells are phenotyped and selected based on performance, e.g., for producing a desired product. Droplet microfluidics enables high-throughput screening, and as such, it lends itself ideally to such experiments, but reliance on a fluorescent readout is limiting. Herein, we report a system integrating droplet electrospray ionization–mass spectrometry (ESI-MS) with a microfluidic voltage-mediated droplet printer to enable online analysis and capture of cell-containing aqueous droplets, ultimately offering the possibility for label-free phenotyping and selection of cells. Escherichia coli (E. coli) cell-containing droplets (20 nL) are split into two volume fractions within a microfluidic splitter chip. One fraction is analyzed using a sheath-flow ESI source with MS, while the sibling fraction is printed onto an agar plate using a custom-built voltage-mediated microfluidic printer. Printed droplets can grow into single microbial colonies that map back to their respective droplet ESI-MS signal with 94–99% accuracy and without carryover. Nearly synchronous and stable system operation is shown for infusion rates in the range of 0.4–1.2 droplets/s, while the achieved droplet spacing and printing precision can enable reliable single-colony retrieval for further analyses or gene sequencing. The system is also shown for phenotyping and selection of an E. coli variant engineered to produce l-lysine among control cells. The method enables screening cell colonies for chemical composition and collecting them for further processing with potential application in synthetic biology and enzyme engineering among others.

Non-heme iron (NHI) enzymes perform diverse oxidative transformations with precise control, which can be challenging to achieve with small molecule catalysts, such as the biosynthesis of tropolone. Among them, Anc3, a reconstructed ancestral α-ketoglutarate (α-KG)-dependent NHI dioxygenase, catalyzes a ring-expansion in fungal tropolone biosynthesis from a cyclohexadienone to afford the tropolone natural product stipitaldehyde (ring-expansion product) alongside 3-hydroxyorcinaldehyde (shunt product). This study reveals how the enzyme environment guides the reaction to the ring-expansion product preferably over the shunt product, where the precise selectivity ratio depends on just a handful of Anc3 residues. In particular, molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) simulations describe how the substrate binds within the NHI active site and can proceed through two distinct mechanisms, ring-expansion or rebound hydroxylation, to yield the two experimentally observed products. Discovery of a linear relationship of ΔEa values and hydrogen bond distances between Arg191 and the Fe(III)–OH group reveals that inhibition of the rebound hydroxylation step increases selectivity toward ring-expansion. Our findings suggest that the rebound hydroxylation rate is further tuned through the Fe(III)–OH bond strength, as influenced by specific secondary sphere coordination effects around the active site. These influences are largely orthogonal to the ring-expansion mechanism, which is shown to prefer to proceed through a radical pathway. In addition, a cationic pathway initiated by electron transfer from substrate to iron is shown to be unfavorable based upon thermodynamic considerations. Altogether, the atomistic details and reaction mechanisms delineated in this work have the potential to guide the tuning of the reaction pathway in related NHI enzymes for selective oxidation reactions.

Nature provides access to biological catalysts that can expand the chemical transformations accessible to synthetic chemists. Among these, α-ketoglutarate, non-heme iron-dependent (NHI) enzymes stand out as scalable biocatalysts for catalyzing selective oxidation reactions. Many NHI enzymes require protein engineering to improve their activity, selectivity, or stability. However, the reliance of this strategy on the innate stability of the enzyme can thwart the success of the engineering campaign. Harnessing innately stable enzymes can overcome these challenges and accelerate biocatalyst engineering. Herein, we highlight the use of ancestral sequence reconstruction (ASR) to mine for thermostable enzymes that can serve as superior starting points for protein engineering. In our effort to develop a biocatalytic route to tropolones, we identified an NHI enzyme that demonstrated poor stability, diminished activity at high substrate concentrations, and a limited substrate scope. We compared the in-lab evolution of the modern NHI enzyme and its ancestor, demonstrating the improved evolvability profile of the latter. By engineering the ancestral protein, we accessed variants with enhanced thermostability and expression, increased rates, and a substrate scope broader than those of their modern counterparts. Altogether, this work provides a strategy to rapidly access enzyme backbones that can accelerate engineering of more robust and synthetically useful NHI enzymes.

The elaboration of amine substrates through C–C bond-forming reactions is important in the synthesis of bioactive small molecules. Pyridoxal-5′-phosphate (PLP)-dependent enzymes have emerged as valuable biocatalysts for this class of reactions, due to their high stereoselectivity and ability to forge new C–C bonds on unprotected α-amino acid substrates. However, the use of abiological primary amines as pronucleophiles with enzymes such as threonine aldolase has been unexplored, moderating the utility of a biocatalytic approach in the synthesis of diverse 1,2-amino alcohols. In this report, we disclose the discovery and engineering of a PLP-dependent aldolase that accepts (2-azaaryl)methanamines in an aldol-type transformation. The 1,2-amino alcohol products generated, which contain representative heteroaromatic pharmacophores, are delivered with control over both the diastereoselectivity and enantioselectivity in the C–C bond-forming event. Protein engineering provided variants with improved binding affinity for the abiological substrate and decreased affinity for the native α-amino acid, overcoming inhibition of the abiotic reaction by components of lysate, a major challenge in reaction discovery with PLP-dependent enzymes such as threonine aldolases. This work represents the first known example of C–C bond formation on nonamino acid substrates with threonine aldolase and provides a platform for further development of complexity-building biocatalytic reactions with abiotic amine substrates.