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Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), resulting in ABE8e. ABE8e contains eight additional mutations that increase activity (kapp) 590-fold compared with ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disrupting regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by introducing additional mutations in the TadA-8e domain. Finally, we show that ABE8e can efficiently edit natural mutations in a GATA1 binding site in the BCL11A enhancer or the HBG promoter in human cells, targets which were poorly edited with ABE7.10. ABE8e broadens the effectiveness and applicability of adenine base editing., Editorial Summary A continuously evolved adenine base editor is compatible with various Cas proteins and mediates efficient A•T-to-G•C base conversions at a wide variety of PAM sites.

Fluorine has become an important element for the design of synthetic molecules for use in medicine, agriculture, and materials. Despite the many advantages provided by fluorine for tuning key molecular properties, it is rarely found in natural metabolism. We seek to expand the molecular space available for discovery through the development of new biosynthetic strategies that cross synthetic with natural compounds. Towards this goal, we engineered a microbial host for organofluorine metabolism and show that we can achieve the production of the fluorinated diketide 2-fluoro-3-hydroxybutyrate at approximately 50 % yield. This fluorinated diketide can be used as a monomer in vivo to produce fluorinated poly(hydroxyalkanoate) (PHA) bioplastics with fluorine substitutions ranging from around 5-15 %. This system provides a platform to produce mm flux through the key fluoromalonyl coenzyme A (CoA) building block, thereby offering the potential to generate a broad range of fluorinated small-molecule targets in living cells.

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.

The catalytic diversity of living systems offers a broad range of opportunities for developing new methods to produce small molecule targets such as fuels, materials, and pharmaceuticals. In addition to providing cost-effective and renewable methods for large-scale commercial processes, the exploration of the unusual chemical phenotypes found in living organisms can also enable the expansion of chemical space for discovery of novel function by combining orthogonal attributes from both synthetic and biological chemistry. In this context, we have focused on the development of new fluorine chemistry using synthetic biology approaches. While fluorine has become an important feature in compounds of synthetic origin, the scope of biological fluorine chemistry in living systems is limited, with fewer than 20 organofluorine natural products identified to date. In order to expand the diversity of biosynthetically accessible organofluorines, we have begun to develop methods for the site-selective introduction of fluorine into complex natural products by engineering biosynthetic machinery to incorporate fluorinated building blocks. To gain insight into how both enzyme active sites and metabolic pathways can be evolved to manage and select for fluorinated compounds, we have studied one of the only characterized natural hosts for organofluorine biosynthesis, the soil microbe Streptomyces cattleya. This information provides a template for designing engineered organofluorine enzymes, pathways, and hosts and has allowed us to initiate construction of enzymatic and cellular pathways for the production of fluorinated polyketides.

Stitching in Fluoroacetate Polyketide synthase enzymes stitch together an impressively diverse series of organic compounds from simple acetate and propionate building blocks. Walker et al. (p. 1089 ) now show that these biochemical pathways can be engineered to incorporate fluoroacetate—a primary product of the only known native enzymatic fluorination route—into tri- and tetraketides. In Escherichia coli cells, this process shows potential as a versatile means of inserting fluorine substituents into a range of complex molecules for use in pharmaceutical and agrochemical research.