We used BE-PACE to evolve new 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 GC, 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.Genome editing has revolutionized the life sciences and entered clinical trials to treat genetic diseases. 1 The use of programmable nucleases to generate double-stranded DNA breaks (DSBs) followed by homology-directed repair can introduce a wide variety of modifications but is inefficient in non-dividing cells, and is typically accompanied by an excess of unwanted insertions and deletions (indels), translocations, or other chromosomal rearrangements. 2 Base editing directly modifies target DNA bases in living cells and has become widely used to correct or install point mutations in organisms ranging from bacteria to human embryos. 3 Base editors use a catalytically impaired Cas9 to open a single-stranded DNA loop at a specified genomic site (Fig. 1a). Bases within the editing window (typically ~5 nt wide) in this region are modified by a tethered base-modification enzyme that only accepts single-stranded DNA.

N‐Nitroso‐containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N‐nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l‐alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N‐nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non‐proteinogenic amino acid l‐2,3‐diaminopropionic acid (l‐Dap) is synthesized and loaded onto a free‐standing peptidyl carrier protein (PCP) domain in l‐alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.

N-nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (Nnitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein, we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product L-alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the nitrogen source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid L-2,3diaminopropionic acid (L-Dap) is synthesized and loaded onto a peptidyl carrier protein (PCP) domain in L-alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. This research framework will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.[a]T.

No abstract

At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.