Terminal Deoxynucleotidyl Transferase-Mediated Tunable Biopart Synthesis and Metabolic Engineering

Abstract The increasing demands of synthetic biology have shifted the central bottleneck from DNA assembly to DNA writing. As engineered systems grow in complexity, progress increasingly depends on the ability to write, test, and rewrite regulatory DNA to support fast design−build−test−learn cycles and precise control of gene expression. In this regard, enzymatic DNA synthesis offers a promising route toward next-generation DNA writing technologies for on-demand benchtop implementation. In particular, terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, provides a unique mechanism for generating sequence diversity without predefined templates. Here, we establish a programmable TdT-based enzymatic platform for the in-house synthesis of random-sequence oligonucleotide libraries with tunable base composition. Systematic kinetic characterization of TdT revealed a substrate-dependent bias, which we mitigated through optimization of divalent cofactors and dNTP input ratios, enabling user-defined nucleotide distributions. Using this platform, we expanded and screened composition-controlled libraries of promoters, 5′-UTRs, and transcription terminators in Escherichia coli, identifying functional bioparts spanning wide dynamic ranges of transcriptional and translational activity. Integration of TdT-synthesized regulatory libraries into a streamlined enzymatic workflow enabled rapid cloning and screening within hours. As a proof of concept, targeted promoter diversification of a lycopene biosynthetic operon yielded a 124.7-fold range in production and identified variants achieving up to 1.43-fold higher titers than those of the parental strain. These results establish TdT-mediated enzymatic DNA synthesis as a controllable DNA writing modality for rapid regulatory-part engineering in synthetic biology and metabolic engineering.