Engineering a Virus‐Derived X Family DNA Polymerase FvPolX for de novo DNA Synthesis

ABSTRACT Enzymatic de novo DNA synthesis has gained increasing attention over the past decades, having emerged as a hotspot in synthetic biology. However, current research remains largely focused on animal‐derived X‐family DNA polymerases (PolXs), such as the extensively studied terminal deoxynucleotidyl transferases (TdTs) from vertebrates and the recently identified RvPolX from the invertebrate Ramazzottius varieornatus . In contrast, microbial PolXs have largely been overlooked, despite their potential in catalytic diversity and industrial applicability. Here, we report the discovery and rational engineering of FvPolX, a PolX enzyme derived from Faustovirus . Through protein engineering, we developed the FvPolX ^{R184L/T186G/N267S} variant, which demonstrates a dramatic enhancement in catalytic activity toward both canonical dNTPs and noncanonical 3′‐ONH ₂ ‐dNTPs—transforming the enzyme from nearly inactive to highly efficient. Its template‐independent DNA synthesis efficiency surpassed that of wild‐type TdTs and is comparable to that of engineered TdT variants for adding 3′‐ONH ₂ ‐dNTPs. Additionally, we constructed a truncated version of FvPolX (s149FvPolX ^R184L/N267S ) containing only the palm and thumb subdomains. This minimal double mutant retained high catalytic activity in template‐independent DNA synthesis and efficiently incorporated both canonical dNTPs and 3′‐ONH ₂ ‐dNTPs. Together, these findings expand the enzymatic toolbox for de novo DNA synthesis by diversifying polymerase sources and exploring structural minimization.