A Synthetic Minimal Genome Design Framework for Bacteriophages: Application to Streptococcus mutans and Broad-Host-Range Expression in Escherichia coli

Minimal genome design, reducing a system to its smallest functional part set, is a core synthetic-biology strategy. Bacteriophages are attractive but underused chassis, yet wild-type phages are difficult to engineer. We present a comparative genomics-guided framework for synthetic minimal phage genome design, demonstrated on Streptococcus mutans, the primary agent of dental caries. Three S. mutans phages (M102AD, φAPCM01, SMHBZ8) were compared by synteny analysis, and their 123 proteins an-notated with InterProScan, EggNOG-mapper and HHpred. Conserved essential genes were selected by HHpred score, assembled as standardized transcriptional units, codon-optimized for E. coli K-12, and cloned in silico into the broad-host-range vector pBBR1MCS-2. Multi-host codon adaptation index (CAI) analysis assessed chassis flexibility. As a result, seventeen genes were assembled into a 15,792 bp synthetic insert; the final 20,948 bp construct raised mean CAI from 38.7% to 46.9% and cut rare-codon usage to 0.7%. Prokka confirmed 21 intact coding sequences, and CAI analysis predicted strong expression in Klebsiella pneumoniae (60.7%) and Pseudomonas aeruginosa (53.1%). This framework yields a validated, broad-host-range synthetic multi-gene phage-expression construct ready for experimental testing. One part (calcium-binding protein, CAI 34.4%) remains under-optimized, and Gibson Assembly, not restriction cloning, is recommended. The design logic is transferable to other phage-host systems.