Orchestrating polyketide synthase-derived aldehyde metabolism for Streptomyces biomanufacturing

Advances in metabolic engineering have enabled the integration of diverse non-native reactions into microbial hosts, providing sustainable alternatives to petrochemical manufacturing. We previously developed a modular polyketide synthase (PKS)-based platform in Streptomyces albus for producing 17 diols, amino alcohols, and hydroxy acids. In this platform, terminal thioester reductases (TRs) release aldehyde intermediates that are subsequently converted by post-PKS enzymes to control product chemistry. Here we found that PKS-TR strains accumulated hundreds of genome-wide single-nucleotide variants, including missense mutations on conserved PKS and TR residues leading to impaired enzyme activity. Based on the hypothesis of aldehyde toxicity, we coupled TR catalysis with downstream alcohol dehydrogenases (ADHs) through gene co-integration or PKS-TR-ADH fusion to limit aldehyde accumulation. These strategies reduced genomic variability and increased diol production, reaching a total diol titer of 14.2 g/L in bioreactors, including 10.8 g/L 1,3-butanediol. Extending this approach, engineered strains incorporating PKS-TR co-integration with transaminases or the padA aldehyde dehydrogenase achieved 79% higher total amino alcohol titers and 145% higher 2-methyl-3-hydroxyhexanoic acid titer than previous reports, while eliminating diol byproducts from the optimized hydroxy acid pathway. These results establish TR-mediated aldehyde release as a programmable branch point and orchestrated downstream catalysis as a general strategy for improving the stability, productivity, and selectivity of PKS-based Streptomyces biomanufacturing.