Rewiring Carbon Metabolism in Bacillus methanolicus via Heterologous Phosphoketolase Expression Enhances Biomass Yield From Methanol and Reduces CO 2 Loss

ABSTRACT Methylotrophic microbes are attractive alternatives to traditional heterotrophic production platforms, yet their efficiency is constrained by carbon loss through pyruvate decarboxylation and the oxidative branch of the RuMP cycle. The phosphoketolase (PKT) pathway provides a carbon‐conserving alternative by cleaving fructose‐6‐phosphate and/or xylulose‐5‐phosphate into acetyl‐phosphate, which can subsequently be converted to acetyl‐coA without pyruvate decarboxylation. The remaining carbon intermediates are recycled through central metabolism to regenerate RuMP cycle intermediates without direct CO 2 release. Here, we engineered this strategy in Bacillus methanolicus , a thermophilic methylotroph with strong industrial potential. We first established a versatile expression toolkit comprising inducible and constitutive promoters, benchmarked using an sfGFP reporter. Leveraging this system, we heterologously expressed the phosphoketolase B ( pktB ) gene from Methylotuvimicrobium buryatense 5GB1C which increased methanol‐to‐biomass yields by 18%–24% relative to controls and reduced biogenic CO 2 production by 9%–12%. Chromosomal integration of pktB preserved these gains, demonstrating stability without reliance on plasmid‐based expression. Together, these results show that PKT‐driven metabolic rewiring enhances substrate yields in B. methanolicus and provides a scalable strategy to improve methylotrophic bioprocesses. This work expands the metabolic engineering toolbox for methylotrophs and highlights carbon‐conserving pathway design as a key lever for advancing single carbon (C1) biomanufacturing.