A characterized promoter suite enables metabolic rewiring of the acetogen Acetobacterium woodii toward ethanol production.

Metabolic reprogramming of the acetogen Clostridium sp. AWRP via adaptive laboratory evolution for improved formate-based growth

Formate is a promising, sustainable feedstock for microbial conversion into value-added products via the Wood–Ljungdahl pathway. Based on the identification of three distinct formate dehydrogenases in Clostridium sp. AWRP, this study investigated the potential of this acetogen for formate-based growth. Although the wild-type strain exhibited an extended lag phase due to formate toxicity, adaptive laboratory evolution yielded the adapted strain F30T, which demonstrated a significantly enhanced growth rate and tolerance to formate concentrations up to 300 mM. Whole-genome and transcriptomic analyses of the F30T strain revealed a sophisticated, multi-layered strategy for mitigating organic acid stress and optimizing energy conservation. Adaptive laboratory evolution resulted in six key non-synonymous mutations, including those in ackA and adhE1 , and triggered the differential expression of 948 genes out of 4199 genes under the formate-supplemented condition. Specifically, F30T achieved intracellular pH buffering and supplemental ATP generation by upregulating the arginine deiminase pathway, histidine biosynthesis, and ethanolamine utilization. To maintain growth near the thermodynamic limit, the strain implemented a stringent energy-saving program by downregulating the F₀F₁-type ATP synthase and the methyl-branch of the Wood–Ljungdahl pathway, while simultaneously enhancing stress resilience through molecular chaperone upregulation. In a pH-stat fed-batch system using formic acid, the adapted F30T strain showed a higher biomass yield while achieving a similar acetate yield from formate as the wild-type strain. This study demonstrated that adaptive laboratory evolution is a highly effective strategy for enhancing the formate utilization and stress tolerance of Clostridium sp. AWRP. Through integrated genomic and transcriptomic analyses, the molecular basis of the F30T strain’s improved performance was elucidated, identifying key mutations and coordinated metabolic and regulatory mechanisms that maintain cellular homeostasis and maximize energy efficiency. These findings provide valuable molecular insights to aid the targeted design and optimization of formate-utilizing acetogens for use in microbial cell factories aimed at sustainable C1-based bioproduction.

Seven mutations unlock strict synthetic methylotrophy in engineered Pseudomonas putida

Methanol is a reduced, soluble one-carbon (C1) feedstock for sustainable bioproduction, but converting this potential into robust microbial growth remains difficult. Several synthetic C1 assimilation routes depend on autocatalytic cycles, whose operation requires coordinated control of redox balance, toxic intermediates, substrate regeneration, and host regulation. Here, we implemented the serine-threonine cycle (STC) in the soil bacterium Pseudomonas putida and used growth-coupled selection with adaptive laboratory evolution (ALE) to transition from mixotrophic C1 incorporation to strict methylotrophy. The evolved strain grew with methanol as the sole carbon and energy source under atmospheric CO2 with a doubling time of ca. 40 h. Whole-genome sequencing, reverse genetics, biosensors, isotope labelling, and comparative RNA sequencing showed that evolution repeatedly targeted native pyrroloquinoline quinone (PQQ)-dependent methanol oxidation, membrane-bound transhydrogenase activity, glycine regeneration, STC enzyme balance, and global regulatory nodes. Additional ALE under glycine-methanol co-feeding increased growth rates and exposed further targets for improving cycle flux. These results establish P. putida as a chassis for strict synthetic methylotrophy and define actionable engineering routes toward C1 biomanufacturing. GRAPHICAL ABSTRACT

Alcohol oxidase-free methanol assimilation boosts carbon efficiency and product yield in industrial yeasts.

Methanol is an attractive single-carbon feedstock for scalable bioprocesses that avoids the consumption of agricultural products. Native yeast methylotrophy relies on alcohol oxidase (Aox), which dissipates reducing power and limits carbon conversion efficiency. Here, we replace Aox with the native, NAD+-dependent alcohol dehydrogenase 2 and eliminate the methanol dissimilatory branch by deleting formaldehyde dehydrogenase (Fld) in the yeasts Komagataella phaffii and Ogataea parapolymorpha. Engineered strains of both species sustain methylotrophic growth despite each modification being individually growth-defective. FLD deletion eliminates competition for cytosolically produced formaldehyde and enforces flux coupling between methanol utilization and NADH formation. Adaptive laboratory evolution over 30-50 generations yielded growth rates comparable to industrial yeast strains. In methanol-limited chemostats, evolved Adh-Δfld strains reduced specific CO₂ production 2-fold while increasing biomass yield 1.4-fold. The biomass-per-methanol yields of 0.48gg-1 for K. phaffii and 0.43gg-1 for O. parapolymorpha are the highest reported for each species. The Adh-based metabolic framework also enabled gram-per-liter itaconic acid production with higher product yield and lower CO₂ evolution than Aox-based strains, underscoring its utility for growth-coupled bioproduction. Adh-coupled methanol oxidation represents a generalizable strategy to increase carbon efficiency in other C1 utilization pathways and yeast species, providing a foundation for more sustainable biomanufacturing processes with yeasts.

Rewiring Carbon Metabolism in Bacillus methanolicus via Heterologous Phosphoketolase Expression Enhances Biomass Yield From Methanol and Reduces CO2 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 CO2 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 CO2 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.

Synthetic consortia towards photosynthetically derived acetate for heterotrophic production of 1-butanol

Synthetic consortia is an emerging field in biotechnology, offering advantages such as division of labor between the individual members and reduced risk of contamination. Consortia combining phototrophic and heterotrophic bacteria are attractive, as phototrophs can utilize CO₂ and light energy to sustain growth. Several studies have explored such systems where sucrose is most commonly used as the provided carbon source. An alternative is acetate, a smaller two-carbon molecule produced as a by-product by many microorganisms, including the model cyanobacterium Synechocystis PCC 6803 (thereafter Synechocystis). Although acetate production during phototrophic growth is typically low, previous studies have demonstrated that metabolic engineering—specifically the introduction of phosphoketolase (PK) and overexpression of phosphotransacetylase (Pta)—enabled the development of a high-producing strain (WT_PKPa_RBS_BsPta_Δacs) capable of secreting significant levels of acetate into the medium (Roussou and Lindblad 2026). In this study, Escherichia coli and Pseudomonas taiwanensis were engineered to produce 1-butanol, an industrially relevant bulk chemical, and cultivated using acetate as the sole carbon source. These strains were then individually co-cultivated with the previously engineered Synechocystis strain, WT_PKPa_RBS_BsPta_Δacs, forming two distinct consortia that were maintained for 42 days. Growth dynamics were successfully monitored, and acetate concentrations in the consortia were lower than in a corresponding axenic Synechocystis culture. At the same time, 1-butanol production was detected in the two co-cultures, demonstrating the feasibility of coupling photosynthetically derived acetate to heterotrophic production of a value-added bulk chemical. •Photosynthetic/heterotrophic synthetic consortia for 1-butanol production. • Engineered Synechocystis PCC 6803 cells produce acetate from CO2. • Modified Escherichia coli and Pseudomonas taiwanensis cells grow on acetate and produce 1-butanol.

Highly efficient production of 1,3-propanediol by CRISPR-Cas12a-edited Klebsiella pneumoniae using glycerol and lignocellulosic hydrolysate as co-substrates.