Allaart, M. T. | Search bioRxiv

bioRxiv2025

Metabolic engineering of Methanothermobacter thermautotrophicus ΔH for recombinant acetoin production

Thermophilic methanogens of the genus Methanothermobacter are established biocatalysts in power-to-gas applications, converting H2 and CO2 into CH4 through the process of methanogenesis. Further expanding this platform for the bioproduction of value-added compounds (power-to-x) has the potential to increase the economic viability of such processes. This requires a genetic toolset that enables the controlled expression of recombinant pathways. Here, we report the fully autotrophic inducible recombinant bioproduction of acetoin from H2 and CO2 in Methanothermobacter thermautotrophicus {Delta}H. To facilitate inducible gene expression, we implemented an anhydrotetracycline (aTc)-inducible promoter system, expanding our available set of promoters. The aTc-inducible system enabled controlled expression of a codon-optimized acetoin-production operon comprising the acetolactate synthase- and acetolactate decarboxylase-encoding genes from Streptococcus thermophilus. Batch cultivation at 42{degrees}C demonstrated aTc-dependent acetoin formation, yielding up to 0.45 {+/-} 0.08 mM acetoin. Fed-batch bioreactor experiments confirmed growth-coupled, recombinant acetoin production, while eliminating the non-specific acetoin accumulation that we observed during non-growth phases in batch cultivation. Continuous cultivation in a chemostat resulted in stable acetoin production rates of 1.28 {+/-} 0.07 {micro}mol L-{superscript 1} h-{superscript 1} at 42{degrees}C. Elevated temperatures led to reduced acetoin production, suggesting diminished activity or thermal instability of the heterologous enzymes. This study demonstrates the feasibility of value-added bioproduction in Methanothermobacter and establishes an inducible expression system suitable for pathway engineering in thermophilic methanogens. Together with genome-scale modeling and emerging enzyme engineering strategies, these results lay the foundation for developing robust, CH4-co-producing power-to-x bioprocesses with Methanothermobacter species.

microbiology↗

bioRxiv2025

D- and L-lactate consumers in the human gut are taxonomically, biochemically, and energetically different

The human gut microbiota routinely produces D- and L-lactate during fermentation; however, the microbial fate of these stereoisomers, particularly the neurotoxic D-lactate, remains poorly understood. Given that D-lactate is an unavoidable byproduct of digestion, understanding its microbial turnover is essential for linking gut metabolism to host health. Here, we used chemostat bioreactors (pH 7.0, 37{degrees}C, and a solids retention time [SRT] of 4 d) to simulate gut-relevant, transient nutrient conditions and to enrich for lactate-consuming communities. DL-lactate-consuming consortia were enriched from a human-derived microbiota and then inoculated into duplicate bioreactors, which were fed exclusively D- or L-lactate. After steady-state was reached, the fed lactate stereoisomers were switched to assess community resilience. Regardless of the fed stereoisomer, the fermentation product spectra were consistent and dominated by acetate, propionate, and CO2. However, microbial communities and biomass yields diverged sharply, with a high relative abundance of Anaerotignum in D-lactate enrichments and Acidipropionibacterium and Propionibacterium in L-lactate enrichments. Notably, the biomass yield for D-lactate feeding was less than half that for L-lactate feeding, suggesting that the two isomers are metabolized through distinct biochemical pathways despite similar product spectra. Metagenomic and metaproteomic analyses confirmed this divergence at the pathway level. Our findings reveal how the stereoisomer identity of microbes shapes their niche specialization in the gut, with implications for understanding the ecology and clinical impact of lactate metabolism.

bioengineering↗