The microbial workhorse at the center of the study is Methylorubrum extorquens PA1, a facultative methylotroph that naturally grows on methanol and can also assimilate ethanol. Methylotrophs have long fascinated metabolic engineers because they can consume cheap, non-food carbon sources that do not compete with agriculture. Methanol, in particular, can be generated from methane, carbon dioxide and biomass, making it an attractive platform molecule for industrial biotechnology. Ethanol, too, is produced at enormous scale from fermentation and is easy to store, transport and handle. If a robust microbial chassis could be engineered to funnel these two-carbon and one-carbon molecules into complex aromatics, the result would be a flexible carbon-recycling platform suited to a circular economy.

Yet turning that vision into reality has proven difficult, and the new study illustrates precisely why. When the researchers profiled the metabolism of M. extorquens PA1 growing on ethanol, they discovered a critical bottleneck: the bacterium was inefficiently converting acetate into acetyl-CoA, the universal metabolic currency that fuels biosynthesis. As a result, acetate accumulated outside the cells and growth on ethanol suffered. In essence, the microbe was letting a large fraction of the carbon it consumed leak out of its metabolic network before that carbon could be routed toward anything useful. Identifying this choke point was the first major insight of the work, and it framed every engineering decision that followed.

The team’s solution was to overexpress an endogenous enzyme called acetyl-CoA synthase, known as MeACS1, which directly activates acetate into acetyl-CoA. This single intervention suppressed the wasteful extracellular acetate accumulation and measurably improved the bacterium’s growth on ethanol. The result underscores a broader principle in metabolic engineering: before a production pathway can be installed, the native assimilation routes feeding it must be able to keep up with demand. In methylotrophs engineered for methanol utilization, the analogous challenge often lies in formaldehyde detoxification and the serine cycle; for ethanol, the study suggests that relieving the acetyl-CoA bottleneck is likely the critical step for efficient assimilation of this C2 feedstock.

With the entry point strengthened, the researchers turned to the synthetic part of the project: building a biosynthetic module that could deliver cinnamic acid. Cinnamic acid is produced in plants from the amino acid L-phenylalanine by phenylalanine ammonia-lyase enzymes, which catalyze the non-oxidative deamination that strips the amino group and releases the aromatic acid. The team imported the gene for one such enzyme, EncP from the marine bacterium Streptomyces maritimus, into M. extorquens PA1. Heterologous phenylalanine ammonia-lyases have been deployed in engineered microbes before, but never in this methylotrophic context, and the enzyme gave the bacterium a direct route from its own phenylalanine pool to the target product.

Supplying enough L-phenylalanine then became the next rate-limiting problem. Phenylalanine is synthesized through the shikimate pathway, a seven-step route that connects central carbon metabolism to the aromatic amino acids. Its entry substrates are phosphoenolpyruvate, drawn from glycolysis, and erythrose-4-phosphate, drawn from the pentose phosphate pathway. The researchers attacked both sides of the precursor question. They co-expressed two feedback-resistant enzymes from Escherichia coli: a mutant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, the first and most heavily regulated enzyme of the shikimate pathway, and a fusion of chorismate mutase with prephenate dehydrogenase that pushes flux from chorismate toward phenylalanine. By using feedback-resistant variants, the engineers ensured that accumulating aromatic intermediates could not slam the brakes on the very pathway they were trying to boost.

On the erythrose-4-phosphate side, the team overexpressed the endogenous transketolase of M. extorquens, MeTkt, an enzyme that helps channel carbon through the sugar-phosphate rearrangements that generate this key precursor. Together, these layered interventions shunted more carbon from ethanol assimilation through the shikimate pathway and into L-phenylalanine, and from there through the bacterial ammonia-lyase into cinnamic acid. The resulting strain, designated PAc1/pTE102-CA3, produced 1.75 milligrams per liter of cinnamic acid from ethanol in simple test-tube cultures. That figure may sound modest by industrial standards, but it represents 2.6 times the yield the same engineered network could achieve from methanol, an important demonstration that ethanol is a superior feedstock for this particular chassis.

The team then moved from test tubes to controlled fed-batch cultivation, the standard workhorse format of industrial fermentation, in which ethanol was supplied incrementally to avoid overwhelming the cells. Under these conditions the engineered bacterium accumulated 52.34 milligrams per liter of cinnamic acid, roughly a thirty-fold improvement over the tube cultures, while maintaining stable growth and without notable acetate accumulation. That combination of productivity and physiological stability is significant: many engineered strains collapse under the metabolic burden of aromatic overproduction, or excrete organic acids that poison the culture. Here, the early repair of the acetyl-CoA bottleneck appears to have paid dividends, keeping the carbon balanced as the production pathway drew heavily on central metabolism.

Why does cinnamic acid matter? The compound and its derivatives sit at the base of a large family of plant phenylpropanoids, including p-coumaric acid, caffeic acid, ferulic acid, cinnamaldehyde and a suite of polymers and pharmaceutical intermediates. A microbial route from ethanol to cinnamic acid could be extended, in principle, by adding downstream enzymes to reach these higher-value molecules. The study therefore positions M. extorquens PA1 not merely as a curiosity of methylotrophic metabolism but as a credible chassis for ethanol-based aromatic biosynthesis more broadly. It also provides a foundation for engineering other C2-assimilating bacteria, an emerging class of platform organisms that can feed on acetate and ethanol generated from gas fermentation and electrochemical carbon capture.

The work, supported by the RIKEN Center for Sustainable Resource Science, RIKEN’s Special Postdoctoral Researcher Program and a JST ACT-X grant, remains at an early stage, and the authors are candid that further engineering will be needed to push titers toward commercial relevance. Even so, the study delivers a clear technical roadmap: fix the acetyl-CoA entry point, install a proven ammonia-lyase, deregulate and amplify the shikimate and phenylalanine branches, and reinforce erythrose-4-phosphate supply. Each step is modular, and each can be iterated as new tools for editing methylotroph genomes mature. As biorefineries search for routes that decouple chemical manufacturing from fossil carbon, this small soil bacterium, now taught to drink ethanol and exude an aromatic acid, offers a compelling proof of principle for the carbon-recycling economy of the future.

Subject of Research: Metabolic engineering of Methylorubrum extorquens PA1 for cinnamic acid production from ethanol

Article Title: Metabolic engineering of Methylorubrum extorquens PA1 for cinnamic acid production from ethanol

Article References: Fujiwara, R., Mitsui, R., Akai, G., Tanaka, Y., & Shirai, T. (2026). Metabolic engineering of Methylorubrum extorquens PA1 for cinnamic acid production from ethanol. Biotechnology for Biofuels and Bioproducts. https://doi.org/10.1186/s13068-026-02833-8

Image Credits: AI Generated

DOI: 10.1186/s13068-026-02833-8

Keywords: Methylorubrum extorquens, metabolic engineering, cinnamic acid, ethanol, methylotroph, shikimate pathway, acetyl-CoA synthase, phenylalanine ammonia-lyase, biorefinery, biotechnology, RIKEN, renewable chemicals

The core problem the researchers tackled is one that plagues nearly all conventional microbial production platforms: during growth, a substantial fraction of the carbon a cell assimilates is ultimately released as biogenic carbon dioxide. In methanol-grown organisms, this loss occurs through central metabolic reactions, most notably the decarboxylation of pyruvate to acetyl-coenzyme A, a step shared by virtually every industrial chassis in use or under development, as well as through the oxidative branch of the ribulose monophosphate cycle that methylotrophs use to assimilate formaldehyde derived from methanol. Every molecule of carbon dioxide vented in this way represents feedstock that was purchased, delivered, and then effectively thrown away, capping biomass yields and undermining the economics of industrial methylotrophic bioprocesses. Reducing carbon loss at these junctions is therefore an established priority for developing low-oxidative, carbon-efficient biocatalysts.

The solution explored in the study exploits an enzyme known as phosphoketolase, or PKT, which cleaves the sugar phosphates fructose-6-phosphate and xylulose-5-phosphate into acetyl-phosphate plus either erythrose-4-phosphate or glyceraldehyde-3-phosphate. Acetyl-phosphate is valuable because it can be converted to acetyl-coenzyme A, the essential biosynthetic precursor, either in a single step by phosphotransacetylase or in two steps via acetate kinase and acetyl-CoA synthetase, entirely without passing through pyruvate decarboxylation. The side products, erythrose-4-phosphate and glyceraldehyde-3-phosphate, are recycled back into the ribulose monophosphate and pentose phosphate pathways. In effect, the enzyme provides a carbon-conserving detour around the cell’s biggest carbon dioxide leak. Previous work had shown that PKT-based pathways can dramatically enhance biomass yield and carbon conversion efficiency in several microbial hosts, including the methanotroph Methylotuvimicrobium buryatense 5GB1C, where overexpression of its native pktB gene substantially improved methane conversion efficiency.

Bacillus methanolicus emerged as an ideal test chassis for several reasons. It is a facultative ribulose monophosphate methylotroph that grows rapidly on methanol, doubling in roughly 1.4 hours under optimal conditions, faster than other native methylotrophs such as Methylobacterium extorquens AM1 at 3.6 hours, the yeast Pichia pastoris at 4.6 hours, and engineered Escherichia coli strains at 4.3 hours. It is also industrially interesting in its own right, capable of producing more than 50 grams per liter of glutamate under optimized conditions as well as lysine. Crucially, while B. methanolicus lacks any native phosphoketolase gene, it already encodes the downstream machinery needed to convert acetyl-phosphate to acetyl-CoA. That combination made it the perfect organism to test whether a methylotroph without native PKT genes could benefit from the carbon-conserving pathway, a question that had remained open.

To carry out the engineering, the team first built a suite of new genetic tools for the organism. They adapted an anhydrous tetracycline-inducible expression system, originally developed for Bacillus subtilis, by fusing the B. subtilis xylA promoter to the Tn10 tet operator and placing the tetR repressor under the control of a strong native B. methanolicus methanol dehydrogenase promoter. When validated with a green fluorescent protein reporter, the system proved highly tunable: fluorescence rose with increasing inducer concentration, and after induction with 0.8 micromolar anhydrous tetracycline, reporter expression climbed roughly 19-fold over five hours, from about 2,450 to about 46,000 normalized relative fluorescent units, before declining as free inducer was depleted. The researchers also characterized four native constitutive promoters of varying strengths, driving reporter expression from the tuf promoter at the highest levels, followed by pdxK, icd, and the much weaker dppE promoter, giving the community a graded palette of expression tools for this organism.

With the expression toolkit in hand, the team introduced a codon-optimized pktB gene from M. buryatense into B. methanolicus. Under inducible expression, the engineered strain produced 0.49 grams of dry cell weight per gram of methanol consumed, compared with 0.41 grams for the empty vector control, a 20 percent improvement in methanol-to-biomass yield. Constitutive expression from native promoters delivered comparable gains: the pdxK-pktB and icd-pktB strains each reached 0.52 grams of dry cell weight per gram of methanol consumed, and the tuf-pktB strain reached 0.50 grams, against a control baseline of 0.42 grams, improvements of roughly 18 to 24 percent. The pdxK-pktB strain, which showed the greatest yield enhancement without the most severe growth penalty, was selected for deeper analysis. Notably, all pktB-expressing strains displayed some growth defect relative to controls, a trade-off the researchers attribute to disruption of the carefully balanced flux through the ribulose monophosphate cycle.

Biochemical and molecular analyses confirmed the enzyme was doing its intended job. Whole-cell lysates from the pdxK-pktB strain produced 0.86 millimolar acetyl-phosphate in a hydroxamate activity assay, nearly double the 0.44 millimolar measured in the control strain, demonstrating functional PKT activity inside the thermophilic host. Reverse transcription PCR showed pktB transcripts at 13.13 times the level of the rpoB housekeeping gene during mid-log growth. Most importantly, when cultures were grown in sealed bottles and headspace gas analyzed by gas chromatography, the pktB-expressing strain released 0.44 moles of carbon dioxide per mole of methanol consumed versus 0.49 for the control, a 9 percent reduction in total biogenic carbon dioxide production, directly linking the yield gain to reduced carbon loss as hypothesized.

To eliminate the burden of maintaining plasmids, which themselves caused growth defects in control strains, the researchers deployed a recently developed temperature-sensitive chromosomal integration method, inserting the pdxK-pktB expression cassette into the chromosome by homologous recombination. The resulting integrated strain, BMGA3::pktB, grew more slowly and consumed methanol more slowly than the wild type, yet still delivered a 19 percent biomass yield enhancement, producing 0.82 grams of dry cell weight per gram of methanol consumed versus 0.69 grams for wild type. Carbon dioxide output fell 12 percent relative to wild type, from 0.49 to 0.44 moles per mole of methanol consumed. Intriguingly, pktB expression in the integrated strain was far lower than in the plasmid strain, just 0.16 times the housekeeping gene, suggesting that once enzyme expression exceeds a certain threshold, other factors such as substrate limitation, protein misfolding, or the enzyme’s thermolability may cap the achievable yield gain at around 24 percent.

The study also revealed unexpected shifts in byproduct metabolism. Whereas pktB overexpression in the enzyme’s native host M. buryatense had previously increased acetate excretion, the integrated B. methanolicus strain excreted no detectable acetate, while the wild type released roughly 3 millimolar. The authors propose two possible explanations: the slower growth rate of the engineered strain may simply reduce overflow metabolism, or carbon flux through acetyl-phosphate may be preferentially channeled toward acetyl-CoA rather than acetate. Distinguishing between these scenarios will require future metabolic flux analyses and intracellular metabolite measurements. Either way, the result hints that the engineered strain channels more carbon toward acetyl-CoA, potentially driving greater flux through the oxidative branch of a partially non-cyclic TCA cycle that fluxomic studies have shown operates at low levels during methanol growth.

Beyond the specific yield numbers, the work carries a broader lesson for metabolic engineering: the outcome of installing a carbon-conserving pathway depends not only on the enzyme’s catalytic properties but on the metabolic architecture of the host into which it is placed. This is, to the authors’ knowledge, the first report of introducing a phosphoketolase into a native methylotroph that lacks a PKT system, and the phenotypes observed differ meaningfully from those in the enzyme’s original host. The researchers suggest that adaptive laboratory evolution of the integrated strain could resolve the growth defect and optimize flux through the combined PKT-ribulose monophosphate network, while adding xylose utilization genes could boost regeneration of ribulose-5-phosphate and further support the pathway. As methanol, increasingly available from renewable electricity and captured carbon dioxide, gains traction as a feedstock, phosphoketolase-based carbon conservation stands out as a broadly applicable strategy for squeezing more product out of every carbon atom.

Subject of Research: Metabolic engineering of Bacillus methanolicus with heterologous phosphoketolase to enhance biomass yield from methanol and reduce CO2 loss

Article Title: Rewiring Carbon Metabolism in Bacillus methanolicus via Heterologous Phosphoketolase Expression Enhances Biomass Yield From Methanol and Reduces CO2 Loss

Article References: Rewiring Carbon Metabolism in Bacillus methanolicus via Heterologous Phosphoketolase Expression Enhances Biomass Yield From Methanol and Reduces CO2 Loss. (n.d.). https://doi.org/10.1111/1751-7915.70430

Image Credits: AI Generated

DOI: 10.1111/1751-7915.70430

Keywords: Bacillus methanolicus, phosphoketolase, methanol, methylotroph, RuMP cycle, carbon conservation, biomass yield, CO2 reduction, metabolic engineering, synthetic biology, biomanufacturing, acetyl-CoA