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