Methanol Assimilation with CO₂ Reduction in Butyribacterium methylotrophicum and Development of Genetic Toolkits for Its Engineering

Abstract

CO 2 -derived methanol is an attractive raw material for biobased production of value-added chemicals. Here, we investigated the native methylotrophButyribacterium methylotrophicum, which could synchronously assimilate methanol and CO 2 to butyric acid anaerobically. Supplementation with an approximate amount of bicarbonate could improve methanol metabolism of B. methylotrophicum, and 2.04 g/L butyric acid was finally obtained from 100 mM methanol and 20 mM bicarbonate. The genes involved in methanol metabolism were further identified through homologous alignment and transcriptome analysis. The methyltransferase cluster along with genes of the carbonyl branch of the Wood−Ljungdahl pathway (WLP) was found to be transcriptionally activated for the assimilation of methanol and CO 2 . To engineer B. methylotrophicum, an efficient electrotransformation protocol and several functional promoters were subsequently developed. Following a systematic investigation of various parameters, the electrotransformation efficiency was increased to 3.2 × 10 3 transformants/μg DNA. The activities of four heterologous promoters including P thl , P araE , P ptb , and P adc were comparatively determined. With these genetic toolkits, transformants overexpressing genes associated with methyltransferase system or butyric acid synthesis were obtained, where methanol consumption was increased by 16.9 and 14%, and butyric acid production was increased by 13.8 and 28.6%, respectively, in methanol and CO 2 medium. These results exhibit the great potential of B. methylotrophicum as a chassis for C1 bioconversion.

Engineering Acetogenic Bacteria for Efficient One-Carbon Utilization

“…When bicarbonate is present in excess in the medium, CO 2 reduction consumes more NADH, resulting in lower NADH availability for reassimilating acetate to produce butyrate. This effect was confirmed in B. methylotrophicum , where supplementation with 20 mM bicarbonate and 100 mM methanol significantly improved butyrate production, making it the major product ( Wang et al, 2021 ). In contrast, 40 mM and 60 mM bicarbonate induced the accumulation of a large amount of acetate.…”

“…E. limosum was reported to produce butyrate only during CO-fed syngas fermentation, and not when using H 2 /CO 2 , as in the latter case, most of the carbon is directed to acetate synthesis for ATP generation ( Litty and Müller, 2021 ). Butyrate production in acetogens not only provides the ATP needed for cell growth but also balances the redox, indicating that butyrate yield and selectivity depend on NADH availability ( Flaiz et al, 2021 ; Wang et al, 2021 ). The production of butyrate has been reported to be enhanced by the supplementation of additional reducing equivalents or methanol to provide more NADH ( Flaiz et al, 2021 ; Fu et al, 2021 ).…”

Molecular understanding ofEubacterium limosumchemostat methanol metabolism

“…39 Alternatively in that same organism, overexpressing the butyrate production pathway relative to pta increased the butyrate:acetate production ratio. 11 In this way, it may actually be more likely the methanol:co-substrate uptake ratio is partially controlled by the butyrate:acetate production ratio, not completely the other way around.…”

“…In acetogens, methanol enters the Wood-Ljungdahl Pathway (WLP) as methyl-THF via a methyltransferase, 10 where mtaB catalyses the transfer of the methyl group to mtaC and is then transferred to the THF carrier catalysed by mtaA. 11,12 It then results in partial oxidation ( i.e ., reversal of the WLP methyl branch) to generate reducing equivalents and satisfy CO 2 demand for the carbonyl branch. 13,14 However, sustained growth has not been shown with methanol as a solesubstrate, and a more oxidised co-substrate, such as CO 2 or formate, is required.…”

Toward Low-Carbon-Footprint Glycolic Acid Production for Bioplastics through Metabolic Engineering in Escherichia coli

“…Recent global weather changes urge the development of novel techniques to capture and utilize CO 2 from point sources to restrict its release . Recently, researchers have dedicated efforts toward carbon-neutral or carbon-negative production of biofuels and chemicals through genetically engineered microorganisms. , Yang et al first attempted to produce GA from CO 2 via genetically engineered cyanobacteria by employing RuBisCo to boost oxygenation activity, resulting in 2.8 g/L GA production after 18 days . Since microalgae showed significant GA secretion under aerobic conditions with CO 2 during photosynthesis, Kang et al developed a continuously two-stage cultivation using engineered Chlamydomonas reinhardtiito achieve a GA productivity of 82 mg/L/d .…”