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.
Methanol, a nonfood C1 feedstock that could be produced either from fossil or potentially renewable raw materials has recently attracted much attention as a very promising feedstock alternative to sugar-based raw materials for biomanufacturing. Methylotrophic cell factories that could efficiently convert methanol to value-added products are highly desired for methanol-based biomanufacturing. Pichia pastoris shows significant industrial promise for methanol bioconversion due to its advantage in the methanol utilization rate compared to other native or synthetic methylotrophs. Here, we review the current understanding of methanol metabolism of P. pastoris, discuss the important factors that influence the methanol utilization ability of P. pastoris, and summarize the recent advances in the application of engineered P. pastoris to produce various chemicals from methanol. We also discuss future challenges and possible solutions to develop P. pastoris as an efficient cell factory used for methanol-based biomanufacturing.
Background Methanol, a promising non-food fermentation substrate, has gained increasing interest as an alternative feedstock to sugars for the bio-based production of value-added chemicals. Butyribacterium methylotrophicum, one of methylotrophic-acetogenic bacterium, is a promising host to assimilate methanol coupled with CO2 fixation for the production of organic acids, such as butyric acid. Although the methanol utilization pathway has been identified in B. methylotrophicum, little knowledge was currently known about its regulatory targets, limiting the rational engineering to improve methanol utilization. Results In this study, we found that methanol assimilation of B. methylotrophicum could be significantly improved when using corn steep liquor (CSL) as the co-substrate. The further investigation revealed that high level of lysine was responsible for enhanced methanol utilization. Through the transcriptome analysis, we proposed a potential mechanism by which lysine confers improved methylotrophy via modulating NikABCDE and FhuBCD transporters, both of which are involved in the uptake of cofactors essential for enzymes of methanol assimilation. The improved methylotrophy was also confirmed by overexpressing NikABCDE or FhuBCD operon. Finally, the de novo synthetic pathway of lysine was further engineered and the methanol utilization and butyric acid production of B. methylotrophicum were improved by 63.2% and 79.7%, respectively. After an optimization of cultivation medium, 3.69 g/L of butyric acid was finally achieved from methanol with a yield of 76.3%, the highest level reported to date. Conclusion This study revealed a novel mechanism to regulate methanol assimilation by lysine in B. methylotrophicum and engineered it to improve methanol bioconversion to butyric acid, culminating in the synthesis of the highest butyric acid titer reported so far in B. methylotrophicum. What’s more, our work represents a further advancement in the engineering of methylotrophic-acetogenic bacterium to improve C1-compound utilization. Graphical Abstract
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