Engineering photo-methylotrophic Methylobacterium for enhanced 3-hydroxypropionic acid production during non-growth stage fermentation.

This study explored the potential of methanol as a sustainable feedstock for biomanufacturing, focusing on Methylobacterium extorquens, a well-established representative of methylotrophic cell factories. Despite this bacterium's long history, its untapped photosynthetic capabilities for production enhancement have remained unreported. Using genome-scale flux balance analysis, it was hypothesized that introducing photon fluxes could boost the yield of 3-hydroxypropionic acid (3-HP), an energy- and reducing equivalent-consuming chemicals. To realize this, M. extorquens was genetically modified by eliminating the negative regulator of photosynthesis, leading to improved ATP levels and metabolic activity in non-growth cells during a two-stage fermentation process. This modification resulted in a remarkable 3.0-fold increase in 3-HP titer and a 2.1-fold increase in its yield during stage (II). Transcriptomics revealed that enhanced light-driven methanol oxidation, NADH transhydrogenation, ATP generation, and fatty acid degradation were key factors. This development of photo-methylotrophy as a platform technology introduced novel opportunities for future production enhancements.

Metabolomic analysis improves bioconversion of methanol to isobutanol in Methylorubrum extorquens AM1.

BACKGROUND: Methylorubrum extorquens AM1 can be engineered to convert methanol to value-added chemicals. Most of these chemicals derive from acetyl-CoA involved in the serine cycle. However, recent studies on methylotrophic metabolism have suggested that C3 pyruvate is a good potential precursor for broadening the types of synthesized products. METHODS AND RESULTS: In the present study, we found that isobutanol was a model chemical that could be generated from pyruvate through a 2-keto acid pathway. Initially, the engineered M. extorquens AM1 could only produce a trace amount of isobutanol at 0.62 mgL-1 after introducing the heterologous 2-ketoisovalerate decarboxylase and alcohol dehydrogenase. Furthermore, the metabolomic analysis revealed that insufficient carbon fluxes through 2-ketoisovalerate and pyruvate were the key limitation steps for efficient biosynthesis of isobutanol. Based on this analysis, the titer of isobutanol was improved by over 20-fold after overexpressing alsS gene encoding acetolactate synthase and deleting ldhA gene for lactate dehydrogenase. Moreover, substituting the cell chassis with the isobutanol-tolerant strain isolated from adaptive evolution of M. extorquens AM1 further increased the production of isobutanol by 1.7-fold, resulting in the final titer of 19 mgL-1 in flask cultivation. CONCLUSION: Our current findings provided promising insights into engineering methylotrophic cell factories capable of converting methanol to isobutanol or value-added chemicals using pyruvate as the precursor.

Rewiring the native methanol assimilation metabolism by incorporating the heterologous ribulose monophosphate cycle into Methylorubrum extorquens.

Methanol is assimilated through the serine cycle to generate acetyl-CoA without carbon loss. However, a highly active serine cycle requires high consumption of reducing equivalents and ATP, thereby leading to the impaired efficiency of methanol conversion to reduced chemicals. In the present study, a genome-scale flux balance analysis (FBA) predicted that the introduction of the heterologous ribulose monophosphate (RuMP) cycle, a more energy-efficient pathway for methanol assimilation, could theoretically increase growth rate by 31.3% for the model alphaproteobacterial methylotroph Methylorubrum extorquens AM1. Based on this analysis, we constructed a novel synergistic assimilation pathway in vivo by incorporating the RuMP cycle into M. extroquens metabolism with the intrinsic serine cycle. We demonstrated that the operation of the synergistic pathway could increase cell growth rate by 16.5% and methanol consumption rate by 13.1%. This strategy rewired the central methylotrophic metabolism through adjusting core gene transcription, leading to a pool size increase of C2 to C5 central intermediates by 1.2- to 3.6-fold and an NADPH cofactor improvement by 1.3-fold. The titer of 3-hydroxypropionic acid (3-HP), a model product in the newly engineered chassis of M. extorquens AM1, was increased to 91.2 mg/L in shake-flask culture, representing a 3.1-fold increase compared with the control strain with only the serine cycle. The final titer of 3-HP was significantly improved to 0.857 g/L in the fed-batch bioreactor, which was more competitive compared with the other 3-HP producers using methane and CO2 as C1 sources. Collectively, our current study demonstrated that engineering the synergistic methanol assimilation pathway was a promising strategy to increase the carbon assimilation and the yields of reduced chemicals in diverse host strains for C1 microbial cell factories.