RESUMEN
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.
Asunto(s)
Metanol , Methylobacterium extorquens , Acetilcoenzima A , Methylobacterium extorquens/genética , PentosasRESUMEN
Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.
Asunto(s)
Escherichia coli/metabolismo , Ingeniería Metabólica , Metanol/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Ciclo del Ácido Cítrico/genética , Variaciones en el Número de Copia de ADN , Evolución Molecular Dirigida , Escherichia coli/genética , Formaldehído/metabolismo , Glucólisis , Mutagénesis , Ribosamonofosfatos/metabolismoRESUMEN
The ribulose monophosphate (RuMP) cycle is a highly efficient route for the assimilation of reduced one-carbon compounds. Despite considerable research, the RuMP cycle has not been fully implemented in model biotechnological organisms such as Escherichia coli, mainly since the heterologous establishment of the pathway requires addressing multiple challenges: sufficient formaldehyde production, efficient formaldehyde assimilation, and sufficient regeneration of the formaldehyde acceptor, ribulose 5-phosphate. Here, by efficiently producing formaldehyde from sarcosine oxidation and ribulose 5-phosphate from exogenous xylose, we set aside two of these concerns, allowing us to focus on the particular challenge of establishing efficient formaldehyde assimilation via the RuMP shunt, the linear variant of the RuMP cycle. We have generated deletion strains whose growth depends, to different extents, on the activity of the RuMP shunt, thus incrementally increasing the selection pressure for the activity of the synthetic pathway. Our final strain depends on the activity of the RuMP shunt for providing the cell with almost all biomass and energy needs, presenting an absolute coupling between growth and activity of key RuMP cycle components. This study shows the value of a stepwise problem solving approach when establishing a difficult but promising pathway, and is a strong basis for future engineering, selection, and evolution of model organisms for growth via the RuMP cycle.