Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production.

Microbial cell factories provide a green and sustainable opportunity to produce value-added products from renewable feedstock. However, the leakage of toxic or volatile intermediates decreases the efficiency of microbial cell factories. In this study, membraneless organelles (MLOs) were reconstructed in Saccharomyces cerevisiae by the disordered protein sequence A-IDPs. A regulation system was designed to spatiotemporally regulate the size and rigidity of MLOs. Manipulating the MLO size of strain ZP03-FM, the amounts of assimilated methanol and malate were increased by 162 % and 61 %, respectively. Furthermore, manipulating the MLO rigidity in strain ZP04-RB made acetyl-coA synthesis from oxidative glycolysis change to non-oxidative glycolysis; consequently, CO2 release decreased by 35 % and the n-butanol yield increased by 20 %. This artificial MLO provides a strategy for the co-localization of enzymes to channel C1 starting materials into value-added chemicals.

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.

Low-carbon and overproduction of cordycepin from methanol using engineered Pichia pastoris cell factory.

Cordycepin, a nucleoside analog, is widely used in medicine and health products. However, the production of cordycepin from Cordyceps militaris faces the challenges of low productivity and high rate of greenhouse gas emissions. In this study, by optimizing the cordycepin biosynthesis pathway through promoter combination, Kozak sequence, and enzyme fusion, enhancing the methanol assimilation capacity in peroxisomes, adjusting the synthesis of NADPH and ATP, and combining the enhanced supply of adenosine and 3'-AMP, the cordycepin high-yield strain Pp29 was constructed, which produced 1551.44 mg/L cordycepin by shake-flask fermentation. In fed-batch fermentation, Pp29 achieved the highest yield (8.11 g/L, 67.64 mg/g DCW, and 1.35 g/L/d) to date in 10 L fermenter, and the CO2-eq emissions were 1.9-17.3 times lower than C. militaris and other yeast systems. This study provide basis for Pichia pastoris to be used as chassis cell for synthesizing cordycepin and other nucleoside analogs by methanol as carbon source.

Metabolic engineering of Escherichia coli to utilize methanol as a co-substrate for the production of (R)-1,3-butanediol.

Due to its abundance, cost-effectiveness, and high reducibility, methanol has gained considerable attention in the biomanufacturing industry as a nonfood feedstock for the production of value-added chemicals. The range of chemicals that can be derived from methanol, however, remains constrained and is currently in the concept validation phase. This study aimed to develop and evaluate a hybrid methanol assimilation pathway in Escherichia coli to improve the production of (R)-1,3-butanediol ((R)-1,3-BDO) by utilizing methanol and sugars as co-substrates. By combining the methanol dehydrogenase (MDH) from the prokaryotes with the dihydroxyacetone synthase (DAS) from the eukaryotes, the hybrid pathway facilitates methanol conversion into the central metabolism while generating NADH at the same time. Through pathway optimization and targeted gene deletions, we have successfully developed an E. coli strain capable of producing 5.79 g/L (R)-1,3-BDO in shake flask experiments and 13.71 g/L (R)-1,3-BDO with a yield of 0.35 C-mol/C-mol in batch fermentation using methanol and glucose as co-substrates. Our study also showed the incorporation of 13C-methanol into cellular intermediates and an increase in NAD(P)H concentration, confirming the role of methanol as a co-substrate and supplier of NADH. In addition, our study also demonstrated the co-utilization of methanol with xylose for the production of (R)-1,3-BDO, expanding the substrate spectrum for sustainable 1,3-BDO production.

Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2.

Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells.