DataSheet1_Engineering and adaptive laboratory evolution of Escherichia coli for improving methanol utilization based on a hybrid methanol assimilation pathway.pdf

Engineering Escherichia coli for efficient methanol assimilation is important for developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer E. coli as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid methanol assimilation pathway which consists of prokaryotic methanol dehydrogenase (Mdh) and eukaryotic xylulose monophosphate (XuMP) pathway enzyme dihydroxyacetone synthase (Das) into E. coli and reprogrammed E. coli metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in the TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved via adaptive laboratory evolution and a final evolved strain can grow on methanol with only 0.1 g/L yeast extract as co-substrate. 13C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of fba, dhak, and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of E. coli based on the hybrid methanol assimilation pathway with Mdh and Das.</p

DataSheet2_Engineering and adaptive laboratory evolution of Escherichia coli for improving methanol utilization based on a hybrid methanol assimilation pathway.xlsx

Engineering Escherichia coli for efficient methanol assimilation is important for developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer E. coli as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid methanol assimilation pathway which consists of prokaryotic methanol dehydrogenase (Mdh) and eukaryotic xylulose monophosphate (XuMP) pathway enzyme dihydroxyacetone synthase (Das) into E. coli and reprogrammed E. coli metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in the TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved via adaptive laboratory evolution and a final evolved strain can grow on methanol with only 0.1 g/L yeast extract as co-substrate. 13C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of fba, dhak, and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of E. coli based on the hybrid methanol assimilation pathway with Mdh and Das.</p

Knockdown of HNRNPM inhibits the progression of glioma through inducing ferroptosis

Ferroptosis acts as an important regulator in diverse human tumors, including the glioma. This study aimed to screen potential ferroptosis-related genes involved in the progression of glioma. Differently expressed genes (DEGs) were screened based on GSE31262 and GSE12657 datasets, and ferroptosis-related genes were separated. Among the important hub genes in the protein-protein interaction networks, HNRNPM was selected as a research target. Following the knockdown of HNRNPM, the viability, migration, and invasion were detected by CCK8, wound healing, and transwell assays, respectively. The role of HNRNPM knockdown was also verified in a xenograft tumor model in mice. Immunohistochemistry detected the expression levels of HNRNPM and Ki67. Moreover, the ferroptosis was evaluated according to the levels of iron, glutathione peroxidase (GSH), and malondialdehyde (MDA), as well as the expression of PTGS2, GPX4, and FTH1. Total 41 overlapping DEGs relating with ferroptosis and glioma were screened, among which 4 up-regulated hub genes (HNRNPM, HNRNPA3, RUVBL1, and SNRPPF) were determined. The up-regulation of HNRNPM presented a certain predictive value for glioma. In addition, knockdown of HNRNPM inhibited the viability, migration, and invasion of glioma cells in vitro, and also the tumor growth in mice. Notably, knockdown of HNRNPM enhanced the ferroptosis in glioma cells. Furthermore, HNRNPM was positively associated with SMARCA4 in glioma. Knockdown of HNRNPM inhibits the progression of glioma via inducing ferroptosis. HNRNPM is a promising molecular target for the treatment of glioma via inducing ferroptosis. We provided new insights of glioma progression and potential therapeutic guidance.</p

Enhanced Stability and Performance of Immobilized Lipase Using Hydrophobically Modified Single-Crystalline Ordered Macro–Microporous CuBTC as a Carrier Material

Single-crystalline ordered macro–microporous CuBTC (SOM–CuBTC) is a promising carrier for lipase immobilization due to enhanced mass transfer and stability toward fatty acids. However, the low yield per mass of the template during the preparation process and the water instability of the SOM–CuBTC carrier have posed significant limitations on its practical applications. In this study, we addressed these challenges by introducing a novel dual-solvent system consisting of dimethyl sulfoxide (DMSO) and ethanol to obtain a stable precursor solution with a concentration approximately 10 times higher than that in previous literature, yielding 21.4 mg of SOM–CuBTC per gram of the polystyrene template. However, the decomposition of SOM–CuBTC in an aqueous system of lipase immobilization was observed. We explored chemical vapor deposition and sol–gel methods for hydrophobic modification on SOM–CuBTC. SOM–CuBTC coated by hydrophobic polydimethylsiloxane (PDMS) via the sol–gel method possessed excellent chemical stability and exhibited great potential for lipase immobilization with a significant increase by 98.7% in the specific activity. The obtained immobilized lipase not only showed improved thermal stability and pH tolerance but also displayed excellent catalytic performance in the synthesis process of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) by acidolysis. This work reveals the great potential of SOM–CuBTC and provides new insights into the rational design of metal–organic frameworks for enzyme immobilization in extensive applications

Relative Significance of the Negative Impacts of Hemicelluloses on Enzymatic Cellulose Hydrolysis Is Dependent on Lignin Content: Evidence from Substrate Structural Features and Protein Adsorption

The biomass recalcitrance of the lignocellulose cell wall constructed by its chemical components, especially hemicelluloses and lignin, has become a bottleneck for the efficient release of glucose. The presence of hemicelluloses has been considered as a major factor limiting the enzymatic digestibility of lignocellulose biomass. However, most of the reported works on the effect of hemicelluloses removal on cellulose hydrolysability were conducted via dilute acid pretreatment at high temperature (>160 °C), and inconsistent conclusions have been found. In the present work, we studied the effects of xylan content on enzymatic digestibility of wheat straw cellulose in the cases of high and low lignin contents. Particularly, xylan removal was achieved by sulfuric acid hydrolysis under mild conditions (120 °C) to minimize lignin melting and migration in the cell wall and lignin structure modification. As revealed by various structure characterizations, when no lignin was removed, xylan removal by dilute acid hydrolysis resulted in reduction of particle size, deformation of the cell shape, etching of the cell lumen surface, some fracture and slight delamination of cell wall, with associated great increase in porosity and specific surface area. These structural modifications greatly improved cellulose digestibility. However, the presence of residual lignin also showed significant negative impacts by physical blocking and nonproductive adsorption of cellulases. In the case of low lignin content (∼4%), cellulose fibers become liberated and significant etching, delamination, fracture and even disappearance of the walls were visualized with xylan removal, which remarkably increased the effective surface area for cellulase binding with cellulose. The finding of this work demonstrates that the limiting action of hemicelluloses seems to be not important to cellulose digestibility as that observed in high-temperature (>160 °C) dilute acid pretreatment. Delignification seems to be more efficient to improve cellulose accessibility for mild-condition (<120 °C) pretreatment. It indicates that the interaction effects between lignin and hemicelluloses as structural factors limiting cellulose digestibility should be considered for investigating the mechanisms of effects of structure features on cellulose accessibility

Image1_Engineering and adaptive laboratory evolution of Escherichia coli for improving methanol utilization based on a hybrid methanol assimilation pathway.TIF

Engineering Escherichia coli for efficient methanol assimilation is important for developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer E. coli as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid methanol assimilation pathway which consists of prokaryotic methanol dehydrogenase (Mdh) and eukaryotic xylulose monophosphate (XuMP) pathway enzyme dihydroxyacetone synthase (Das) into E. coli and reprogrammed E. coli metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in the TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved via adaptive laboratory evolution and a final evolved strain can grow on methanol with only 0.1 g/L yeast extract as co-substrate. 13C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of fba, dhak, and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of E. coli based on the hybrid methanol assimilation pathway with Mdh and Das.</p

Structural Features of Formiline Pretreated Sugar Cane Bagasse and Their Impact on the Enzymatic Hydrolysis of Cellulose

Enzymatic digestibility of sugar cane bagasse could be greatly enhanced by Formiline pretreatment, which comprises a formic acid (FA) delignification followed by an alkaline deformylation. The FA can be easily recovered and recycled for delignification, indicating that this pretreatment is a green process for biomass fractionation. It was found that removing hemicelluloses and lignin during pretreatment contributed to the increase of cellulose accessibility; however, delignification seemed to be more important for exposing cellulose fibers. The compact cell wall structure of raw bagasse was destroyed by removing considerable parts of lignin and hemicelluloses with liberation of cellulose fibers, and the specific surface area of the pretreated substrates increased by more than 2-fold. However, formylation of cellulose took place during FA delignification, which showed significant negative impact on the initial enzymatic hydrolysis rate and enzymatic polysaccharide conversion at 120 h. Removing formyl groups by alkaline post-treatment could well recover the cellulose digestibility but without significant alteration of the substrate structure

Ordered Macro–Microporous ZIF‑8 with Different Macropore Sizes and Their Stable Derivatives for Lipase Immobilization in Biodiesel Production

Enzyme immobilization in hierarchical macro–microporous metal–organic frameworks (MOFs) is greatly desirable but challenging due to the poor stability of MOFs in a practical biocatalysis process. Herein, we prepared a series of single-crystalline ordered macro–microporous zeolitic imidazolate framework-8 (SOM-ZIF-8) with different macropore sizes of 180, 270, and 360 nm for lipase immobilization and investigated their performance in a practical biodiesel production process. Under an ethanol-assisted infiltration strategy, the potential of SOM-ZIF-8 for enzyme immobilization was unleashed from the nonwettability arising from its exceptional surface hydrophobicity, with a significant increase by 123.8% in the loading capacity. However, a fatty acid-induced complete decomposition of ZIF-8 in a nonaqueous system of biodiesel production was observed. To address this challenge, a degradation mechanism coupling the intermolecular autoionization of carboxylic acid was proposed, and a template-assisted pyrolysis approach was adopted to prepare the SOM-ZIF-8-derived carbon material that not only well preserved the 3D-ordered structure of the precursor but also exhibited enhanced chemical stability. Moreover, the carbon derivative exhibited great potential for enzyme immobilization, with a 22.9% increase in loading capacity, a 35.9% increase in specific activity, and a 12.7% increase in activity recovery, compared with SOM-ZIF-8. It is anticipated that this study will shed new light on the realistic design and modification of MOFs for the immobilization of enzymes in biocatalysis and extensive applications

Metabolic Engineering of <i>Escherichia coli</i> for <i>De Novo</i> Production of 1,2-Butanediol

1,2-Butanediol (1,2-BDO) is an important platform chemical widely utilized in the synthesis of polyester polyols, plasticizers, cosmetics, and pharmaceuticals. However, no natural metabolic pathway for its biosynthesis has been identified, and biological production of 1,2-BDO from renewable bioresources has not been reported so far. In this study, we designed and experimentally verified a feasible non-natural synthesis pathway for the de novo production of 1,2-BDO from renewable carbohydrates for the first time. This pathway extends the l-threonine synthesis pathway by introducing two artificial metabolic modules to sequentially convert l-threonine into 2-hydroxybutyric acid and 1,2-BDO. Following key enzyme screening and enhancement of l-threonine synthesis module in the chassis microorganism, the best engineered Escherichia coli strain was able to produce 0.15 g/L 1,2-BDO using glucose as the sole carbon source. This work lays the foundation for the bioproduction of 1,2-BDO from renewable resources