Enhanced Four-Electron Selective Oxygen Reduction Reaction at Carbon-Nanotube-Supported Sulfonic-Acid-Functionalized Copper Phthalocyanine.

In the present work, the oxygen reduction reaction (ORR) is explored in an acidic medium with two different catalytic supports (multi-walled carbon nanotubes (MWCNTs) and nitrogen-doped multi-walled carbon nanotubes (NMWCNTs)) and two different catalysts (copper phthalocyanine (CuPc) and sulfonic acid functionalized CuPc (CuPc-SO3 - )). The composite, NMWCNTs-CuPc-SO3 - exhibits high ORR activity (assessed based on the onset potential (0.57 V vs. reversible hydrogen electrode) and Tafel slope) in comparison to the other composites. Rotating ring disc electrode (RRDE) studies demonstrate a highly selective four-electron ORR (less than 2.5 % H2 O2 formation) at the NMWCNTs-CuPc-SO3 - . The synergistic effect of the catalyst support (NMWCNTs) and sulfonic acid functionalization of the catalyst (in CuPc-SO3 - ) increase the efficiency and selectivity of the ORR at the NMWCNTs-CuPc-SO3 - . The catalyst activity of NMWCNTs-CuPc-SO3 - has been compared with many reported materials and found to be better than several catalysts. NMWCNTs-CuPc-SO3 - shows high tolerance for methanol and very small deviation in the onset potential (10 mV) between the linear sweep voltammetry responses recorded before and after 3000 cyclic voltammetry cycles, demonstrating exceptional durability. The high durability is attributed to the stabilization of CuPc-SO3 - by the additional coordination with nitrogen (Cu-Nx ) present on the surface of NMWCNTs.

Digging and identification of novel microorganisms from the soil environments with high methanol-tolerant lipase production for biodiesel preparation.

Converting renewable biomass into carbon-neutral biofuels is one of the most effective strategies to achieve zero carbon emissions and contribute to environmental protection. Microorganisms from the soil were primarily screened on the rhodamine B-plate for highly-active lipase producing strains and re-screened on a tributyrin-methanol plate using crude lipases produced from the initially screened-out strains. The lipase-producing strains with higher methanol-tolerant lipase were identified based on morphological characteristics and 16S rDNA sequencing. The crude lipases with much higher methanol-tolerance from screened top-4 strains, Stenotrophomonas maltophilia D18, Lysinibacillus fusiformis B23, Acinetobacter junii C69, and A. pittii C95 showed temperature optima of 25 °C, 35 °C, 30 °C, and 30 °C at pH 7.0, respectively, while their pH optima were 8.0, 7.0, 7.5, and 7.5 at each optimum temperature, respectively. After 24-h incubation, they retained more than 85% of their original activities in 25%, 15%, 20%, and 20% of methanol, respectively. They catalyzed the conversion of soybean oil into biodiesel by yields of 63.1%, 35.4%, 74.6%, and 78.5% after 24-h reactions, respectively. In conclusion, the as-isolated microorganisms producing high methanol-tolerant lipase are considered promising to provide robust biocatalyst for efficient biodiesel preparation and other industrial applications.

Nanosheets with High-Performance Electrochemical Oxygen Reduction Reaction Revived from Green Walnut Peel.

The synthesis of metal-free carbon-based electrocatalysts for oxygen reduction reactions (ORR) to replace conventional Pt-based catalysts has become a hot spot in current research. This work proposes an activation-assisted carbonization strategy, to manufacture N-doped ultra-thin carbon nanosheets (GWS180M800) with high catalytic activity, namely, melamine is used as an accelerator/nitrogen source, and walnut green peels biological waste as a carbon source. The melamine acts as a nitrogen donor in the hydrothermal process, effectively enhancing the nitrogen doping rate. The content of pyridine nitrogen groups accounts for up to 48.5% of the total nitrogen content. Electrochemical tests show that the GWS180M800 has excellent ORR electrocatalytic activity and stability, and makes a quasi-four-electron ORR pathway clear in the alkaline electrolyte. The initial potential and half slope potential are as high as 1.01 and 0.82 V vs. RHE, respectively. The GWS180M800 catalyst has a better ability to avoid methanol cross poisoning than Pt/C has. Compared with 20 wt% Pt/C, GWS180M800 has improved methanol tolerance and stability. It is a metal-free biochar ORR catalyst with great development potential and application prospects. This result provides a new space for the preparation of valuable porous nano-carbon materials based on carbonaceous solid waste and provides new ideas for catalyzing a wide range of electrochemical reactions in the future.

Synthesizing value-added products from methane by a new Methylomonas.

AIMS: Methane and methanol are potential carbon sources of industrial micro-organisms in addition to crop-derived bio-carbon sources. Methanotrophs that can utilize these simple, stable and large amounts chemicals are expected to be developed into 'cell factories' for the production of specific chemicals. In this study, a methanotroph that can synthesize lycopene, C30 carotenoid and exopolysaccharides (EPS) with relative better performances from C1 substrates was isolated, and its performances were evaluated. METHODS AND RESULTS: The isolated strain was identified as Methylomonas sp. ZR1 based on 16S rRNA sequence analysis. Its maximum specific growth rate achieved 0·200 h-1 under flask culture conditions, and 0·386 h-1 in bubble column reactors. ZR1 was able to utilize 35 g l-1 of methanol and even exhibited slight growth in the presence of 40 g l-1 of methanol. Furthermore, ZR1 was proved to synthesize lycopene (C40 carotenoids) besides the C30 carotenoids through HPLC-DAD and HPLC-MS/MS analysis methods. Its carotenoid extracts exhibited excellent antioxidative activities measured by the ABTS+ method. Plenty of polysaccharides were also synthesized by ZR1, the components of the polysaccharides were identified as glucose, mannose and galactose with a proportion of 1 : 2 : 1 by GC-MS, and its yield achieved 0·13 g g-1 cell dry weight. CONCLUSIONS: The isolated strain has great potential for the production of value-added bioproducts from C1 compounds because of its excellent C1 substrate utilizing abilities and its abilities to naturally synthesize lycopene, C30 carotenoids and EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated a fast-growing methanotroph, its C1 carbon substrate utilizing ability is excellent in comparison with reported methanotrophs. Furthermore, besides polysaccharides and C30 carotenoids which were commonly synthesized by methanotrophs, our findings suggested that C40 lycopene could also be naturally synthesized from methane by methanotrophs.

Synthesis of Pd3Co1@Pt/C core-shell catalysts for methanol-tolerant cathodes of direct methanol fuel cells.

A composite Pd-based electrocatalyst consisting of a surface layer of Pt (5 wt.%) supported on a core Pd3Co1 alloy (95 wt.%) and dispersed as nanoparticles on a carbon black support (50 wt.% metal content) was prepared by using a sulphite-complex route. The structure, composition, morphology, and surface properties of the catalyst were investigated by XRD, XRF, TEM, XPS and low-energy ion scattering spectroscopy (LE-ISS). The catalyst showed an enrichment of Pt on the surface and a smaller content of Co in the outermost layers. These characteristics allow a decrease the Pt content in direct methanol fuel cell cathode electrodes (from 1 to 0.06 mg cm(-2)) without significant decay in performance, due also to a better tolerance to methanol permeated through the polymer electrolyte membrane.

Bimetallic palladium-copper nanoplates with optimized d-band center simultaneously boost oxygen reduction activity and methanol tolerance.

The methanol-poisoning of electrocatalysts at the cathodic part of direct methanol fuel cells (DMFCs) can severely degrade the overall efficiency. Therefore, engineering cathodic catalysts with outstanding oxygen reduction activity, and simultaneously, superior methanol tolerance is greatly desired. Herein, bimetallic palladium-copper (PdCu) nanoplates with the optimized d-band center are designed as promising cathodic catalysts for DMFCs. It shows outstanding oxygen reduction activity with a mass activity (MA) of 0.522 A mgPd-1 in alkaline electrolyte, overwhelming the benchmarked commercial Pt/C and Pd/C. Meanwhile, it has prominent stability with only 4.0 % loss in MA after continuous 20 K cycles. More importantly, the PdCu nanoplates are almost inert toward methanol oxidation and show excellent anti-methanol capability. The theoretical calculations reveal that the downshift of d-band center in PdCu nanoplates and the electronic interaction between Pd and Cu atoms could effectively lower the methanol adsorption energy, thus leading to enhanced methanol tolerance. This work highlights the important role of tuning the electronic structure and optimized geometry of electrocatalysts to simultaneously boost their oxygen reduction activity, stability, and methanol tolerance for their future application in DMFCs.

Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation.

Methanol tolerance of lipase is one of the important factors affecting its esterification ability in biodiesel preparation. By B factor indicated prediction of Candida antarctica lipase B (CalB) surface amino acids, eight sites (Val139, Ala146, Leu147, Pro218, Val286, Ala287, Val306, and Gly307) with high B value indicating more flexibility were chosen to perform saturation mutagenesis. High-methanol-tolerant variants, CalB-P218W and -V306N, created larger haloes on emulsified tributyrin solid plate including 15% (v/v) methanol and showed 19% and 31% higher activity over wild-type CalB (CalB-WT), respectively. By modeling, a newly formed hydrogen bond in CalB-V306N and hydrophobic force in CalB-P218W contributing more stability in protein may have resulted in increased methanol tolerance. CalB-P218W and -V306N transesterified the soybean oil into biodiesel at 30 °C by 85% and 89% yield, respectively, over 82% by CalB-WT for 24 h reactions. These results may provide a basis for molecular engineering of CalB and expand its applications in fuel industries. The as-developed semi-rational method could be utilized to enhance the stabilities of many other industrial enzymes.

Improvement of methanol tolerance and catalytic activity of Rhizomucor miehei lipase for one-step synthesis of biodiesel by semi-rational design.

Exploiting highly active and methanol-resistant lipase is of great significance for biodiesel production. A semi-rational directed evolution method combined with N-glycosylation is reported, and all mutants exhibiting higher catalytic activity and methanol tolerance than the wild type (WT). Mutant N267 retained 64% activity after incubation in 50% methanol for 8 h, which was 48% greater than that of WT. The catalytic activity of mutants N267 and N167 was 30- and 71- fold higher than that of WT. Molecular dynamics simulations of N267 showed that the formation of new strong hydrogen bonds between glycan and the protein stabilized the structure of lipase and improved its methanol tolerance. N267 achieved biodiesel yields of 99.33% (colza oil) and 81.70% (waste soybean oil) for 24 h, which was much higher than WT (51.6% for rapeseed oil and 44.73% for wasted soybean oil). The engineered ProRML mutant has high potential for commercial biodiesel production.

N-glycosylation as an effective strategy to enhance characteristics of Rhizomucor miehei lipase for biodiesel production.

The construction of methanol-resistant lipases with high catalytic activity is world-shattering for biodiesel production. A semi-rational method has been constructed to enhance the properties of Rhizomucor miehei lipase with propeptide (ProRML) by introducing N-glycosylation sites in the Loop structure. The enzyme activities of the mutants N288 (1448.89 ± 68.64 U/mg) and N142 (1073.68 ± 33.87 U/mg) increased to 56.09 and 41.56 times relative to that of wild type ProRML (WT, 25.83 ± 0.73 U/mg), respectively. After incubation in 50 % methanol for 2.5 h, the residual activities of N314 and N174-1 were 95 % and 85%, which were higher than the WT (27 %). Additionally, the biodiesel yield of all mutants was increased after a one-time addition of methanol for 24 h. Among them, N288 increased the quantity of biodiesel from colza oil from 9.49 % to 88 %, and N314 increased the amount of biodiesel from waste soybean oil from 8.44% to 70%. This study provides an effective method to enhance the properties of lipase and improve its application potential in biodiesel production.

Improved methanol tolerance of Rhizomucor miehei lipase based on N­glycosylation within the α-helix region and its application in biodiesel production.

BACKGROUND: Liquid lipases are widely used to convert oil into biodiesel. Methanol-resistant lipases with high catalytic activity are the first choice for practical production. Rhizomucor miehei lipase (RML) is a single-chain α/ß-type protein that is widely used in biodiesel preparation. Improving the catalytic activity and methanol tolerance of RML is necessary to realise the industrial production of biodiesel. RESULTS: In this study, a semi-rational design method was used to optimise the catalytic activity and methanol tolerance of ProRML. After N-glycosylation modification of the α-helix of the mature peptide in ProRML, the resulting mutants N218, N93, N115, N260, and N183 increased enzyme activity by 66.81, 13.54, 10.33, 3.69, and 2.39 times than that of WT, respectively. The residual activities of N218 and N260 were 88.78% and 86.08% after incubation in 50% methanol for 2.5 h, respectively. In addition, the biodiesel yield of all mutants was improved when methanol was added once and reacted for 24 h with colza oil as the raw material. N260 and N218 increased the biodiesel yield from 9.49% to 88.75% and 90.46%, respectively. CONCLUSIONS: These results indicate that optimising N-glycosylation modification in the α-helix structure is an effective strategy for improving the performance of ProRML. This study provides an effective approach to improve the design of the enzyme and the properties of lipase mutants, thereby rendering them suitable for industrial biomass conversion.

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization.

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Corrigendum: Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR.

[This corrects the article DOI: 10.3389/fbioe.2020.01034.].

Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR.

Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with Tm values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a Tm value of 68.0°C. Although the increase of Tm was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes.

Ultrasound-assisted production of biodiesel using engineered methanol tolerant Proteus vulgaris lipase immobilized on functionalized polysulfone beads.

In the present study, Proteus vulgaris lipase (PVL) was engineered using directed evolution to increase methanol tolerance so that it would be more tolerant and efficient for harsh conditions employed in biodiesel synthesis, which is limiting their industrial use. The influence of ultrasound under different experimental conditions on the biodiesel conversion yield using methanolysis of non-edible neem oil was also emphasized. A special attention was also paid to the immobilization of lipase on Polysulfone (PS) beads and comparative studies with industrially used Burkholderia cepacia lipase. The Engineered Proteus vulgaris lipase showed >80% activity after 3 h when incubated in 50% methanol with simultaneous sonication. The lipase retained improved longevity (~70% residual activity) over wild-type PVL over repeated use.

Methanol Tolerant Pt-C Core-Shell Cathode Catalyst for Direct Methanol Fuel Cells.

Methanol crossover is one of the largest problems in direct methanol fuel cells (DMFCs). Methanol passing from the anode to the cathode through the membrane is oxidized at the cathode, degrading the DMFC performance, and the intermediates of the methanol oxidation reaction (MOR) cause cathode catalyst poisoning. Therefore, it is essential to develop a cathode catalyst capable of inhibiting MOR while promoting the oxygen reduction reaction (ORR), which is a typical cathode reaction in DMFCs. In this study, a carbon-encapsulated Pt cathode catalyst was synthesized for this purpose. The catalyst was simply synthesized by heat treatment of Pt-aniline complex-coated carbon nanofibers. The carbon shell of the catalyst was effective in inhibiting methanol from accessing the Pt core, and this effect became more prominent as the graphitization degree of the carbon shell increased. Meanwhile, the carbon shell allowed O2 to permeate regardless of the graphitization degree, enabling the Pt core to participate in ORR. The synthesized catalyst showed higher performance and stability in single-cell tests under various conditions compared to commercial Pt/C.

Atomically dispersed Ni/NixSy anchored on doped mesoporous networked carbon framework: Boosting the ORR performance in alkaline and acidic media.

Rational and strategic fabrication of cost-effective, active and durable oxygen reduction reaction (ORR) electrocatalyst is the bottle-neck for the commercialization of fuel cells and metal-air batteries. Atomically dispersed nickel (Ni)/nickel sulfide (NixSy) anchored on heteroatom doped networked hierarchical porous carbonaceous sheets are synthesized from nickel nitrate and guanidine thiocyanate. The sample annealed at 750 °C followed by acid-treatment (Ni-GT-750-A) emerges as the best performing pH-universal ORR catalyst with an onset potential of 0.91 (0.1 M KOH) and 0.89 V (0.1 M HClO4) vs. reversible hydrogen electrode (RHE). It also exhibits better current durability (95.0 and 60%) and methanol tolerance (90.6 and 80.3%) in comparison to the commercial catalyst (65.0, 27, -33.0 and 16.5%) in alkaline and acidic media, respectively. An insight into the microstructure and ORR-active chemical sites is obtained with the aid of electron microscopic (FE-SEM and HR-TEM) and physiochemical (sorption isotherm, XRD, Raman and XPS) studies, respectively. The enhanced activity results from the synergistic influence of metallic ORR-active sites in hierarchical porous doped defective carbon support, which provides the well-interlinked conducting channel for electron transfer and additional ORR-active sites. The introduced electrocatalyst establishes Ni decorated doped carbon systems as potential revolutionary substitutes for commercial systems.

The unique Pd@Pt/C core-shell nanoparticles as methanol-tolerant catalysts using sonochemical synthesis.

Over the past decades, there were a few reports on the use of sonochemical method to prepare noble metals catalysts for fuel cells. However, the synthetic processes were conducted under high frequency (200 kHz)/long reaction time in most cases. In this work, Pd and PdxPt nanoparticles were prepared by sonochemical method under low frequency (20 kHz) in a shorter time (20-40 mins). In the first time, a sequentialsonochemical synthesis was explored to achieve a core/shell structure of PdxPt nanoparticles. Consequently, the unique core-shell structure was formed with two shells surrounding the Pd core. The Pd core was firstly grown. In the second step, the Pd2+ ion existing in the Pd core reduced simultaneously with Pt4+ ion in the solution as the first layer of PdPt alloy. Further, the Pt layer was formed subsequently. The Pd-based catalysts exhibited a superior ORR selective activity and exceptional methanol-tolerance property compared with the commercial Pt/C catalyst. In 0.5 M CH3OH + 0.5MH2SO4 solution, the best performance was achieved on Pd3Pt/C catalyst with increased overpotential of 24 mV. However, overpotentials was increased 174 mV on commercial Pt/C catalyst. The excellent performance of the Pd3Pt/C catalyst is ascribed to its combination of preferable growth of the Pd (1 1 1) plane, small particle size (∼4 nm), unique core/shell structure as well as the electronic effects between Pd and Pt. These results have demonstrated that the sequential ultrasonic synthesis is an effective method for the synthesis of binary/trinary catalysts in a green approach.

Rh-doped PdAg nanoparticles as efficient methanol tolerance electrocatalytic materials for oxygen reduction.

Direct methanol fuel cells (DMFCs) have received extensive attention on their high efficiency, high reliability, and no carbon emission. Unfortunately, the poor methanol tolerance and sluggish oxygen reduction reaction (ORR) at cathode have seriously hindered their further development. Herein we report the synthesis of a new class of Rh-doped PdAg alloy nanoparticles (NPs) for boosting ORR activity with high methanol tolerance capacity concurrently. The ORR mass activity of typical Rh4Pd40Ag56 NPs is 4.2 times higher than that of commercial Pt catalyst. Moreover, it shows a great methanol tolerance capability by maintaining 92.4% in ORR mass activity in alkaline solution with 0.1 mol L-1 methanol, against a big decrease of almost 100% for commercial Pt. Even after 30,000 potential cycles with 1.0 mol L-1 methanol, Rh4Pd40Ag56 NPs still retain ORR mass activity of up to 68.3%. DFT calculations reveal that excellent ORR performance with excellent methanol tolerance originates the active d-band-pinning engineering for an efficient site-independent electron-transfer. A generalized d-band mediated fine electron-transfer tuning path has blueprinted for effectively minimizing intrinsic ORR barriers with high current density. The present work highlights the key role of Rh doping in enhancing the ORR activity and methanol tolerance ability of PdAg NPs for future high-performance DMFCs.

Spherical α-MnO2 Supported on N-KB as Efficient Electrocatalyst for Oxygen Reduction in Al-Air Battery.

Traditional noble metal platinum (Pt) is regarded as a bifunctional oxygen catalyst due to its highly catalytic efficiency, but its commercial availability and application is often restricted by high cost. Herein, a cheap and effective catalyst mixed with α-MnO2 and nitrogen-doped Ketjenblack (N-KB) (denoted as MnO2-SM150-0.5) is examined as a potential electrocatalyst in oxygen reduction reactions (ORR) and oxygen evolution reactions (OER). This α-MnO2 is prepared by redox reaction between K2S2O8 and MnSO4 in acid conditions with a facile hydrothermal process (named the SM method). As a result, MnO2-SM150-0.5 exhibits a good catalytic performance for ORR in alkaline solution, and this result is comparable to a Pt/C catalyst. Moreover, this catalyst also shows superior durability and methanol tolerance compared with a Pt/C catalyst. It also displays a discharge voltage (~1.28 V) at a discharge density of 50 mA cm-2 in homemade Al-air batteries that is higher than commercial 20% Pt/C (~1.19 V). The superior electrocatalytic performance of MnO2-SM150-0.5 could be attributed to its higher Mn3+/Mn4+ ratio and the synergistic effect between MnO2 and the nitrogen-doped KB. This study provides a novel strategy for the preparation of an MnO2-based composite electrocatalyst.

Breeding of Methanol-Tolerant Methylobacterium extorquens AM1 by Atmospheric and Room Temperature Plasma Mutagenesis Combined With Adaptive Laboratory Evolution.

Methylobacterium extorquens AM1, which can be used as a methylotrophic cell factory (MeCF) for the production of fine chemicals from methanol, is the most extensively studied model methylotrophic strain. However, its low tolerance for methanol limits the development of bioprocesses and there have been no reports of improved methanol tolerance of M. extorquens AM1. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis, in combination with adaptive laboratory evolution (ALE), is used to generate a mutant with high methanol tolerance (referred to as CLY-2533). The final cell density of CLY-2533 is 7.10 times higher than that of the wild-type strain in medium containing 5% (v/v) methanol. Through comparative genomics analysis and overexpression of the exploited putative genes, seven mutated genes are identified as being closely related to the higher methanol tolerance of CLY-2533. Additionally, the mvt operon, which contains genes related to the biosynthesis of mevalonate acid (MEV), is introduced into CLY-2533. This recombinant strain shows significant improvements in both MEV production and cell growth in 5% methanol medium. These findings will be helpful in rational design of methanol-utilizing strain for an improved host platform for methanol based biomanufacturing.