Characterization and application of a putative transcription factor (SUT2) in Pichia pastoris.

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the - 973 base pair (bp) to - 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, "A" at the 22nd bp upstream of ATG, was determined with 5'-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.

Metabolic and proteomic alteration in phytohormone-producing endophytic Bacillus amyloliquefaciens RWL-1 during methanol utilization.

INTRODUCTION: Methanol utilization by bacteria is important for various industrial processes. Methylotrophic bacteria are taxonomically diverse and some species promote plant growth and induce stress tolerance. However, methylotrophic potential of bacterial endophytes is poorly understood. OBJECTIVE: The current study aimed to evaluate the metabolomic and proteomic changes in endophytic Bacillus amyloliquefaciens RWL-1 caused by its methanol utilization and the resultant influence on its phytohormone production. METHODS: B. amyloliquefaciens RWL-1 was grown in LB medium with different concentrations [0 (control), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4%) of methanol to examine its methylotrophic potential. SDS-PAGE analysis was carried out for bacterial protein confirmation. Moreover, the phytohormones (indole 3 acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA)) produced by RWL-1 in methanol supplemented medium were quantified by GC-MS/SIM (6890N Network GC system, and 5973 Network Mass Selective Detector; Agilent Technologies, Santa Clara, CA, USA), while the antioxidants were estimated spectrophotometrically (T60 UV-VIS spectrophotometer, Leicester, UK). The amino acid quantification was carried out by amino acid analyzer (HITACHI L-8900, Japan). Furthermore, Nano-liquid chromatography (LC)-MS/MS analysis was performed with an Agilent system (Wilmington, DE, USA) for proteomic analysis while mascot algorithm (Matrix science, USA) was used to identify peptide sequences present in the protein sequence database. RESULTS: RWL-1 showed significant growth in media supplemented with 2 and 3.5% methanol, when compared with other concentrations. Mass spectroscopy analysis revealed that RWL-1 utilizes methanol efficiently as a carbon source. In the presence of methanol, RWL-1 produced significantly higher levels of IAA but lower levels of ABA, when compared with the control. Further, enzymatic antioxidants and functional amino acids were significantly up-regulated, with predominant expression of glutamic acid and alanine. Nano-liquid chromatography, quadrupole time-of-flight analysis, and quantitative analysis of methanol-treated bacterial cells showed expression of eight different types of proteins, including detoxification proteins, unrecognized and unclassified enzymes with antioxidant properties, proteases, metabolism enzymes, ribosomal proteins, antioxidant proteins, chaperones, and heat shock proteins. CONCLUSION: Results demonstrate that RWL-1 can significantly enhance its growth by utilizing methanol, and could produce phytohormones when growing in methanol-supplemented media, with increased expression of specific proteins and different biochemicals. These results will be useful in devising strategies for utilizing methylotrophic bacterial endophytes as alternative promoters of plant growth. Understanding RWL-1 ability to utilize methanol. The survival and phytohormones production by Bacillus amyloliquefaciens RWL-1 in methanol supplemented media whistle inducing metabolic and proteomic changes.

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale.

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (kLa) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by MutS P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2 kW m-3. Specific growth rate, and then dextranase production, increased as kLa and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2 L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut+ strain. After a scale-up process, at constant kLa, the specific growth rate was kept at 30 L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30 L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.

Methanol assimilation in Escherichia coli is improved by co-utilization of threonine and deletion of leucine-responsive regulatory protein.

Methane, the main component of natural gas, can be used to produce methanol which can be further converted to other valuable products. There is increasing interest in using biological systems for the production of fuels and chemicals from methanol, termed methylotrophy. In this work, we have examined methanol assimilation metabolism in a synthetic methylotrophic E. coli strain. Specifically, we applied 13C-tracers and evaluated 25 different co-substrates for methanol assimilation, including amino acids, sugars and organic acids. In particular, co-utilization of threonine significantly enhanced methylotrophy. Through our investigations, we proposed specific metabolic pathways that, when activated, correlated with increased methanol assimilation. These pathways are normally repressed by the leucine-responsive regulatory protein (lrp), a global regulator of metabolism associated with the feast-or-famine response in E. coli. By deleting lrp, we were able to further enhance the methylotrophic ability of our synthetic strain, as demonstrated through increased incorporation of 13C carbon from 13C-methanol into biomass.

Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis.

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

Hansenula polymorpha methanol metabolism genes enhance recombinant protein production in Komagataella phaffi.

Recombinant protein production in Komagataella phaffi (K. phaffi), a widely utilized host organism, can be optimized by enhancing the metabolic flux in the central carbon metabolism pathways. The methanol utilization pathway (MUT) during methanol-based growth plays a crucial role in providing precursors and energy for cell growth and development. This study investigated the impact of boosting the methanol dissimilation pathway, a branch of MUT that plays a vital role in detoxifying formaldehyde and providing energy in the form of NADH, in K. phaffi. This was achieved by integrating two orthologous genes from Hansenula polymorpha into the K. phaffi genome: formaldehyde dehydrogenase (HpFLD) and formate dehydrogenase (HpFMDH). The HpFLD and HpFMDH genes were isolated from the Hansenula polymorpha genome and inserted under the regulation of the pAOX1 promoter in the genome of recombinant K. phaffi that already contained a single copy of model protein genes (eGFP or EGII). The expression levels of these model proteins were assessed through protein activity assays and gene expression analysis. The findings revealed that while both orthologous genes positively influenced model protein production, HpFMDH exhibited a more pronounced upregulation in expression compared to HpFLD. Co-expression of both orthologous genes demonstrated synergistic effects, resulting in approximately a twofold increase in the levels of the model proteins detected. This study provides valuable insights into enhancing the production capacity of recombinant proteins in K. phaffi.

Artificial Methylotrophic Cells via Bottom-Up Integration of a Methanol-Utilizing Pathway.

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.

Improving Methanol Utilization by Reducing Alcohol Oxidase Activity and Adding Co-Substrate of Sodium Citrate in Pichia pastoris.

Methanol, which produced in large quantities from low-quality coal and the hydrogenation of CO2, is a potentially renewable one-carbon (C1) feedstock for biomanufacturing. The methylotrophic yeast Pichia pastoris is an ideal host for methanol biotransformation given its natural capacity as a methanol assimilation system. However, the utilization efficiency of methanol for biochemical production is limited by the toxicity of formaldehyde. Therefore, reducing the toxicity of formaldehyde to cells remains a challenge to the engineering design of a methanol metabolism. Based on genome-scale metabolic models (GSMM) calculations, we speculated that reducing alcohol oxidase (AOX) activity would re-construct the carbon metabolic flow and promote balance between the assimilation and dissimilation of formaldehyde metabolism processes, thereby increasing the biomass formation of P. pastoris. According to experimental verification, we proved that the accumulation of intracellular formaldehyde can be decreased by reducing AOX activity. The reduced formaldehyde formation upregulated methanol dissimilation and assimilation and the central carbon metabolism, which provided more energy for the cells to grow, ultimately leading to an increased conversion of methanol to biomass, as evidenced by phenotypic and transcriptome analysis. Significantly, the methanol conversion rate of AOX-attenuated strain PC110-AOX1-464 reached 0.364 g DCW/g, representing a 14% increase compared to the control strain PC110. In addition, we also proved that adding a co-substrate of sodium citrate could further improve the conversion of methanol to biomass in the AOX-attenuated strain. It was found that the methanol conversion rate of the PC110-AOX1-464 strain with the addition of 6 g/L sodium citrate reached 0.442 g DCW/g, representing 20% and 39% increases compared to AOX-attenuated strain PC110-AOX1-464 and control strain PC110 without sodium citrate addition, respectively. The study described here provides insight into the molecular mechanism of efficient methanol utilization by regulating AOX. Reducing AOX activity and adding sodium citrate as a co-substrate are potential engineering strategies to regulate the production of chemicals from methanol in P. pastoris.

Light-Driven CO2 Reduction with a Surface-Displayed Enzyme Cascade-C3N4 Hybrid.

Efficient and cost-effective conversion of CO2 to biomass holds the potential to address the climate crisis. Light-driven CO2 conversion can be realized by combining inorganic semiconductors with enzymes or cells. However, designing enzyme cascades for converting CO2 to multicarbon compounds is challenging, and inorganic semiconductors often possess cytotoxicity. Therefore, there is a critical need for a straightforward semiconductor biohybrid system for CO2 conversion. Here, we used a visible-light-responsive and biocompatible C3N4 porous nanosheet, decorated with formate dehydrogenase, formaldehyde dehydrogenase, and alcohol dehydrogenase to establish an enzyme-photocoupled catalytic system, which showed a remarkable CO2-to-methanol conversion efficiency with an apparent quantum efficiency of 2.48% in the absence of externally added electron mediator. To further enable the in situ transformation of methanol into biomass, the enzymes were displayed on the surface of Komagataella phaffii, which was further coupled with C3N4 to create an organic semiconductor-enzyme-cell hybrid system. Methanol was produced through enzyme-photocoupled CO2 reduction, achieving a rate of 4.07 mg/(L·h), comparable with reported rates from photocatalytic systems employing mediators or photoelectrochemical cells. The produced methanol can subsequently be transported into the cell and converted into biomass. This work presents a sustainable, environmentally friendly, and cost-effective enzyme-photocoupled biocatalytic system for efficient solar-driven conversion of CO2 within a microbial cell.

Coordinate regulation of methanol utilization pathway genes of Komagataella phaffii by transcription factors and chromatin modifiers.

The methylotrophic yeast Komagataella phaffii (a.k.a. Pichia pastoris) harbors a methanol utilization (MUT) pathway, enabling it to utilize methanol as the sole source of carbon. The nexus between transcription factors such as Mxr1p and Trm1p and chromatin-modifying enzymes in the regulation of genes of MUT pathway has not been well studied in K. phaffii. Using transcriptomics, we demonstrate that Gcn5, a histone acetyltransferase, and Gal83, one of the beta subunits of nuclear-localized SNF1 (sucrose non-fermenting 1) kinase complex are essential for the transcriptional regulation by the zinc finger transcription factors Mxr1p and Trm1p. We conclude that interactions among Gcn5, Snf1, Mxr1p, and Trm1p play a critical role in the transcriptional regulation of genes of MUT pathway of K. phaffii.

Discovery of lanthanide-dependent methylotrophy and screening methods for lanthanide-dependent methylotrophs.

The lanthanide elements (Lns) affect the physiology and growth of certain microorganisms known as "Ln-responsive microorganisms." Among them, in 2011, it was first reported that strains of Methylobacterium exhibited high methanol dehydrogenase (MDH) activity when grown in the presence of Lns; the purified Ln-inducible MDH was identified as XoxF-type MDH, whose catalytic function had previously been unknown. XoxF was the first enzyme to be identified as Ln-dependent, and its function in methylotrophy is more fundamental and important than that of the corresponding Ca2+-dependent MDH MxaFI. XoxF is encoded in the genomes of methylotrophic as well as non-methylotrophic bacteria. Thus, Lns are among the most fascinating and important growth factors for bacteria that potentially utilize methanol. Bacteria that require Lns for methanol growth are called "Ln-dependent methylotrophs." Recent findings indicate that these microorganisms comprise an "Ln-dependent ecosystem" that we have not been able to reconstruct under laboratory conditions without Lns. In this chapter, we summarize methods for (1) screening of Ln-responsive microorganisms, (2) purification of native XoxFs from Ln-dependent methylotrophs, and (3) screening of Ln-dependent methylotrophs from natural environments, while providing a history of the discovery of the Ln-dependent methylotrophs.

Lanthanide-dependent methanol dehydrogenase from the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium diazoefficiens strain USDA110.

The legume symbiotic nitrogen-fixing bacterium, B. diazoefficiens strain USDA110, utilizes methanol for growth in the presence of light lanthanides, such as La3+, Ce3+, Pr3+ or Nd3+, and its cells possess significant methanol dehydrogenase (MDH) activity. We purified MDH to homogeneity from B. diazoefficiens strain USDA110 grown in a methanol/Ce3+ medium; the protein was identified as XoxF5-type MDH (blr6213). The purified XoxF contained 0.58 cerium atoms per enzyme subunit. Moreover, the in-solution structure of XoxF was analyzed by small angle X-ray scattering (SAXS) analysis; the radius of gyration (Rg) and maximum particle dimension (Dmax) of XoxF were calculated to be 32.3 and 96.8 Å, respectively, suggesting that XoxF adopts a dimer structure in solution. These results show that B. diazoefficiens strain USDA110 has XoxF, a lanthanides-dependent MDH, which has methanol oxidation activity and is induced by methanol/lanthanaides, and that lanthanide is one of the important factors in methanol utilization by the strain.