Methanol Metabolic Pathway Research Articles

<i>Komagataella phaffii</i> yeast as a model organism in biotechnology and fundamental research

The choice of a model organism in biology is based on various factors, such as practical significance and ease of manipulation. Saccharomyces cerevisiae yeast is one of the most widely used and well-studied eukaryotic models. However, developments in NGS, proteomics, metabolomics and gene editing methods allow other species to become the object of fundamental research. A good example of such emerging model organisms is the yeast Komagataella phaffii.
 K. phaffii belongs to a unique group of eukaryotic methylotrophs that can use methanol as the sole source of carbon and energy. On the other hand, K. phaffii seems to be more characteristic of the common ancient yeast ancestors than the rapidly evolving S. cerevisiae. Comparative studies between S. cerevisiae and K. phaffii will shed light on the mechanisms of evolution of metabolic pathways and regulatory systems. Such studies are accelerated by the practical importance of K. phaffii as a common microbial production host in biotechnology.
 In our studies, we demonstrate that some amino acids greatly affect gene expression in K. phaffii [1]. Transcriptome analysis revealed drastic changes in gene expression when proline was present in the media. About 18.9% of total protein-coding genes were differentially expressed, including genes involved in methanol utilization [2]. Our results show that the unique methanol metabolism pathway is regulated not only by methanol, but also by other carbon sources. Therefore, our findings suggest that the regulation of methanol metabolism pathway is integrated into other cellular regulatory networks. Methanol metabolism, acquired by K. phaffii during evolution, is tightly associated with nitrogen an amino acid metabolism.

Methanol oxidase from Hansenula polymorpha shows activity in peroxisome-deficient Pichia pastoris

Pichia pastoris can upgrade the methanol into various of high-valued biochemical products. This biosynthetic method was achieved mostly based on inherent methanol metabolism pathway of yeast. The alcohol oxidase (AOX) from Pichia pastoris plays a crucial role in methanol metabolism pathway. However, when the cells are exposed to excess methanol, the disruption of the peroxisome and inhibition of AOX enzyme activity happens. In our study，the strain Δpex3 derived from P. pastoris GS115 was constructed as initial genetic engineering strain to simulate the damage of the peroxisome which was often happened under high concentrations of methanol. No AOX activity was detected in Δpex3 with peroxisome defected. Subsequently, the methanol oxidase (MOX) from Hansenula polymorpha was employed to make up for activity loss of AOX. Introduction of MOX in the peroxisome-deficient strain Δpex3, thus new recombinant strain was obtained which named as Δpex3-MOX. Δpex3-MOX showed certain activity of alcohol oxidase, and the harvest biomass was higher compared to Δpex3. The ability of utilizing and toleration higher methanol was partially improved when methanol oxidase (MOX) from H. polymorpha was introduced compared to cells with peroxisome defected.

Methylotrophy in Mycobacteria: Dissection of the Methanol Metabolism Pathway in Mycobacterium smegmatis.

The mycobacteria comprise both pathogenic and nonpathogenic bacteria. Although several features related to pathogenicity in various mycobacterial species, such as Mycobacterium tuberculosis, have been studied in great detail, methylotrophy, i.e., the ability of an organism to utilize single-carbon (C1) compounds as the sole source of carbon and energy, has remained largely unexplored in mycobacteria. Reports are available that suggest that mycobacteria, including M. tuberculosis and M. smegmatis, are capable of utilizing alternative C1 compounds to meet their carbon and energy requirements. However, physiological pathways that are functional in mycobacteria to utilize such carbon compounds are only poorly understood. Here we report the identification and characterization of the gene products required for establishing methylotrophy in M. smegmatis We present N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol oxidase (Mno) as the key enzyme that is essential for the growth of M. smegmatis on methanol. We show that Mno has both methanol and formaldehyde dehydrogenase activities in vitro Further, M. smegmatis is able to utilize methanol even in the absence of the major formaldehyde dehydrogenase MscR, which suggests that Mno is sufficient to dissimilate methanol and the resulting formaldehyde in vivo Finally, we show that M. smegmatis devoid of phosphoenolpyruvate carboxykinase, which has been shown to fix CO2 in M. tuberculosis, does not grow on methanol, suggesting that the final step of methanol utilization requires CO2 fixation for biomass generation. Our work here thus forms the first comprehensive report that explores methylotrophy in a mycobacterial species.IMPORTANCE Methylotrophy, the ability to utilize single-carbon (C1) compounds as the sole carbon and energy sources, is only poorly understood in mycobacteria. Both pathogenic and nonpathogenic mycobacteria, including Mycobacterium tuberculosis, are capable of utilizing C1 compounds to meet their carbon and energy requirements, although the precise pathways are not well studied. Here we present a comprehensive study of methylotrophy in Mycobacterium smegmatis With several genetic knockouts, we have dissected the entire methanol metabolism pathway in M. smegmatis We show that while methanol dissimilation in M. smegmatis differs from that in other mycobacterial species, the concluding step of CO2 fixation is similar to that in M. tuberculosis It is therefore both interesting and important to examine mycobacterial physiology in the presence of alternative carbon sources.

Comprehensive discovery of 13C labeled metabolites in the bacterium Methylobacterium extorquens AM1 using gas chromatography–mass spectrometry

Herein, we report the identification of isotopically labeled metabolite peaks (or the lack of labeling) between sets of GC–MS data from Methylobacterium extorquens AM1. M. extorquens AM1 is one of the best-characterized model organisms for the study of C1 metabolism in methylotrophic bacteria, a diverse group of microbes that can use reduced one-carbon (C1) sources, such as methanol and methane as a sole source for both energy generation and carbon assimilation. Application of a match value (MV) based metric was used to rank the metabolite peaks in the data from those exhibiting the most mass spectral indications of labeling, to those not exhibiting any indications of labeling. The MV-based ranking corresponded well with analyst interpretation of the mass spectra. The MV-based method was initially demonstrated and validated using a mixture of 21 standards with data sets generated for mixtures at natural abundance, a mixture with 6 of the compounds labeled, and a 1:1 mixture of the natural abundance and labeled mixtures. Experimental data from TMS-derivatized extracts from the bacterium M. extorquens AM1 grown with natural abundance or 13C-labeled methanol as the carbon source were analyzed. Of 131 peaks considered for the analysis of M. extorquens AM1, the 40 peaks ranked highest for indications of 13C labeling were all found to be labeled, while those peaks ranked lower progressed from peaks for which labeling was uncertain, to a larger number of peaks that were clearly not labeled. The list of peaks determined to be labeled forms a library of compounds that are known to be labeled following the methanol metabolic pathway in M. extorquens AM1 that can be further investigated in future work, e.g. fluxomic studies.

Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes

Increased copy number of foreign gene can result in the alteration of normal metabolism in Pichia pastoris. To better understand the effect of foreign gene dosage on the cellular physiology of P. pastoris cells, comparative transcriptional analysis was performed among three P. pastoris strains carrying 0, 6, and 18 copies of porcine insulin precursor (PIP) expression cassettes, respectively. mRNA levels of 13 selected genes involved in methanol metabolic pathway, central metabolic pathway, protein folding, and oxidative stress were determined by real-time PCR. Results showed that enhanced PIP copy number resulted in an increase in PIP mRNA and also in folding stress on the yeast cells' endoplasmic reticulum. The metabolism of 6-copy P. pastoris strain was not significantly changed as compared to 0-copy strain (control). In contrast, physiology of 18-copy strain was remarkably affected, characterized by the upregulation of antioxidative genes and readjusted expression level of methanol metabolic pathway genes. These data suggested that high copy P. pastoris strain might be suffering from protein folding-related oxidative stress and insufficient supply of carbon and energy sources.

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions.

Coping with Polychlorinated Biphenyl (PCB) Toxicity: Physiological and Genome-Wide Responses of Burkholderia xenovorans LB400 to PCB-Mediated Stress

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.

Production of IFNα-2a in Hansenula polymorpha

A DNA-sequence coding for IFNα-2a was expressed in the methylotrophic yeast Hansenula polymorpha from a strong inducible promoter element derived from the MOX gene, a key gene of the methanol metabolism pathway. For secretion the coding sequence was fused to the KEX2 recognition site of the S. cerevisiae-derived MFα1 prepro-leader. To a large extent the secreted molecules were found to be incorrectly processed from the precursor molecule exhibiting N-terminal extensions of the mature protein. Correct processing was achieved when co-expressing a S. cerevisiae-derived KEX2 gene from its native promoter. Undesirable proteolytic cleavage at additional dibasic sites of the protein sequence could be minimised when optimising fermentation conditions. A pH/pO 2-controlled C-source feeding mode was applied to fermentations on a 1.5–10 l scale. In cultures of a transformant strain harbouring 30 copies of the IFN expression cassette a productivity of 350 mg/l could be obtained. Various capture procedures were found to be impaired when using a standard synthetic medium for culturing. Binding to ion exchange and hydrophobic interaction matrices was regained when using a modified YPG-based culture medium.

Heterologous protein production in methylotrophic yeasts.

The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied examples of the recent past.

Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha.

We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.

Selection of Promoter Fragments Involved in Peroxisomal Biosynthesis and Metabolic Pathway Using Glucose Oxidase as a Marker

This work is concerned with the development of Hansenula polymorpha as a host for the expression of heterologous gene, glucose oxidase (GOD), from Aspergillus niger as well as searching for any promoter which may act in relation to peroxisomes. Expression of GOD was used to isolate promoter fragments from the genome of H. polymorpha. A promoterless plasmid (MERPROp 10) was constructed and Bam II-Bg/II digested total H. polymorpha DNA was ligated to the unique BamHI site of this promoterless plasmid. Transformants which contain methanol inducible promoters were screened using the plate assay which was designed for glucose oxidase enzyme (Godp) secretion. Positive colonies were hybridised against three methanol metabolic pathway promoters, MOX (Methanol oxidase), DHAS (Dihydroxyacetone synthase) and FMDH (Formate dihydrogenase). Then some of the best promoter fragments were chosen for further analyisis. Western blots to ensure secretion of Godp and Northern blots to show production of GOD mRNA under inducive and repressive conditions for the methanol metabolic pathway were carried out. Two of the inserts were sequenced and their sequences were compared to the sequences in UWGCG database The first insert is in the construct MEHPROp10-21 which is 137 base pairs (bp). This insert does not show any significant homology to the sequences in the database and contains multiple T residues. The second insert is in MEHPROPp10-43 which is 1160 bp. This insert contains two different fragments which were ligated fortuitously during ligation of totol DNA fragments. One of the inserts is a mitochondrial DNA and the other is genomic. The genomic DNA fragment of this insert is 260 bp long and shows 81 % identity with the Candida albicans carboxypeptidase Y gene and 78 % identity with the Saccharomyces cerevisiae carboxypeptidase Y gene.

High-level secretion of hirudin by Hansenula polymorpha —authentic processing of three different preprohirudins

A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MF alpha 1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.