Oleg Mosin

Department of Biotechnology. M.V. State Academy of fine Chemical Technology, Moscow.

Fultative methylotrophs can be found abundantly among methylotrophic organisms employing the Calvin cycle, the serine pathway, or the XuMP cycle for the assimilation of C1-compounds (O. Mosin, 1998). It is only in recent years, however, that scientists have succeeded in the isolation of a number of versatile RuMP cycle bacteria. These facultative RuMP cycle methylotrophs are found almost exclusively among Gram-positive bacteria. Representatives are various bacilli, coryneform bacteria, and actinomycete species (Dijkhuizen et al., 1992; Dijkhuizen, 1993).

Most of these methylotrophs grow on methylated amines and only few use methanol as sole carbon- and energy source for growth. Currently scientists are engaged in a detailed physiological, biochemical and genetic analysis of pathways of primary metabolism in the actinomycetes. These bacteria are a very versatile methanol-utilizing organisms, employing the fructose-bisphosphate aldolase cleavage variant of the RuMP cycle of formaldehyde fixation (Hazeu et al., 1983; de Boer et al., 1990).

Little is known at the moment about primary metabolism in actinomycetes. Over the years attention of scientisys has been devoted to the analysis of the genetics of pathways for secondary metabolite synthesis, and the screening and testing for new applications of the enormous variety of secondary metabolites (e.g. antibiotics) produced by many actinomycetes (L. Dijkhuizen, 1998).

Secondary metabolites, however, are derived from intermediates of central metabolic pathways, including those of glucose utilization and aromatic ammo acid biosynthesis. Knowledge about primary metabolism is considered to be important, especially for the further improvement of processes for the fermentative production of primary and secondary metabolites (Dijkhuizen, Harder, 1992; Dijkhuizen, 1993).

Methanol metabolism

Methanol oxidation in Gram-positive methylotrophic bacteria involves enzymes clearly different from those in Gram-negative bacteria (a periplasmic PQQ-dependent methanoi dehydrogenase; EC 1.1.99.8) and in yeasts (a peroxisomal alcohol oxidase; EC 1.1.3.13). All thermotolerant, methanol-utilizing strains of Bacillus methanolicus studied were found to possess a cytoplasmic NAD-dependent methanoi dehydrogenase (MDH; EC 1.1.1.1) (Arfman et al., 1989; Dijkhuizen, Arfman, 1990), which is strongly stimulated by a specific (activator) protein (Arfman et al., 1991). No NAD-dependent MDH activity could be detected in A. methanolica. Instead, methanoi oxidation in this organism resulted in concomitant reduction of N,N-dimethyl-4-nitrosoaniline (NDMA). The corresponding cytoplasmic enzyme has been designated methanol NDMA oxidoreductase (MNO) (Bystrykh et al., 1993). NDMA is known to reoxidize pyridine nucleotides which are tightly bound to the active centers of dehydrogenases (Dunn, Bernhard, 1971; Kovaf et al, 1984).

Analysis of the quaternary protein stuctures of the purified B. methanolicus MDH (subunit M, 43,000) (Vonck et al., 1991) and the A. methanolica MNO enzyme (subunit M,. 50,000) (Bystrykh et al., 1993b) by electron microscopy and image processing revealed similar decameric structures with five-fold symmetry. The three proteins are also similar with respect to their metal composition (1-2 Zn- and Mg-ions per subunit) and the presence of a bound NAD(P)(H) cofactor in each subunit. The amino acid sequences of these enzymes, deduced from the cloned genes (de Vries et al., 1992), show that these proteins share a high degree of identity and belong to the Family III alcohol dehydrogenases. The classical dinucleotide binding fold for NAD(P)(H) is not present in these proteins.

In addition to the methanol NDMA oxidoreductase activity of MNO, also dye (DCPIP and MTT)-linked methanol dehydrogenase activities can be detected reproducibly in crude extracts of A. methanolica (van Ophem et al., 1991; Bystrykh et al., 1995). These dye-linked methanol dehydrogenases appear to represent the overall activities of multienzyme systems. The biochemistry of methanol oxidation in Gram-positive bacteria is complex and both MDH of B. methanolicus (Arfman et al., 1991) and MNO of A. methanolica (Bystrykh et al., 1995) in vivo require additional proteins, most likely participating in the transfer of reducing equivalents from NAD(P)H cofactors to NAD coenzyme and/or to the electron transport chain. Tthe gene encoding the activator protein of B. methanolicus has been cloned and characterized (L. Dijkhuizen). No clear similarities were observed, however, with any other protein sequence available in databases (H.J. Kloosterman, 1997).

Glucose metabolism

Studies of glucose metabolism in A. methanolica revealed the presence of the normal set of glycolytic pathway and pentose phosphate cycle enzymes, with a few exceptions. During growth on glucose, glycolysis involved a PPi-dependent phosphofructokinase (PFK) which was completely insensitive to allosteric control (Alves et a!., 1994). The amino acid sequence deduced from the cloned gene, nevertheless, revealed a strong similarity with ATP-dependent PFK enzymes from various other sources (Bacillus stearolhermophilus) (A. Alves, 1994).

Screening of other actinomycetes revealed the presence of similar PPi-dependent PFK enzymes in other members of the family Pseudonocardiacea but not in other actinomycetes, e.g. Streptomyces coelicolor A3, which thus may reflect an evolutionary signature. Protein purification studies revealed a second remarkable feature, namely the presence of a 2,3-bisphosphoglycerate activated 3-phosphoglycerate mutase in A methanolica, which is normally present in eukaryotes only.

Glucose metabolism is regulated at the level of the PPi-dependent PFK enzyme synthesis, at the phosphoglycerate mutase activity and pyruvate kinase activity steps. The later step involves an allosteric enzyme regulated via feedback inhibition by ATP and Pi, and activated by AMP (Alves et al., 1994).

Mixed substrate experiments in batch cultures with glucose plus methanol resulted in simultaneous utilization of these substrates (Lubbert Dijkhuizen, 1996). The presence of glucose repressed synthesis of the RuMP cycle enzymes HPS and HPI, and methanol was only utilized as an energy source. Similar results were found following addition of formaldehyde to a culture growing on glucose. The synthesis of enzymes involved in methanol dissimilation and assimilation in A. methanolica was regulated differently. Methanol and/or formaldehyde induce the synthesis of these enzymes, but under carbon-excess conditions their inducing effect on HPS and HPI synthesis is overruled completely by glucose (L. Dijkhuizen). Repression of HPS and HPI was of minor significance following addition of methanol to glucose-, acetate- and ethanol-limited chemostat cultures (de Boer et al., 1990).

Biosynthesis of aromatic ammo acids

Using brief ultrasonication treatments to obtain single cells of the actinomycete A. methanolica and simple protocols for the identification of metabolic lesions, the scientists were succeeded in the isolation and characterization of a large number of auxotrophic mutants, covering virtually every step in aromatic amino acid biosynthesis (Euverink et al., 1995, O.V.Mosin, 1998).

Characterization of these mutants revealed that synthesis of L-phenylalanine and L-tyrosine proceeds via single pathways, involving phenylpyruvate and L-arogenate as intermediates, respectively.

Dehydroquinate (DHQ) dehydratase mutants invariably were also blocked in DHQ synthase, suggesting common control elements or gene clustering (Euverink et al., 1992). No mutants were obtained in 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase and prephenate aminotransferase, suggesting the presence of isoenzymes, as has been reported for various other organisms. This was subsequently confirmed in biochemical studies (G.J.W. Euverink).

L-Phenylalanine aminotransferase (aroAT) catalyzes the last step in L-phenylalanine biosynthesis. Enzyme purification studies showed the presence of a minor aroATI activity, coeluting with branchd chain aminotransferase, and a major aroATII activity using both L-phenylalanine and L-tyrosine as substrates (Abou-Zeid et al., 1995). L-phenylalanine auxotroph, strain GH141, was subsequently identified as deficient in the aroATII enzyme. In this strain the minor aroATI activity is responsible for L-phenylalanine biosynthesis; its low specific activity explains the leaky phenotype. Interestingly, strain GH141 also had lost the ability to grow on L-tyrosine as carbon source. Apparently the aroATII protein of A. methanollca is functioning in both L-phenylalanine biosynthesis and in L-tyrosine catabolism (Abou-Zeid et al., 1995; Euverink et al., 1995b).

Three species of prephenate aminotransferase (PpaATI-III), the first enzyme of L-tyrosine biosynthesis, were chromatographically resolved. PpaATI and PpaATII coeluted with aroATI (branched chain aminotransferase) and with aspartate aminotransferase, respectively. PpaATIII appeared highly specific for prephenate and thus appears to be the main in vivo PpaAT activity (Abou-Zeid et al., 1995). The presence of these three isoenzymes with PpaAT activity explains the failure to isolate L-tyrosine auxotrophic mutants in this step.

The product of the PpaAT activity with prephenate, arogenate, is subsequently converted into L-tyrosine by an NAD(P)-dependent L-arogenate dehydrogenase. Numerous mutants blocked in this step have been isolated, confirming the presence of a single pathway from prephenate towards L-tyrosine in A. methanolica (Abou-Zeid et al., 1995; Euverink et al., 1995b).

The single DAHP synthase enzyme species that can be detected in wild type A, methanolica is sensitive to cumulative feedback inhibition by all three aromatic amino acids. Partially purified enzyme showed apparent K values of 3, 160 and 180 mM for L-tryptophan, L-phenylalanine and L-tyrosine, respectively. The aromatic amino acids displayed competitive inhibition with respect to E4P. L-Tryptophan and E4P showed uncompetitive and competitive inhibition towards PEP, with apparent K, values of 11 and 530 mM, respectively (de Boer et al., 1989). Chorismate mutase functions in L-phenylalanine and L-tyrosine biosynthesis. The activity of the single chorismate mutase detectable in extracts of the wild type organism was inhibited by both L-phenylalanine and L-tyrosine (apparent K values of 60 and 35 mМ, respectively) (de Boer et al, 1989).

Prephenate dehydratase, an enzyme specifically involved in L-phenylalanine biosynthesis, was purified to homogeneity and characterized as a 150 kDa homotetrameric protein with a subunit size of 34 kDa (L. Dijkhuizen, 1996). Kinetic studies showed that this enzyme is allosterically inhibited by L-phenylalanine and activated by L-tyrosine (Euverink et al., 1995).

L-Phenylalanine caused an increase in the S for prephenate and a decrease in the F. L-Tyrosine caused a decrease in the S for prephenate and an increase in the V (Euverink et al., 1995a). Anthranilate synthase, the first enzyme in the L-tryptophan specific branch, was strongly inhibited by L-tryptophan (L. Dijkhuizen). Addition of the aromatic amino acids, either separately or in combinations, did not result in significant repression of the synthesis of these enzymes (de Boer et al., 1989).

o-Fluoro- and p-fluorophenylalanine inhibited the activities of chorismate mutase and prephenate dehydratase in vitro (de Boer et al., 1990b). Many analog-resistant mutant methylotrophic strains had become unable to grow on L-phenylalanine as carbon source and most likely had lost phenylalanine(analog) transport systems. Several mutants were found to possess either a chorismate mutase or a prephenate dehydratase enzyme which had become completely insensitive to L-phenyalanine(analog) inhibition. Some prephenate dehydratase mutants were still activated by tyrosine, while others had become insensitive to both phenylalanine and tyrosine (Euverink et al., 1995).

Gene cloning systems for A. methanolica

A. methanolica was found to possess a conjugative plasmid (рМЕАЗОО) which is able to integrate into the chromosome at a specific site (Vrijbbed et al., 1994). Recently scientists have completed the nucleotide sequence analysis of рМЕА3ОО-plasmide, revealing a total of twenty open reading frames with relatively little untranslated intervening sequences (L. Dijkhuizen, 1996). The overall G+C content of рМЕАЗОО iwas found to be 69%. This high value is characteristic for actinornycetes.

Suitable рМЕАЗОО derivatives were used by scientists as a basis for the construction of E. coli - A. methanollca shuttle vectors, based on the pHSS6 coffil replicon (L. Dijkhuizen, 2000). It was reported by L. Dijkhuizen, that cloning of genes involved in glucose, methanol utilization, and in aromatic amino acid biosynthesis.

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