Regulation of methabolism in facultive methylotrophs

REGULATION OF METABOLISM IN THE FACULTATIVE METHYLOTROPHS. 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). Llittle 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 identificati

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