Carnitine in yeast and filamentous fungi

[摘要] ENGLISH ABSTRACT:In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure thatactivated cytoplasmic or peroxisomal acetyl-groups are made available formitochondrial energy production when the cells utilise non-fermentable carbonsources. The first pathway is the glyoxylate cycle, where two activated acetyl-groupsare incorporated into each cycle, which releases a C4 intermediate. This intermediateis then transported to the mitochondria where it can enter the tricarboxylic acid cycle.The second pathway is the carnitine shuttle. Activated acetyl-groups react withcarnitine to form acetylcarnitine, which is then transported to the mitochondria wherethe acetyl group is transferred.In this study it was shown that the deletion of the glyoxylate cycle specific citratesynthase, encoded by CIT2, results in a strain that is dependent on carnitine forgrowth on non-fermentable carbon sources. Using a /::cit2 strain, mutants affected incarnitine-dependent metabolic activities were generated. Complementation of themutants with a genomic library resulted in the identification of four genes involved inthe carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitineacetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitineacetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase,encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded byYAT2. All three carnitine acetyltransferases are essential in a carnitine-dependentstrain.The dependence on exogenous carnitine of the /::cit2 strain when grown on nonfermentablecarbon sources suggested that S. cerevisiae does not biosynthesisecarnitine. Measurements using electrospray mass spectrometry confirmed thishypothesis. As a result an investigation was initiated into carnitine biosynthesis inorder to genetically engineer a S. cerevisiae strain that could endogenouslybiosynthesise carnitine.The filamentous fungus, Neurospora crassa, was one of the first organisms usedin the seventies to identify the precursor and intermediates of carnitine biosynthesis.However, it was only about twenty years later that the first genes encoding theseenzymes where characterised. Carnitine biosynthesis is a four-step process, whichstarts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysineby the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysineis cleaved to trimethylamino-butyraldehyde by thehydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehydeis dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) bytrimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaineis converted to t-carnltine by y-butyrobetaine hydroxylase (BBH).The N. crassa TMLH homologue was identified in the genome database basedon the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing,which then revealed that the gene indeed had seven introns. Functional expressionof the gene in S. cerevisiae and subsequent enzymatic analysis revealed that thegene coded for a TMLH. It was therefore named cbs-1 for carnitine biosynthesisgene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLHenzyme. Following this, a genomic copy of the N. crassa BBH homologue was clonedand functionally expressed in S. cerevisiae. Biochemical analysis revealed that theBBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene wasnamed cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependentScii? strain on non-fermentable carbon sources when y-butyrobetaine waspresent. This is the first report of an endogenously carnitine biosynthesising strain ofS. cerevisiae.The cloning of the remaining two biosynthesis genes presents particularchallenges. To date, the HTMLA has not been characterised on the molecular levelmaking the homology-based identification of this protein in N. crassa impossible.Although the TMABA-DH has been characterised molecularly, the protein sequenceis conserved for its function as a dehydrogenase and not conserved for its function incarnitine biosynthesis, as in the case of TMLH and BBH. The reason for this isprobably due to the fact that the enzyme is involved in other metabolic processes.The use of N. crassa carnitine biosynthesis mutants would probably be one way inwhich to overcome these obstacles.The !1cit2 mutant proved useful in studying carnitine related metabolism. Wetherefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. InS. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Rasproteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels.The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase(PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. Thecatalytic subunits phosphorylate a variety of regulators and enzymes involved inmetabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt?mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation ofwild-type cells grown on glycerol. However, the enhancement of proliferation wasmuch better for the !1cit2 strain grown on glycerol. In this respect, the retrograderesponse may play a role. Overexpression of RAS2 resulted in elevated levels ofintracellular citrate and citrate synthase activity. It therefore appears that thesuppression of !1cit2 by RAS2 overexpression is a result of the general upregulationof the respiratory capacity and possible leakage of citrate and/or citrate synthasefrom the mitochondria. The phenotype of RAS2 overexpression contrasts with thehyperactive RAS2val19 allele, which causes a growth defect on glycerol. However,both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but theRAS2val19dependent activation is more severe. Finally, this study implicated theRas/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in aMpk1 strain, the growth effect is blocked. However, the enhanced proliferation wasstill observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed.Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentablecarbon sources, a notion that is supported by the literature.

[发布机构] Stellenbosch University