The present review focuses on microbial type I fatty acid synthases (FASs), demonstrating their structural and functional diversity. Depending on their origin and biochemical function, multifunctional type I FAS proteins form dimers or hexamers with characteristic organization of their catalytic domains. A single polypeptide may contain one or more sets of the eight FAS component functions. Alternatively, these functions may split up into two different and mutually complementing subunits. Targeted inactivation of the individual yeast FAS acylation sites allowed us to define their roles during the overall catalytic process. In particular, their pronounced negative cooperativity is presumed to coordinate the FAS initiation and chain elongation reactions. Expression of the unlinked genes, FAS1 and FAS2, is in part constitutive and in part subject to repression by the phospholipid precursors inositol and choline. The interplay of the involved regulatory proteins, Rap1, Reb1, Abf1, Ino2/Ino4, Opi1, Sin3 and TFIIB, has been elucidated in considerable detail. Balanced levels of subunits α and β are ensured by an autoregulatory effect of FAS1 on FAS2 expression and by posttranslational degradation of excess FAS subunits. The functional specificity of type I FAS multienzymes usually requires the presence of multiple FAS systems within the same cell. De novo synthesis of long-chain fatty acids, mitochondrial fatty acid synthesis, acylation of certain secondary metabolites and coenzymes, fatty acid elongation, and the vast diversity of mycobacterial lipids each result from specific FAS activities. The microcompartmentalization of FAS activities in type I multienzymes may thus allow for both the controlled and concerted action of multiple FAS systems within the same cell

6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P . patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'-and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Succharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS-and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. putulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.

ABSTRACT

The yeast gene, ACPI, encoding the mitochondrial acyl carrier protein, was deleted by gene replacement. The resulting acpl-deficient mutants had only 5-10% of the wild-type lipoic acid content remaining, and exhibited a respiratorydeficient phenotype. Upon meiosis, the lipoate deficiency cosegregated with the acpl deletion. The role of ACPI in longchain fatty acid synthesis was studied in fasl and fas2 null mutants completely lacking cytoplasmic fatty acid synthase. When grown on odd-chain (13:0 and 15:0) fatty acids, these cells showed less than 1% of C-16 and C-18 acids in their total lipids. Mitochondrial ACP is therefore suggested to be involved with the biosynthesis of octanoate, a precurser to lipoic acid.

A systematic search for upstream controlling elements necessary for efficient expression of the yeast fatty acid synthase genes FAS1 and FAS2 revealed identical activation sites, UASFAS, in front of both FAS genes. The individual element confers, in a heterologous yeast test system, an approximately 40‐fold stimulation of basal gene expression. The UASFAS motifs identified have the consensus sequence TYTTCACATGY and function in either orientation. The same sequence motif is found in the upstream regions of all so far characterized yeast genes encoding enzymes of phospholipid biosynthesis. In gel retardation assays, a protein factor, Fbf1 (FAS binding factor), was identified which interacted with UASFAS. The UASFAS motif proved to be an inositol/choline responsive element (ICRE) conferring strict repression by exogenous inositol and choline on a heterologous reporter gene. Its core sequence perfectly matches the CANNTG motif typical of basic helix‐loop‐helix DNA‐binding proteins. In contrast to the individual UASFAS element, the intact yeast FAS promoters are not significantly influenced by inositol and choline, and thus allow nearly constitutive fatty acid synthase production. Available evidence suggests that additional cis‐ and trans‐acting elements, other than UASFAS and Fbf1, are involved in this constitutive FAS gene expression.