The Gluconeogenic Enzyme Fructose-1,6-Bisphosphatase Is Dispensable for Growth of the Yeast Yarrowia lipolytica in Gluconeogenic Substrates

Abstract

The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K m (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.The growth of yeasts and other microorganisms in nonsugar carbon sources is dependent on gluconeogenesis. This pathway implicates specific gluconeogenic enzymes, such as fructose-1,6-bisphosphatase (Fbp) and phosphoenolpyruvate carboxykinase (Pck), that bypass the two physiologically irreversible steps in the glycolytic pathway, namely, phosphofructokinase and pyruvate kinase. In addition, under certain growth conditions, the enzymes from the glyoxylate cycle, isocitrate lyase (Icl) and malate synthase, are also required for the function of gluconeogenesis (Fig. 1). Growth in any nonsugar carbon source requires Fbp, which catalyzes the hydrolysis of fructose-1,6-P 2 to fructose-6-phosphate, and therefore, Fbp is a key enzyme in gluconeogenesis. The simultaneous function of specific gluconeogenic enzymes and their glycolytic counterparts would lead to futile cycles, i.e., reactions that waste ATP without yielding a net product (42). Presumably due to evolutionary pressure, a series of control mechanisms have been selected to minimize the functioning of those cycles. In the yeast Saccharomyces cerevisiae, the activities of phosphofructokinase and pyruvate kinase are controlled by a series of activators and inhibitors whose concentrations vary upon growth on sugars or nonsugar carbon sources (12,61,65). The activity of gluconeogenic enzymes is determined by the interplay of a variety of mechanisms. The transcription of the genes encoding them is subjected to catabolite repression, a complex regulatory phenomenon that involves many proteins (13,32,41). In addition, in S. cerevisiae, most of the gluconeogenic enzymes undergo catabolite inactivation (27, 48, 50), a proteolytic degradation that destroys them after glucose addition. Control by metabolites appears to be restricted to Fbp that ...

アフラトキシンB₁がMAPキナーゼ<I>PTC1</I>変異株に及ぼす遺伝子発現変化の解析

“…Moreover, HXT2 showed the same expression pattern as HXT5. Hexose is converted into glucose-6-P and is merged into glucose metabolism such as gluconeogenesis and glycolysis [18]. Not only was the hexose transporter induced, but glucose sensor genes (except for HXT1) were also induced by AFB 1 in this study.…”

Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

“…In other organisms, the YK23 orthologues might function as alternative FBPases, because they exhibit the substrate affinity and catalytic efficiency similar to that of metal-dependent FBPases. Recently, it has been shown that the FBP1 deletion strain of the non-conventional yeast Yarrowia lipolytica can grow on gluconeogenic substrates (like ethanol) suggesting the presence of another FBPase (76). The Y. lipolytica genome encodes a YK23 orthologue, YALI0D9229 (Q6C9Q2, 42% sequence identity to YK23), which has all residues important for the FBPase activity of YK23 conserved (Arg (Fig.…”

The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica

“…The glyoxylate shunt was also omitted from the reaction network. Although this pathway is only incompletely repressed on glucose in Y. lipolytica (Flores and Gancedo, 2005;Jardón et al, 2008), Blank et al (2005) found no evidence of flux through the pathway in Y. lipolytica during growth on glucose. Consistent with these results, we found that the estimated flux through the glyoxylate shunt was negligible when the pathway was included in the model (data not shown).…”

“…Inclusion of either an oxaloacetate carrier reaction (Palmieri et al, 1999;Luévano-Martínez et al, 2010) or a reversible malate transport reaction did not have any significant effect on the flux estimation, and these reactions were therefore excluded. Of the gluconeogenic enzymes, PEP carboxykinase was omitted from the model while fructose-1,6-bisphosphatase was included, as the former is glucose-repressed (albeit incompletely) in Y. lipolytica while the latter is not (Jardón et al, 2008). The glyoxylate shunt was also omitted from the reaction network.…”