Effect of HXT 1 and HXT 7 hexose transporter overexpression on wild-type and lactic acid producing Saccharomyces cerevisiae cells

Background: Since about three decades, Saccharomyces cerevisiae can be engineered to efficiently produce proteins and metabolites. Even recognizing that in baker’s yeast one determining step for the glucose consumption rate is the sugar uptake, this fact has never been conceived to improve the metabolite(s) productivity. In this work we compared the ethanol and/or the lactic acid production from wild type and metabolically engineeredS. cerevisiae cells expressing an additional copy of one hexose transporter. Results: Different S. cerevisiae strains (wild type and metabolically engineered for lactic acid production) were transformed with the HXT1 or the HXT7 gene encoding for hexose transporters. Data obtained suggest that the overexpression of an Hxt transporter may lead to an increase in glucose uptake that could result in an increased ethanol and/or lactic acid productivities. As a consequence of the increased productivity and of the reduced process timing, a higher production was measured. Conclusion: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is a well established concept. A high production relies on a multi-factorial system. We showed that by modulating the first step of the pathway leading to lactic acid accumulation an improvement of about 15% in lactic acid production can be obtained in a yeast strain already developed for industrial application. Background Natural Saccharomyces cerevisiae cells have long been utilized as very efficient biocatalysts, thanks to their native enzymatic capabilities. Ethanol, single cell proteins, flavours and fragrances are among the most traditional examples. Since about three decades budding yeast can also be engineered and has been used to efficiently produce simple as well as complex molecules. Prominent examples are proteins with pharmaceutical applications, industrial enzymes, organic acids, new bio-fuels, biopolymers, vitamins and steroids, in a single fermentation step [1-8]. Glucose, either derived from starch and/or cellulosic materials, is the main carbon and energy source today available. An economically sustainable bioprocess leading to the production of a homologous or heterologous low molecular compound requires a high yield (grams of product obtained by gram of substrate), high production titer (g/ L) and high productivity (g/L/h) values. It has been shown that high yields and high production titres can be obtained by recombinant redirection of the carbon flow towards the desired compound. In this respect, prominent examples are the production of lactic-, pyruvicand malicacid, glycerol and resveratrol [9-13]. Theoretically, high productivities could be obtained by increasing the carbon consumption rates itself (i.e., essentially the glycolytic flux rate). It should be also underlined that an increased productivity, and therefore a reduction of the process duration, is not only implying a reduction in terms of costs. Cell factory viability (and therefore production) has also to be taken into account: very often the production process involves a stressful environment, leading to cell death during * Correspondence: danilo.porro@unimib.it Università degli Studi di Milano-Bicocca, Department of Biotechnology and Bioscience, P.zza della Scienza 2, 20126 Milano, Italy Rossi et al. Microbial Cell Factories 2010, 9:15 http://www.microbialcellfactories. com/content/9/1/15 © 2010 Rossi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. fermentation, as in the case of ethanol production, gradually reducing cell viability and thereby biocatalyst concentration [14]. Glucose transport, hexose phosphorylation, phosphofructokinase and pyruvate kinase activities have all been proposed to play central roles in the control of glycolysis flux rates [15-20]. Individual or simultaneous overproduction of glycolytic enzymes resulted either in no increases in glycolytic flux or in only incremental increases [21-25]. Furthermore, attempts to correlate glycolytic flux with enzyme levels under different physiological conditions have generally failed [26-28]. This is likely because the control of glucose catabolism is distributed over several different metabolic controls; in this context, glucose transport has been suggested to be one of the most important players [29,30]. Glucose transport in S. cerevisiae relies on a multi-factorial uptake system. More in details, the uptake of glucose by S. cerevisiae is controlled by multiple hexose transporters (Hxts) [31]. At least 20 HXT genes encoding these transporters have been identified [31,32]. Many and different studies were done to determine the respective biochemical features of these transporters (affinity and capacity), as well as to construct strains deleted in one or more HXT genes and to construct chimera proteins combining affinity and capacity of different transporters [33-35]. Remarkably, Otterstedt et al. [33] showed that a simple manipulation of the glucose uptake can strongly alter the mode of metabolic control. Essentially, the various hexose transporters differ considerably in substrate specificity and affinity. Hxt1 and Hxt3 are low-affinity transporters (Km for glucose, ~50 to 100 mM), Hxt4 is a moderately low-affinity transporter, and Hxt2, Hxt6, and Hxt7 are high-affinity transporters (Km for glucose, ~1 to 4 mM) [36,37]. Hxt5 has been shown to be a transporter with intermediate to high affinity [38,39]. Both highand low-affinity carriers have been shown to have a higher affinity for glucose than for fructose [37]. Analyses of the effect of HXT gene inactivation have shown that the hexose carriers Hxt1 to Hxt7 are the main transporters [37]. In this respect, it has been already shown that the ethanol (and CO2) productivity and yield (grams of ethanol produced per gram of glucose consumed) can be improved by overexpression of HXT1 transporter in S. cerevisiae [40-43]. S. cerevisiae has long been studied for the production of organic acids like lactic, ascorbic, pyruvic and malic [[4,8,11] and [44]]. Indeed, yeast can grow and survive at low pH values, avoiding the accumulation of the respective salts [4,11]. Here we show improved lactic acid productivities, induced by an increase of the glucose consumption rate. Hxt1 and Hxt7 have been selected for this study. In spite of their different biochemical properties, the overexpression of HXT1 or HXT7 genes does lead to very similar results in the tested conditions. Moreover, we demonstrate that the increase of the glucose consumption rate has a positive effect not only in respect to microbial productivity and metabolite production, but also on biomass accumulation. Said phenomenon is more or less evident in respect to the yeast background.