Abstract
The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).
Highlights
The oleaginous yeast Yarrowia lipolytica is an industrial micro-organism used for production of complex biological compounds such as tailor-made lipids [1,2], biofuel precursors [3] or industrial and therapeutic enzymes [4,5], as well as more compact molecules such as acidifying agent citric acid [6] and low-calorie sweeteners, including erythritol and mannitol [7,8]
In this study we investigated the role of hexose transporters and hexokinase in the preferential uptake of glucose over fructose in Y. lipolytica
We first examined the kinetic properties of Yht1, Yht3 and Yht4 in a heterologous host and through overexpression of hexose transporters in combination with native YlHxk1 or heterologous SpHxk1, we showed rearrangement of glucose and fructose utilization profiles
Summary
The oleaginous yeast Yarrowia lipolytica is an industrial micro-organism used for production of complex biological compounds such as tailor-made lipids [1,2], biofuel precursors [3] or industrial and therapeutic enzymes [4,5], as well as more compact molecules such as acidifying agent citric acid [6] and low-calorie sweeteners, including erythritol and mannitol [7,8]. Substrates can be chosen among hydrophobic carbon sources (n-alkanes, oils and fats) and hydrophilic ones, such as glycerol, monosaccharides and organic acids. From the latter, glycerol is a superior carbon source for Y. lipolytica, due to its rapid and preferred use over glucose when supplied in a mixture [9,10,11]. Many yeast species, including Saccharomyces cerevisiae, show a preferential uptake of glucose over fructose in mixed sugar media [18]. In order to enter the metabolism, monosaccharides need first to be taken up by the cell, a step which requires transporters, to be activated through phosphorylation by hexose kinases
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