The first question we asked in this study was whether the recently reported respiratory phenotype of engineered S. cerevisiae
) could be transferred to other strains of this yeast, and the answer is clearly yes. We chose to study the transferability of the respiratory phenotype to a mutant strain lacking HXT1
derived from the enological strain V5, which was obtained by sporulation of an industrial wine yeast strain (19
). Many laboratory strains perform poorly under enological conditions and cannot, as discussed by Luyten et al. (19
), completely consume the sugars present in grape must. In contrast, the V5 strain performs well under such conditions, which are characterized by the occurrence of a large range of sugar concentrations.
It is of industrial relevance to test if it is possible to transfer the TM6*
construct to a genetically simpler mutant background than a complete hexose transporter null strain. Since deletion of the genes HXT1-HXT7
has been reported to abolish hexose transport in laboratory yeast strains (33
) and more recently also in the enological V5 strain (19
), this background was chosen. After transformation of the TM6*
chimeric gene to the V5 hxt1
strain, the resulting V5.TM6*P strain grew well on glucose as the sole carbon and energy source.
The respiratory phenotype of the V5.TM6*P strain was clearly demonstrated by the occurrence of only one growth phase during batch cultivations in defined minimal medium with glucose as the sole carbon and energy source. The ethanol yield did not increase with increasing external glucose concentrations; instead, it was independent of the external glucose concentration, as illustrated by comparing growth on 2%, 5%, and 10% (data not shown) glucose. As for the KOY.TM6*P strain, only negligible amounts of by-products other than ethanol were produced.
Despite the fact that the glucose flux was reduced to 30% that of its parental strain, which is also in accordance with the relationship between KOY.TM6*P and its parental strain (27
), the V5.TM6*P strain completed its growth cycle at least as rapidly as the wild-type strain. In other words, the TM6*
strain consumed the external carbon supply (glucose) as fast as the wild-type strain (taking both glucose and ethanol into account). What may be even more interesting to potential industrial users is that the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain, and the log phase was equally short for the two strains. Even more relevant is that the biomass yield was increased by 50% in the mutant compared to its parental strain (Fig. ). Biomass formation rather than the formation of by-products like ethanol, glycerol, and acetate is considered a necessity for efficient heterologous protein production.
Depending on the kind of industrial process to be employed, different substrates will be used, including molasses, in which the major sugar component is sucrose and which gives rise to both glucose and fructose after hydrolysis. It was therefore relevant to test the respiratory phenotype on fructose, particularly since the same hexose transporters are used for both glucose and fructose (32
). Growth of both mutants, V5.TM6*P and KOY.TM6*P, gave rise to higher ethanol yields on fructose than on glucose. About a fourfold increase in ethanol yield was obtained on fructose (Fig. and data not shown), while both wild-type strains produced equal amounts of ethanol on fructose and glucose. However, the mutants still produced only 50% of the ethanol produced by the corresponding wild types. The difference in ethanol yield between glucose and fructose for the TM6*
strains may be explained by different uptake kinetics for the two sugars. This has previously been seen during studies of native Hxts (32
). However, fructose also causes a different degree of catabolite repression. The likely reason is Hxk1, whose expression is repressed by glucose but which is more a fructo- than a glucokinase (6
). This opens possibilities to further reduce ethanol production on fructose as the carbon and energy source if this would be industrially advantageous.
Although the reduced glucose uptake capacity of the TM6*
strains is a strong candidate for the low-ethanol-producing phenotype (8
), this may not be the sole mechanism. An increased respiratory capacity, as shown for the KOY.TM6*P strain compared to its wild type, may additionally amplify the phenotype (27
). The respiratory capacity may be further increased during conditions of coexistence of glucose and ethanol. For example, different respiratory rates were reported for a hexokinase mutant, the hxk2Δ
mutant, with by far the highest rate on a mixture of glucose and ethanol, in line with data reported from Rigoulet's laboratory (1
). The hxk2Δ
mutant was also argued to coconsume glucose and ethanol, a feature which in the present study is clearly illustrated for the V5.TM6*P strain and was also recently shown for the KOY.TM6* strain (9
). Since the ethanol yield is equal on 2% and 5% external glucose, larger absolute ethanol amounts result when the external glucose supply is increased. This means that coconsumption with glucose of the small amounts of ethanol produced is clearly visible with 5% glucose (Fig. ), in contrast to the case with 2% glucose. The regulatory mechanism for the coconsumption of glucose and ethanol with a simultaneous increased respiratory rate may have been developed during evolution under conditions when external sugar concentrations became low while ethanol was simultaneously accumulated.
The KOY.TM6*P strain has been shown by expression analysis at the mRNA level not to exert a completely glucose derepressed phenotype during growth on glucose (9
). The observation that the TM6*
strains maintain characteristics typical of glucose-grown S. cerevisiae
cells was also strengthened in this study by 2D-PAGE analysis. Attempts to correlate glycolytic flux with levels of enzymes during different physiological conditions have generally failed (5
). In this study, we also did not find evidence for altered protein levels controlling the glycolytic flux, with the obvious exception of the hexose transport machinery.
In spite of many years of research, the initial signal for glucose repression remains to be deduced. We believe that the TM6*
strains, together with other strains with different hexose transport capacities (8
), will be valuable tools in this search. The respiratory phenotypes of the TM6*
strains in this study, together with the high respiratory capacity and considerably less glucose repression of the KOY.TM6*P strain (9
) with high external glucose, indicate that external glucose is not the initial signal for the different targets of glucose repression, in line with data from other laboratories (32
). Although published data are often conflicting, some potential candidates for the initial glucose repression signal are intracellular glucose, the ATP/AMP ratio (42
), hexokinase 2 (different roles have been suggested [6
]), intracellular (glycolytic) metabolite concentrations (2
), and (indirectly) the glycolytic rate (32
). However, the rate per se cannot possibly be the initial glucose repression signal. Instead, the glycolytic rate may be responsible for signaling via some protein modification(s) or low-molecular-weight metabolites. In all this, the activity of the hexokinase reaction seems to play a central role in mediating glucose repression by both the short-term (minutes) response via any of the hexokinases and the long-term (hours) response via Hxk2 (6
). Since glucose repression seems to correlate with the activity of the hexokinase reaction, it seemed logical to analyze metabolites such as glucose (not performed in this study), G6P, and ATP. However, no significant differences in ATP concentrations were observed between V5.TM6*P and the V5 wild type. G6P concentrations in the mutant varied from about 6 mM down to levels below 1 mM, while the corresponding values in the wild type were more stable and ranged from 2 to 3 mM. It is therefore difficult to imagine that any of these metabolites are the sole triggering agents for glucose repression. The large variability in G6P concentration observed for the mutant might seem strange, but it should be noted that the lowest concentrations were recorded during a phase of simultaneous consumption of glucose as well as ethanol. Most probably, the main control of the glycolytic rate in the mutant strain(s) is exerted by the sugar uptake system (8
), but other control mechanisms may be operative as well. For instance, the low concentration of FBP may contribute, since this is a known allosteric activator of the pyruvate kinase reaction (15
). However, the identity of the initial glucose repression signal(s) will be further elucidated by the use of the TM6*
strains and other strains covering a range of glycolytic rates at high external sugar concentrations (8