Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.     

Allelism of IMP1 and GAL2 genes of Saccharomyces cerevisiae.

Cloning and characterization of the previously described Saccharomyces cerevisiae IMP1 gene, which was assumed to be a nuclear determinant involved in the nucleomitochondrial control of the utilization of galactose, demonstrate allelism to the GAL2 gene. Galactose metabolism does not necessarily involve the induction of the specific transport system coded by GAL2/IMP1, because a null mutant takes up galactose and grows on it. Data on galactose uptake are presented, and the dependence on ATP for constitutive and inducible galactose transport is discussed. These results can account for the inability of imp1/gal2 mutants to grow on galactose in a respiration-deficient background. Under these conditions, uptake was affected at the functional level but not at the biosynthetic level.     

Characterizing the memory of the GAL regulatory network in Saccharomyces cerevisiae

Genetic regulatory networks respond dynamically to perturbations in the intracellular and extracellular environments of an organism. The GAL system in the yeast Saccharomyces cerevisiae has evolved to utilize galactose as an alternative carbon and energy source, in the absence of glucose in the environment. We present a dynamic model for GAL system in Saccharomyces cerevisiae, which includes a novel mechanism for Gal3p activation upon induction with galactose. The modification enables the model to simulate the experimental observation that in absence of galactose, oversynthesis of Gal3p can also induce the GAL system. We then characterize the memory of the GAL system as the domain of attraction of the steady states.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.     

Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae

Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon alpha, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer.     

Iron requirement for GAL gene induction in the yeast Saccharomyces cerevisiae

Iron is an essential nutrient. Its deficiency hinders the synthesis of ATP and DNA. We report that galactose metabolism is defective when iron availability is restricted. Our data support this connection because 1) galactose-mediated induction of GAL promoter-dependent gene expression was diminished by iron limitation, and 2) iron-deficient mutants grew slowly on galactose-containing medium. These two defects were immediately corrected by iron replacement. Inherited defects in human galactose metabolism are characteristic of the disease called galactosemia. Our findings suggest that iron-deficient galactosemic individuals might be more severely compromised than iron-replete individuals. This work shows that iron homeostasis and galactose metabolism are linked with one another.