Analysis of cloned SUC2 gene expression in continuous culture of recombinantSaccharomyces cerevisiae (original) (raw)
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Differential expression of the invertase-encoding SUC genes in Saccharomyces cerevisiae
Gene, 1992
Invertase (INV) is encoded in Saccharomyces cerevisiae by a family of genes, comprising SUCI-SUC5 and SUC7. Production of INV is highly variable, dependent on the strain and SUC gene present in the cell. The differences in INV production derive from the structure of the genes or are dependent on the genetic background of the strain. Centromeric plasmids (based on YCpSO) carrying one of the SUC genes (except SUC7) were introduced into a strain (SEY2101) lacking SUC genes. The INV produced by the transformants was dependent on the individual SUC genes, and correlated with INV mRNA levels. Plasmids in which SUC2 had been placed under control of promoters from the other SUC genes, were used to transform SEY2101 cells. The amounts of INV produced by cells carrying hybrid SUC genes were in agreement with the levels expected if the promoter controlled the expression of the SUC2 structural region. It is suggested that the differences in expression are a function of the transcription efficiency of the different SUC gene promoters, based on the divergence of 5' sequences.
Biotechnology and Bioengineering, 1991
The effects of growth rate on cloned gene product synthesis in recombinant Saccharomyces cerevisiae have been studied in continuous culture. The plasmid employed contains a yeast GAL10-CYC1 hybrid promoter directing expression of the E. coli lacZ gene. β-Galactosidase production was therefore controlled by the yeast galactose regulatory circuit, and the induction process and its effects were studied at the various dilution rates. At all dilution rates plasmid stability decreased with induction of lacZ gene expression. In some instances, two induced “steady states” were observed, the first 10–15 residence times after induction and the second after 40–50 residence times. The second induced steady state was characterized by greater biomass concentration and lower β-galactosidase specific activity relative to the first induced “steady-state.” β-Galactosidase specific activity and biomass concentration increased as dilution rate was reduced, and despite lower flow rate and plasmid stability, overall productivity (activity/L/hr) was substantially higher at low dilution rate. Important factors influencing all of the trends were the glucose and galactose (inducer) concentrations in the vessel and inducer metabolism.
Metabolic Engineering, 2011
Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5 0 coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.
Saccharomyces cerevisiae, industrial yeast isolate, has been of great interest in recent years for fuel ethanol production. The ethanol yield and productivity depend on many inhibitory factors during the fermentation process such as temperature, ethanol, compounds released as the result of pretreatment procedures, and osmotic stress. An ideal strain should be able to grow under different stress conditions occurred at different fermentation steps. Development of tolerant yeast strains can be achieved by reprogramming pathways supporting the ethanol metabolism by regulating the energy balance and detoxicification processes. Complex gene interactions should be solved for an in-depth comprehension of the yeast stress tolerance mechanism. Genetic engineering as a powerful biotechnological tool isrequired to design new strategies for increasing the ethanol fermentation performance. Upregulation of stress tolerance genes by recombinant DNA technology can be a useful approach to overcome inhibitory situations. This review presents the application of several genetic engineering strategies to increase ethanol yield under different stress conditions including inhibitor tolerance, ethanol tolerance, thermotolerance, and osmotolerance.
Biotechnol Bioeng, 1990
Protein localization i n Saccharomyces cerevisiae was studied with two plasmid systems used as a model: one containing the SUC2 structural gene fused with the MFtx1 (a-factor) promoter and signal-sequence, the other containing the entire SUC2 gene. Special emphasis was placed on the effect of promoterisignal-sequence (SUC2 vs. MFal) on the efficiency of invertase transport. The MFal and SUC2 signal sequences were capable of transporting, respectively, 83% and 77% of cloned invertase out of the cytoplasm. However, the SUC2 promoter was easier to control since a six-fold enhancement of the transported invertase activity associated with derepression was achieved in response to a glucose concentration change from 10 to 2 g/L. Cloning on a multicopy plasmid resulted in a four-fold increase in total specific invertase activity over the wild type yeast strain (which harbors a single copy of the SUC2 gene on the chromosome), whereas the chromosomal site was more efficient for invertase localization yielding over 90% of the invertase transported out of the cytoplasm. Transient experiments done with the SUC2 signal-sequence-containing plasmid showed that the specific invertase activity i n the periplasmic space reached a maximum three hours after derepression, then decreased very slowly with an accompanying gradual increase in invertase activity in the growth medium.
Biotechnology and Bioengineering, 1989
Protein localization i n Saccharomyces cerevisiae was studied with two plasmid systems used as a model: one containing the SUC2 structural gene fused with the MFtx1 (a-factor) promoter and signal-sequence, the other containing the entire SUC2 gene. Special emphasis was placed on the effect of promoterisignal-sequence (SUC2 vs. MFal) on the efficiency of invertase transport. The MFal and SUC2 signal sequences were capable of transporting, respectively, 83% and 77% of cloned invertase out of the cytoplasm. However, the SUC2 promoter was easier to control since a six-fold enhancement of the transported invertase activity associated with derepression was achieved in response to a glucose concentration change from 10 to 2 g/L. Cloning on a multicopy plasmid resulted in a four-fold increase in total specific invertase activity over the wild type yeast strain (which harbors a single copy of the SUC2 gene on the chromosome), whereas the chromosomal site was more efficient for invertase localization yielding over 90% of the invertase transported out of the cytoplasm. Transient experiments done with the SUC2 signal-sequence-containing plasmid showed that the specific invertase activity i n the periplasmic space reached a maximum three hours after derepression, then decreased very slowly with an accompanying gradual increase in invertase activity in the growth medium.
Biotechnology and Bioengineering, 1995
The effects of temperature on the kinetics and efficiency of secretion of cloned invertase were investigated in a recombinant yeast system. This system consisted of the baker's yeast Saccharomyces cerevisiae (SEY2102) transformed with the 2p-based plasmid pBR58 which contains the entire SUC2 gene including the promoter, signal sequence, and structural gene. The recombinant yeast produces the naturally secreted yeast enzyme invertase. In transition experiments done at temperatures ranging from 25" to 45°C. the maximum invertase level and secretion rate exhibited maxima of 5.5 UlmL . OD and 4.6 U/mL . OD per hour, respectively, at 35°C. Experiments involving the use of cycloheximide showed that it took approximately 15 min for secreted invertase to move through the secretion pathway, which held 0.4 U/mL. OD of specific activity. 0 1995 John Wiley & Sons, Inc.
Microbial Cell Factories Switching the mode of sucrose utilization by Saccharomyces cerevisiae
Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H + symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.
Enzyme and Microbial Technology, 2002
The production of ethanol from starch has been investigated in a genetically modified Saccharomyces cerevisiae strain, YPB-G, which secretes a bifunctional fusion protein that contains both the Bacillus subtilis ␣-amylase and the Aspergillus awamori glucoamylase activities. The effects of a number of environmental factors on starch degradation, ethanol production, and plasmid stability have been assessed in batch culture. These include initial glucose supply, colony selection methodology prior to inoculation, and medium formulation. Cultures containing 40 g/l starch were observed to degrade starch effectively and produce higher amounts of ethanol in shorter periods. The provision of glucose in the growth medium during the early phases of fermentation resulted in faster growth and higher ethanol productivities. YE-Salts medium was found to support plasmid-containing cells throughout the whole fermentation; only 15% of the recombinant cells had lost the plasmid content by the end of the fermentation of 120 h. Fed-batch cultures produced high yields of ethanol on starch (0.46 g ethanol/g substrate) through the longer production period.