lacZ Research Articles

Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing.

The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase activity in the yeast strain Kluyveromyces lactis GG799, which expresses a native invertase and a heterologous FFT from Aspergillus terreus. To maximize transferase activity while minimizing native invertase activity in a scaled-up process, we evaluated two reactor systems in terms of oxygen input capacity in relation to the cell dry weight. In the 0.5-L reactor, we found that galactose was superior to lactose for the induction of the LAC4 promoter, and we optimized the induction time and induction to carbon source ratio using a response surface model. Based on the critical parameter of oxygen supply, we scaled up the process to 7 L using geometric similarity and a higher oxygen transport rate, which boosted the transferase activity by 159%. To favor the forward reaction even more, we deleted the native invertase gene by CRISPR/Cas9 genome editing and compared the ΔInv mutant to the original production strain in batch and fed-batch reactions. In fed-batch mode, we found that the ΔInv mutant increased the transferase activity by a further 66.9%. The enhanced mutant strain therefore provides the basis for a highly efficient and scalable fed-batch process for the production of FOS.

Characterization of Gene Repression by Designed Transcription Activator-like Effector Dimer Proteins

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.

A practical guide to mechanistic systems modeling in biology using a logic-based approach.

Mechanistic computational models enable the study of regulatory mechanisms implicated in various biological processes. These models provide a means to analyze the dynamics of the systems they describe, and to study and interrogate their properties, and provide insights about the emerging behavior of the system in the presence of single or combined perturbations. Aimed at those who are new to computational modeling, we present here a practical hands-on protocol breaking down the process of mechanistic modeling of biological systems in a succession of precise steps. The protocol provides a framework that includes defining the model scope, choosing validation criteria, selecting the appropriate modeling approach, constructing a model and simulating the model. To ensure broad accessibility of the protocol, we use a logical modeling framework, which presents a lower mathematical barrier of entry, and two easy-to-use and popular modeling software tools: Cell Collective and GINsim. The complete modeling workflow is applied to a well-studied and familiar biological process-the lac operon regulatory system. The protocol can be completed by users with little to no prior computational modeling experience approximately within 3h.

Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible Pgrac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the β-galactosidase (bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The Pgrac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated Pgrac01-bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, Pgrac100-bgaB and Pgrac212-bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the Pgrac212-bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the Pgrac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis.

Subpopulation behaviors in lactose metabolism by Streptococcus mutans.

When Streptococcus mutans is transferred from a preferred carbohydrate (glucose or fructose) to lactose, initiation of growth can take several hours, and substantial amounts of glucose are released during growth. Here, S. mutans strains UA159 and GS-5 were examined for stochastic behaviors in transcription of the lac operon. Using a gfp reporter fusion, we demonstrated that induction of the lac operon occurs in only a fraction of the population, with prior exposure to carbohydrate source and strain influencing the magniture of the sub-population response. Lower glucokinase activity in GS-5 was associated with release of substantially more glucose than UA159 and significantly lower lac expression. Mutants unable to use lactose grew on lactose as the sole carbohydrate when strains with an intact lac operon were also present in the cultures, indicative of the potential for population cheating. Utilizing a set of engineered obligate cheating and non-cheating strains, we confirmed that cheating can sustain a heterogeneous population. Futher, obligate cheaters of GS-5 competed well with the non-cheaters and showed a high degree of competitive fitness in a human-derived consortium biofilm model. The results show that bet-hedging behaviors in carbohydrate metabolism may substantially influence the composition and pathogenic potential of oral biofilms.

Improved pEKEx2-derived expression vectors for tightly controlled production of recombinant proteins in Corynebacterium glutamicum

The Escherichia coli/Corynebacterium glutamicum shuttle vector pEKEx2 is an IPTG-inducible expression vector that has been used successfully for the synthesis of numerous proteins in C. glutamicum. We discovered that the leaky gene expression observed for pEKEx2-derived plasmids relates to reduced functionality of the plasmid-encoded repressor LacI carrying a modified C-terminus, while duplicate DNA sequences in the pEKEx2 backbone contribute to plasmid instability. We constructed the pEKEx2-derivatives pPBEx2 and pPREx2, which harbor a restored lacI gene and which lack the unnecessary duplicate DNA sequences. pPREx2 in addition enables fusion of target genes to a C-terminal Strep-tag II coding region for easy protein detection and purification. In the absence of inducer, the novel vectors exhibit tight gene repression in C. glutamicum, as shown for the secretory production of Fusarium solani pisi cutinase and the cytosolic production of green fluorescent protein and C. glutamicum myo-inositol dehydrogenase. Undesired heterogeneity amongst clones expressing cutinase from pEKEx2 was attributed to the loss of a vector fragment containing the cutinase gene, which likely occurred via homologous recombination of the identical flanking DNA sequences. Such loss was not observed for pPBEx2. Using pPREx2, IolG-Strep was successfully produced and purified to homogeneity by Strep-Tactin affinity chromatography, obtaining 1.5 mg IolG with a specific activity of 27 μmol·min−1·(mg protein)−1 from 100 mL culture. The tight gene repression in the absence of inducer and the improved plasmid stability make expression vectors pPBEx2/pPREx2 attractive alternatives to the available molecular tools for genetic manipulation and high-level production of recombinant proteins in C. glutamicum.

Molecular mechanisms controlling fructose-specific memory and catabolite repression in lactose metabolism by Streptococcus mutans.

Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and long-term memory, where the bacteria can pause for >11 h before initiating growth on lactose. Here, we explored mechanisms contributing to unusual aspects of regulation of the lac operon. The fructose-phosphate metabolites, F-1-P and F-6-P, could modulate the DNA-binding activities of the lactose repressor. Recombinant LacR proteins bound upstream of lacA and Gal-6-P induced the formation of different LacR-DNA complexes. Deletion of lacR resulted in strain-specific growth phenotypes on lactose, but also on a number of mono- and di-saccharides that involve the glucose-PTS or glucokinase in their catabolism. The phenotypes were consistent with the novel findings that loss of LacR altered glucose-PTS activity and expression of the gene for glucokinase. CcpA was also shown to affect lactose metabolism in vivo and to bind to the lacA promoter region in vitro. Collectively, our study reveals complex molecular circuits controlling lactose metabolism in S. mutans, where LacR and CcpA integrate cellular and environmental cues to regulate metabolism of a variety of carbohydrates that are critical to persistence and pathogenicity of S. mutans.

Molecular cloning and characterization of long-chain acyl-CoA synthetase 9 from the mesocarp of African oil palm (Elaeis guineensis Jacq.)

The oil palm (Elaeis guineensis Jacq.) is the prime source of vegetable oil for many tropical countries for its extremely high oil content in fruit. In previous researches, LACS9 has been proved that it plays an important role in plant cells by regulating lipid metabolic pathways. However, its function in oil palm has not yet been fully elucidated. In this paper, a full-length cDNA encoding EgLACS9 was cloned from the mesocarp of oil palm, and subcellular localization confirmed that it was located in the endoplasmic reticulum (ER). Meanwhile, overexpression of EgLACS9 in the LACS-deficient S. cerevisiae strain YB525 indicated that EgLACS9 can rescue the deficiency of LACS gene and take up activating exogenous fatty acids. Especially, three kinds of fatty acid, including C16:0, C18:0 and C18:1, displayed a significant decrease in EgLACS9-overexpressing cells. Moreover, EgLACS9 was also overexpressed in embryoids induced from oil palm. In three obtained transgenic embryoid lines, the content of long-chain fatty acids (including C20:1, C20:2 and C24:0) was increased substantially. Meanwhile, the levels of C18:1 and C18:2 were significantly reduced. Together, these results fully elucidated the function of EgLACS9 in regulation of fatty acids accumulation in oil palm.

Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis.

Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membranestructure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the+1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

Optogenetic control of the lac operon for bacterial chemical and protein production.

Control of the lac operon with isopropyl β-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.

Operon Construction Containing alsS, ilvC, ilvD and kivd Genes in Esch-erichia coli for Isobutanol Production from Glucose

Isobutanol is a biofuel considered to be a potential gasoline substitute. However, isobutanol production is difficult because there is no native organism that can produce isobutanol. A biosynthetic pathway to produce isobutanol had been designed to utilize pyruvate produced from glucose breakdown by glycolysis in Escherichia coli (E. coli). This biosynthetic pathway con-sists of acetolactate-synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxy-acid dehydratase (DHAD), alpha-ketoisovalerate decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes. Since E. coli does not have ALS and KDC, the genes coding for the protein is needed to be cloned and overexpressed in E. coli. KARI and DHAD were overexpressed to increase the accumulation of keto acid to increase isobutanol production. Plasmid contains an operon controlled by lac pro-moter and lac operator consisting of alsS (coded ALS from Bacillus subtilis), ilvC (coded KARI from E. coli MG1655) and ilvD (coded DHAD from E. coli MG1655) genes, obtained from previous research, and operon sequences have been confirmed by DNA sequencing. kivd gene (coding KDC from Lactococcus lactis) was obtained from iGEM 2013 kit. kivd was amplified by PCR and inserted into pJET 1.2 blunt. kivd gene was then added into 3’ end of previous operon using restriction-ligation tech-nique. The plasmid constructed was then transfered into E. coli DH5α using heat shock. The recombinant genes were expressed using IPTG (isopropyl-β-D-1-thiogalactopyranoside) induction. The SDS PAGE results were inconclusive, however isobutanol was detected by Gas Chromatography Mass Spectrometry – Selected Ion Monitoring (GC-MS-SIM) from 48 hours fermenta-tion culture at 30 oC (1,17%). An operon regulated by the lac promoter-operator containing four genes for the biosynthesis of isobutanol has been constructed and cloned in E. coli. The isobutanol production was not optimal due to weak expression and repression by glucose, which was used as substrate.

Bioluminescence burst caused by a process in carbohydrate metabolism in a luciferase reporter strain of Escherichia coli

We previously reported that Escherichia coli strains carrying a firefly luciferase reporter gene (luc+) showed a posttranslationally-generated bioluminescence burst upon entry into the stationary phase. In this paper, we studied the mechanism underpinning this burst by using a series of “Keio” gene deletion strains. When luc+ driven by the lac gene promoter (lacp::luc+) was introduced into a group of Keio strains, the resulting reporter strains showed significantly altered timing and/or sizes of the burst. Remarkably, a reporter strain that lacked phosphoglucose isomerase (PGI), which catalyzes the second step of glycolysis, showed no burst, while the onset of the stationary phase of this strain was the same as that of the wild-type (WT) reporter strain. Consistently, the WT reporter strain showed no burst, when grown on arabinose or xylose instead of glucose as the carbon source. These results suggest that a process in carbohydrate metabolism is involved in the mechanism of generation of the burst. We measured temporal changes in intracellular NADPH concentrations but could not detect a significant increase or decrease relative to the occurrence of the burst. Functional implications and possible applications of the burst are discussed.

Function perturbation impact on asymptotical stability of probabilistic Boolean networks: Changing to finite-time stability

Asymptotical stability is a special property of probabilistic Boolean networks (PBNs) comparing with deterministic Boolean networks which are always finite-time stable. This paper analyzes the influence of deterministic function perturbation on the asymptotical stability of PBNs. Firstly, the stability of PBNs is recalled and the concepts of positive-probability fixed point and positive-probability cycle are given. Secondly, the topological structure of asymptotically stable PBNs is analyzed, based on which, some criteria are proposed to guarantee that an asymptotically stable PBN changes to finite-time stability with probability one (FTSPO) subject to function perturbation. Finally, the results are applied to the lac operon in the Escherichia coli.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BackgroundAuto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level.ObjectivesIn this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium.MethodsFirst, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression).ResultsAuto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture.ConclusionThe medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering.

Bacillus subtilis is an ideal host for secretion and expression of foreign proteins. The promoter is one of the most important elements to facilitate the high-level production of recombinant protein. To expand the repertoire of strong promoters for biotechnological applications in Bacillus species, 14 highly transcribed genes based on transcriptome profiling of B. pumilus BA06 were selected and evaluated for their promoter strength in B. subtilis. Consequently, a strong promoter P2069 was obtained, which could drive the genes encoding alkaline protease (aprE) and green fluorescent protein (GFP) to express more efficiency by an increase of 3.65-fold and 18.40-fold in comparison with the control promoter (PaprE), respectively. Further, promoter engineering was applied to P2069, leading to a mutation promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-type promoter (P2069). Moreover, the IPTG-inducible expression systems were constructed using the lac operon based on the strong promoters of P2069 and P2069M, which could work well both in B. subtilis and B. pumilus. In this study, highly efficient expression system for Bacillus was constructed based on transcriptome data and promoter engineering, which provide not only a new option for recombinant expression in B. subtilis, but also novel genetic tool for B. pumilus.

Summerfield scaling model and conduction processes defining the transport properties of silver substituted half doped (La–Ca) MnO3 ceramic

Conductivity spectra of La0.5Ca0.3Ag0.2MnO3 (LCAM) oxide have been investigated in the temperature range [80 K–680 K]. The studied system displays a semiconductor behavior over the explored temperature and frequency ranges. From direct current study, the transport of charge carries is assisted via disorder energy below θD/4. For the temperature range [θD/4- θD/2], the transport properties are governed by activation of Mott-variable range hopping (Mott-VRH) process. Beyond θD/2, the conduction phenomena are assisted through the thermally activation of the small polaron hopping (SPH) mechanism. At high frequencies, the conductivity spectra reveal a dispersive region. They are explained by the double Jonscher response. The latter side was characterized by the appearance of the overlapping-large-polaron-tunneling (OLPT), the correlated barrier hopping (CBH) and the non small polaron tunneling (NSPT) conduction processes. It is found that the calculated disorder energy is frequency independent. At elevated frequencies, the hopping energy is predicted to decrease with increasing frequency which can be related to the change in the Polarons radius. In the temperature range [80 K–220 K], the conductivity spectra follow double Jonscher law. Using the scaling model, the spectra merge into a single master curve, which confirmed the validity of the time temperature superposition principle (TTSP). A confident divergence from the Summerfield is noticed at high frequencies. It proves that the charge mobility is temperature dependant. The Summerfield correction scaling is effectuated by introducing a scaling positive parameter (α = 1.2). An excellent coalescence of conductivity spectra is observed.

Perturbation Analysis for Finite-Time Stability and Stabilization of Probabilistic Boolean Networks.

This article analyzes the function perturbation impact on the finite-time stability and stabilization of the probabilistic Boolean networks (PBNs). First, the concept of stability in the distribution of PBNs is divided into two disjoint concepts, that is, finite-time stability with probability one (FTSPO) and asymptotical stability with probability one (ASPO), and a new criterion is proposed for the verification of ASPO. Second, by constructing a parameterized set, it is shown that PBNs subject to function perturbation keep FTSPO if and only if the perturbed point does not belong to the parameterized set, while PBNs become ASPO if and only if the perturbed point belongs to the parameterized set. Third, as an application of perturbed stability analysis, the robust state-feedback stabilization is discussed for probabilistic Boolean control networks (PBCNs) with function perturbation. Finally, the obtained results are applied to a WNT5A network and lac operon in the Escherichia coli, respectively.

Genome re-sequencing and reannotation of the Escherichia coli ER2566 strain and transcriptome sequencing under overexpression conditions

BackgroundThe Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure.ResultsThe reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3′ non-coding region) positioned 19-bp away from the lacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing.ConclusionThe ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of the E. coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications.

Robustness for Stability and Stabilization of Boolean Networks With Stochastic Function Perturbations

In genetic regulatory networks (GRNs), gene mutations often occur in a stochastic manner. As an important model of GRNs, gene mutations of Boolean networks are always described as function perturbations. This article studies the robust stability and stabilization of Boolean networks with stochastic function perturbations. A kind of parameterized set is constructed, and it is revealed that under the stochastic function perturbations, the property of finite-time stability remains unchanged when the perturbed set and the parameterized set are disjoint. In addition, it is proved that the finite-time stability is reduced to stability in distribution when the intersection of perturbed set and complement set of parameterized set is nonempty. As an application, the robust stabilization problem of Boolean control networks with stochastic function perturbations is discussed, and several necessary and sufficient conditions are presented for the robustness of feedback stabilizers. Finally, the obtained results are used to study the Drosophila melanogaster segmentation polarity gene network and the lac operon in the bacterium Escherichia coil.

Role of Noise-Induced Cellular Variability in Saccharomyces cerevisiae During Metabolic Adaptation: Causes, Consequences and Ramifications

The concept of genetic determinism as illustrated by the metaphors such as ‘genetic blueprint’, or ‘genetic program’ had its beginning immediately after the rediscovery of growth lag observed when bacteria were exposed to a mixture of glucose and galactose or lactose. This concept got reinforced with the discovery of the mechanisms of how lactose activates the transcription of lac operon of E. coli. According to this doctrine, genetically identical cells exposed to the same environment respond in equal measure. However, studies carried out in the past two decades in organisms ranging from prokaryotes to eukaryotes, have clearly established that genetically identical cells need not necessarily respond in an identical fashion when exposed to a given environment. It has now become amply clear that organisms can stochastically switch from one physiological state to the other, thereby resulting in a phenotypically heterogeneous population. Such exhibition of heterogeneity by a population has been, in several contexts, shown to be beneficial in a temporally changing environment. In this review, we have discussed how individual cells of a genetically identical population of Saccharomyces cerevisiae remain fit by exploiting this fascinating phenomenon of stochastic switching from one metabolic state to the other when exposed to glucose and galactose, as a source of carbon and energy. We suggest that this inherent stochastic switching seems to have been exploited for an adaptive response in a fluctuating environment.