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Tech Talk

"Imagine, for example, that you wanted to engineer bacteria to perform a complex synthesis of a natural product of medicinal value, but that you needed to separate the reactions into different sets of steps because of cross reactions of intermediates of the first set of enzymes with the second set. One option would be to create two strains of bacteria expressing enzymes that perform both sets of syntheses and to make the two strains cooperate by having the first strain send a signal to the second one telling it not to start the second series of steps until the first set of reactions is complete (for example, all available reactants for the first steps are used up). After the second group of reactions is complete, the second strain could send a signal to the first strain telling it to switch back into synthesis mode. Couple all of this to a substrate delivery system and you would have a 'just in time' cooperative chemical synthesis machine."[1]

"We identified two design principles: the closer the enzyme is to the beginning of the pathway, the shorter the response time of the activation of its promoter and the higher its maximal promoter activity. Mathematical analysis suggests that this 'just-in-time' transcription program is optimal under constraints of rapidly reaching a production goal with minimal total enzyme production." [2]

"Similarly, the budding yeast Saccharomyces cerevisiae exhibits cycles in the form of glycolytic and respiratory oscillations. Such cycles were first documented over 40 years ago and can occur with a variety of period lengths both in cell-free extracts and during continuous culture. A recent study has described a ~40-min respiratory oscillation that produces a genome-wide, low-amplitude oscillation of transcription during continuous culture."[3]

"However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. ... This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance."[4]

"Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell– cell communication."[5]

"Our design of this oscillatory circuit, termed the metabolator, integrates transcriptional regulation with metabolism. This is in contrast with the yeast glycolytic oscillator which does not involve transcriptional regulation, as well as previous synthetic gene expression oscillators, which were independent of metabolism"[6]

"A very large fraction of the genes involved in nutrition that are cell cycle regulated are involved in transport of essential minerals and organic compounds across the cell membrane. Some of the compounds that are moved by these transporters are amino acids (GAP1), ammonia (AUA1 and MEP3), sugars (e.g., HXT1 and RGT2), and iron (FET3 and FTR1). We also identified the acid phosphatases (e.g., PHO3 and PHO8). Nearly all of these genes reach peak expression late in the cell cycle during M and M/G1."[7]

"Periodic mRNA fluctuation was also observed in functional classifications of genes not previously associated with the cell cycle. For example, transcripts for the FAA1, FAA3, and ELO1 enzymes, which participate in fatty acid biosynthesis, peaked during G1. Many of the nuclear-encoded mitochondrial enzymes required for glycolysis path and oxidative phosphorylation were induced in early G1 with very similar patterns of mRNA fluctuation. None of the transcripts for these mitochondrial genes peaked outside of G1."[8]

"Yeast cells growing under continuous conditions at high cellular density employ a robust metabolic cycle for energy generation in which a respiratory burst alternates with a non-respiratory, reductive phase. Two related studies have recently shown that global transcriptional co-regulation of genes defines the phases of this metabolic network in time and synchronizes cell division with metabolism."[9]

"Perhaps the major function of this system is to partition potentially damaging processes from sensitive biosynthetic events, where bursts of pro- and anti-oxidants are produced out-of-phase every cycle. These bursts are essential for network and population coherence, signalling the events that lead to the redox state changes of the cell. ROS feed onto some of the most promiscuous transcription factors (the YAP family of sensors and Skn7 two component system), indeed thiol-specific reagents or NO+ release produce damped oscillations. Although it is difficult to specifically alter ROS generation and thiol redox states independently as they are so intimately coupled, it is apparent that the cellular network coherence is stubbornly-defended and maintained during experimental perturbation. It will be a major challenge to elucidate the molecular mechanisms involved as the phenomena involve a large part of the cellular network, perhaps more challenging will be to produce more detailed formal models."[10]

"The basic idea of our paper is that time dependent gene expression enables cells to adapt their metabolic capabilities in an optimal way to varying external conditions."[11]

1. Michnick SW. A luxRury of synthetic signals. Nat Biotechnol. 2006 Jun;24(6):658-60. DOI:10.1038/nbt0606-658 |
2. Zaslaver A, Mayo AE, Rosenberg R, Bashkin P, Sberro H, Tsalyuk M, Surette MG, and Alon U. Just-in-time transcription program in metabolic pathways. Nat Genet. 2004 May;36(5):486-91. DOI:10.1038/ng1348 |
3. Tu BP, Kudlicki A, Rowicka M, and McKnight SL. Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes. Science. 2005 Nov 18;310(5751):1152-8. DOI:10.1126/science.1120499 |
4. Farmer WR and Liao JC. Improving lycopene production in Escherichia coli by engineering metabolic control. Nat Biotechnol. 2000 May;18(5):533-7. DOI:10.1038/75398 |
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10. Lloyd D and Murray DB. The temporal architecture of eukaryotic growth. FEBS Lett. 2006 May 22;580(12):2830-5. DOI:10.1016/j.febslet.2006.02.066 |
11. Klipp E, Heinrich R, and Holzhütter HG. Prediction of temporal gene expression. Metabolic opimization by re-distribution of enzyme activities. Eur J Biochem. 2002 Nov;269(22):5406-13. DOI:10.1046/j.1432-1033.2002.03223.x |
12. Bier M, Bakker BM, and Westerhoff HV. How yeast cells synchronize their glycolytic oscillations: a perturbation analytic treatment. Biophys J. 2000 Mar;78(3):1087-93. DOI:10.1016/S0006-3495(00)76667-7 |
13. Richard P. The rhythm of yeast. FEMS Microbiol Rev. 2003 Oct;27(4):547-57. DOI:10.1016/S0168-6445(03)00065-2 |
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15. Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, and Stephanopoulos G. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol. 2006 Aug;72(8):5266-73. DOI:10.1128/AEM.00530-06 |
16. Rosenfeld N, Elowitz MB, and Alon U. Negative autoregulation speeds the response times of transcription networks. J Mol Biol. 2002 Nov 8;323(5):785-93. DOI:10.1016/s0022-2836(02)00994-4 |
17. Xu Z and Tsurugi K. A potential mechanism of energy-metabolism oscillation in an aerobic chemostat culture of the yeast Saccharomyces cerevisiae. FEBS J. 2006 Apr;273(8):1696-709. DOI:10.1111/j.1742-4658.2006.05201.x |
18. Ronen M and Botstein D. Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source. Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):389-94. DOI:10.1073/pnas.0509978103 |