Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli

Author Manuscript February 6, 2011Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene—the first committed Taxol intermediate—approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid–forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products

Economic Impact of Dengue Illness and the Cost-Effectiveness of Future Vaccination Programs in Singapore

Dengue illness is a tropical disease transmitted by mosquitoes that threatens more than one third of the worldwide population. Dengue has important economic consequences because of the burden to hospitals, work absenteeism and risk of death of symptomatic cases. Governments attempt to reduce the disease burden using costly mosquito control strategies such as habitat reduction and spraying insecticide. Despite such efforts, the number of cases remains high. Dengue vaccines are expected to be available in the near future and there is an urgent need to evaluate their cost-effectiveness, i.e. whether their cost will be justified by the reduction in disease burden they bring. For such an evaluation, we estimated the economic impacts of dengue in Singapore and the expected vaccine costs for different prices. In this way we estimated price thresholds for which vaccination is not cost-effective. This research provides useful estimates that will contribute to informed decisions regarding the adoption of dengue vaccination programs

Optimization in computational systems biology

Optimization aims to make a system or design as effective or functional as possible. Mathematical optimization methods are widely used in engineering, economics and science. This commentary is focused on applications of mathematical optimization in computational systems biology. Examples are given where optimization methods are used for topics ranging from model building and optimal experimental design to metabolic engineering and synthetic biology. Finally, several perspectives for future research are outlined

Statistical Analysis of Molecular Signal Recording

A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.United States. Defense Advanced Research Projects Agency. Living Foundries ProgramGoogle (Firm)New York Stem Cell Foundation. Robertson Neuroscience Investigator AwardNational Institutes of Health (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Transformative R01 1R01GM104948)National Institutes of Health (U.S.) (Single Cell Grant 1 R01 EY023173)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (CAREER Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS1042134)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

Algal Photosynthesis as the Primary Driver for a Sustainable Development in Energy, Feed, and Food Production

High oil prices and global warming that accompany the use of fossil fuels are an incentive to find alternative forms of energy supply. Photosynthetic biofuel production represents one of these since for this, one uses renewable resources. Sunlight is used for the conversion of water and CO2 into biomass. Two strategies are used in parallel: plant-based production via sugar fermentation into ethanol and biodiesel production through transesterification. Both, however, exacerbate other problems, including regional nutrient balancing and the world's food supply, and suffer from the modest efficiency of photosynthesis. Maximizing the efficiency of natural and engineered photosynthesis is therefore of utmost importance. Algal photosynthesis is the system of choice for this particularly for energy applications. Complete conversion of CO2 into biomass is not necessary for this. Innovative methods of synthetic biology allow one to combine photosynthetic and fermentative metabolism via the so-called Photanol approach to form biofuel directly from Calvin cycle intermediates through use of the naturally transformable cyanobacterium Synechocystis sp. PCC 6803. Beyond providing transport energy and chemical feedstocks, photosynthesis will continue to be used for food and feed applications. Also for this application, arguments of efficiency will become more and more important as the size of the world population continues to increase. Photosynthetic cells can be used for food applications in various innovative forms, e.g., as a substitute for the fish proteins in the diet supplied to carnivorous fish or perhaps—after acid hydrolysis—as a complex, animal-free serum for growth of mammalian cells in vitro

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells