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Leguia M.,University of California at Berkeley | Leguia M.,Synthetic Biology Engineering Research Center | Brophy J.,University of California at Berkeley | Densmore D.,Boston University | And 3 more authors.
Methods in Enzymology | Year: 2011

The primary bottleneck in synthetic biology research today is the construction of physical DNAs, a process that is often expensive, time-consuming, and riddled with cloning difficulties associated with the uniqueness of each DNA sequence. We have developed a series of biological and computational tools that lower existing barriers to automation and scaling to enable affordable, fast, and accurate construction of large DNA sets. Here we provide detailed protocols for high-throughput, automated assembly of BglBrick standard biological parts using iterative 2ab reactions. We have implemented these protocols on a minimal hardware platform consisting of a Biomek 3000 liquid handling robot, a benchtop centrifuge and a plate thermocycler, with additional support from a software tool called AssemblyManager. This methodology enables parallel assembly of several hundred large error-free DNAs with a 96+% success rate. © 2011 Elsevier Inc. All rights reserved.

Zhang F.,University of California at Berkeley | Zhang F.,Joint BioEnergy Institute | Zhang F.,Lawrence Berkeley National Laboratory | Rodriguez S.,University of California at Berkeley | And 4 more authors.
Current Opinion in Biotechnology | Year: 2011

Production of biofuels from renewable resources such as cellulosic biomass provides a source of liquid transportation fuel to replace petroleum-based fuels. This endeavor requires the conversion of cellulosic biomass into simple sugars, and the conversion of simple sugars into biofuels. Recently, microorganisms have been engineered to convert simple sugars into several types of biofuels, such as alcohols, fatty acid alkyl esters, alkanes, and terpenes, with high titers and yields. Here, we review recently engineered biosynthetic pathways from the well-characterized microorganisms Escherichia coli and Saccharomyces cerevisiae for the production of several advanced biofuels. © 2011 Elsevier Ltd.

Beller H.R.,Joint BioEnergy Institute | Lee T.S.,Joint BioEnergy Institute | Katz L.,Synthetic Biology Engineering Research Center
Natural Product Reports | Year: 2015

Covering: 2005 to 2015 Although natural products are best known for their use in medicine and agriculture, a number of fatty acid-derived and isoprenoid natural products are being developed for use as renewable biofuels and bio-based chemicals. This review summarizes recent work on fatty acid-derived compounds (fatty acid alkyl esters, fatty alcohols, medium- and short-chain methyl ketones, alkanes, α-olefins, and long-chain internal alkenes) and isoprenoids, including hemiterpenes (e.g., isoprene and isopentanol), monoterpenes (e.g., limonene), and sesquiterpenes (e.g., farnesene and bisabolene). © 2015 The Royal Society of Chemistry.

Zhang F.,Joint BioEnergy Institute | Zhang F.,Lawrence Berkeley National Laboratory | Zhang F.,University of California at Berkeley | Keasling J.,Joint BioEnergy Institute | And 3 more authors.
Trends in Microbiology | Year: 2011

Many metabolic pathways in microbial hosts have been created, modified and engineered to produce useful molecules. The titer and yield of a final compound is often limited by the inefficient use of cellular resources and imbalanced metabolism. Engineering sensory-regulation devices that regulate pathway gene expression in response to the environment and metabolic status of the cell have great potential to solve these problems, and enhance product titers and yields. This review will focus on recent developments in biosensor design, and their applications for controlling microbial behavior. © 2011.

Poust S.,University of California at Berkeley | Phelan R.M.,Joint BioEnergy Institute | Deng K.,Joint BioEnergy Institute | Katz L.,Synthetic Biology Engineering Research Center | And 3 more authors.
Angewandte Chemie - International Edition | Year: 2015

The gem-dimethyl groups in polyketide-derived natural products add steric bulk, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem-dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem-dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gemdimethyl group producing PKS modules. Experiments showed that both PKSs are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module 8, use of dimethylmalonyl-ACP appeared to be the sole route to form a gem-dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation. © 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

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