Jay D. Keasling - US grants

The University of California Berkeley will receive ARI Facilities support to create modern facilities for researchers working in a unique, integrated Program in Biological Chemistry and Engineering within the College of Chemistry. The renovated facilities will promote a strong interaction among bioinorganic chemists, biochemical engineers, and bioorganic chemists working in the areas of biotechnology and environmental research. The renovations activity funded by this award will be directed at the improvement of 1,305 square meters in 30-year old Latimer Hall and will consolidate researchers. Program activity will be enhanced as researchers are currently located in geographically distributed campus laboratories that are between 32 and 77 years old. A 7 month- long space assessment precedes the project which will involve gutting much of the interior space, installation of modular laboratory units and new fume hoods and upgrades to the mechanical, electrical and plumbing services for increased safety and improved efficiency. Provision of adequate facilities will enable the basic discovery processes to be linked more closely with the development process. Biochemical engineers will study separation techniques for the recovery of biological products, biomimetic adsorbents for metal removal and recovery and new approaches to bioremediation. Bioinorganic chemists will study metal ion transport that is essential to life processes, the use of chelating agents in the sequestration of heavy metals, and lanthanide complexes for possible use in enhancing MRI. A total of 8 professors and 117 graduate students and postdoctoral fellows will benefit from these improvements.

9409603 Keasling The goals of this project are to understand how low-copy plasmids are maintained in bacteria by quantifying the cell-cycle replication patterns, the degree of segregative stabilization, and the metabolic burden of naturally-occurring replication and segregation elements. This information is to be used to design and engineer chromosome-like vehicles in applications involving complex cellular manipulations and requiring a high degree of regulation. Escherichia coli is to be used as the subject for these investigations, because metabolism and macromolecular synthesis are well understood in this organism. ***

9502495 Keasling This project is on research to develop a methodology whereby bacterial metabolism can be redirected to synthesize all precursors necessary or the production of a desired, complex compound from an inexpensive carbon source, and to control metabolism in such a way that a defined composition can be achieved. The general aims of the research are: (1) to introduce multiple metabolic pathways from various organisms into a single bacterium: (2) to determine how to balance production of enzyme subunits at stoichiometric levels sufficient for a functioning complex: (3) to develop a mathematical model to predict how to balance more than one flux necessary for a desired product: and (4) to coordinately express the genes encoding the enzymes for the pathway at the desired level. In addition to this research activity, the investigator will enhance education by developing relevant laboratory experiments. ***

This proposal seeks to develop the experimental and theoretical
methods to introduce multiple, heterologous, biodegradation
pathways into a single organism and optimize the flux through
those pathways for the remediation of toxic or recalcitrant
organic contaminants. The project will focus the biodegradation
of parathion as a model compound since: (1) it has been widely
used as a pesticide; (2) it could potentially serve as a source
for carbon, phosphorus, and sulfur for cell growth; (3) no single
organism has been isolated that can use parathion as a sole carbon
and phosphorus source; and (4) it is similar in structure to a
number of other important environmental contaminants, such as
nerve agents and other pesticides. An engineered form of
Pseudomonas putida will be used as a model organism.

9912472 Jenkins Enhanced Biological Phosphorous Removal (EBPR) is an activated sludge wastewater treatment process modification in which anaerobic/aerobic cycling of activated sludge produces a higher than normal biomass phosphorous content. This research seeks to (1) characterize the key organisms whose presence is necessary for efficient EBPR operation, (2) determine how these organisms regulate and control the genes and gene products associated with EBPR metabolism and (3) determine the relationship between community structure, metabolic activity and process performance. Specifically, the research will include: survey of prototype plants, correlation of population dynamics with process performance in laboratory scale reactors, cloning of genes for polyphosphate and polyhydroxyalkanoate metabolisms, determination of gene expression patterns in mixed community cultures focusing especially on the shifts between anaerobic and aerobic phases, and correlation of various process upsets with changes in community structure and with changes in intracellular metabolism in a stable community structure. The observations will be integrated to develop recommendations for potential control strategies for full-scale EBPR systems. In addition to the application of EBPR, this research will improve the understanding of the microbial ecology of activated sludge and of the methods that microorganisms use to survive shortages of carbon, phosphorous and oxygen. ***

The goal of this project is to develop the experimental methodologies to engineer synthetic novel operons for coordinated expression of multiple genes in bacteria. The specific aims are: (1) to develop a dual-gene expression system that allows for convenient insertion of hairpins, RNase cleavage sites, and genes;
(2) to design synthetic hairpins to be used to stabilize one or more coding regions of an operon; (3) to confirm that the synthetic inserts form the structures predicted by RNA folding algorithms; (4) to test the effects of hairpin strength and endoribonuclease cleavage site location, and gene location in an operon on mRNA stability and protein production; (5) to test the effect of RNase E sites in the genes and RNase III sites in the
hairpins on the stability of the transcript for these genes; and (6) to test the synthetic operon with a dual-enzyme metabolic pathway that has an easily assayable intermediate.

The goal of this project is to develop a host strain and
expression system that will allow high-level production of natural
and un-natural terpenoids in Escherichia coli. The specific aims
of this project are to: (1) maximize the production of the
isoprenoid precursor isopentyl diphosphate in E. coli by
expressing the genes for either the mevalonate-dependent or the
mevalonate-independent synthesis pathway using the metabolic
engineering tools developed in the Principal Investigator's
laboratory; (2) maximize production of the primary precursors to
the terpenoids: geranyl diphosphate, farnesyl diphosphate, and
geranylgernyldiphosphate; (3) introduce into E. coli the genes for
specific classes of terpenoids and optimize production of these
"natural" terpenoids; and (4) use laboratory evolution of terpene
cyclases to produce novel terpenoids or to change the distribution
of products made by terpenoid biosynthetic enzymes. This project
is part of an Interagency Activity in Metabolic Engineering and is
to be funded by the Office of Naval Research (ONR) and three
Programs within NSF.

The Biochemical Engineering Conference serves as the premier meeting for the Biochemical Engineering community. The focus of this Conference will include important core areas of Biochemical Engineering (fermentation/cell culture and downstream process development) as well as exciting new frontiers of Biochemical Engineering (nanotechnologies, the "omics" of proteomics, genomics and physiomics, marine and environmental biotechnology). The Conference will illustrate how the same engineering fundamentals (unit operations, mass and energy transport, thermodynamics, and kinetics) that form the basis of traditional areas of Biochemical Engineering are being applied to drive advances in both areas. In addition to the regular sessions, two workshops are planned. A workshop entitled "Biochemical Engineering Education: fundamentals versus state-of-the-art" will focus on the often-competing needs to incorporate engineering fundamental into the curriculum as well as state-of-the-art applications. This workshop will feature representatives from industry and academe. A second workshop entitled "New Directions in Biochemical Engineering Research" will focus on potentially new areas for research in Biochemical Engineering. The discussion will have as panelists both academic and industrial representatives. Each panelist will be asked to prepare a single transparency and speak for no longer than 5 minutes. Following the presentations, input from the audience will be requested.

0332182 Jenkins A broad objective of the proposed work is to determine how microbialcommunity structure and function is linked to successful EBPR process performance. To achieve this objective, the key organisms whose presence is necessary for efficient EBPR operation must be studied. In particular, it is necessary to investigate how these organisms regulate and control the genes and gene products involved in EBPR metabolism. Specific goals of this work are to:
oClone the genes involved in PHA and glycogen cycling directly from mixed
communities in PAO-enriched sludges;
oElucidate the mechanisms by which carbon and P cycling occurs in PAOs;
oDevelop an immunochemical method to detect polyP metabolism in sludge;
oIntegrate observations and recommend potential control strategies for full-scale
EPBR systems.

DESCRIPTION (provided by applicant): The study of metabolism has made great strides elucidating the metabolic pathways that sustain life in the cell. This investigation has produced a complex map of metabolites and enzymes that describe the chemical transformations that are possible in an organism. With the sequencing of entire genomes it is possible to foresee the conclusion of this phase of study such that the function of each gene product encoded in a genome will be described and every metabolite will be cataloged. At that point, we will have a static view of the chemistry possible in an exquisitely dynamic organism. To gain insight into the dynamic structure of metabolism, new tools must be developed to measure transient levels of metabolites in a living cell. The research proposed here will develop tools for the real-time monitoring of intra-cellular metabolites in vivo using native and engineering biosensors founded on transcriptional regulators. Although the tools developed here will be of value for the study of metabolism in general, we will focus our research on the development of biosensors for the purpose of directing the evolution of metabolism. The identification of chemical compounds has traditionally relied on expensive and cumbersome analytical instruments that require considerable expertise to operate. The data generated by these machines must be interpreted by a trained scientist to accurately determine the presence or absence of a specific compound in a complex sample. Microorganisms face a similar problem as they must constantly sample their chemical environme nt to identify the most efficient source of carbon, nitrogen and other cellular building materials. Microbes accomplish this task through a multitude of biological sensing systems (biosensors) that determine which metabolic pathway should be expressed at any given time. Exploiting these sensor-actuator systems to detect target compounds will not only provide the tools for the next phase of metabolism study, but will also deliver methodology to direct the metabolic machinery of the cell to make target compounds. These systems also may be used to detect toxic chemicals in the environment such as groundwater contamination or chemical warfare agents. We propose the development of chemical sensor/actuator systems to detect target analytes. We will demonstrate the utility of these biosensors as tools for metabolic engineering, as well as develop methodogy applicable for building any desired biosensor. To demonstrate that a biosensor can be used to direct the evolution of a metabolic pathway, we will first use a native transcriptional regulator to report on the relative concentrations of intracellular target compounds and we will use this regulator to direct the evolution of a biosynthetic pathway to make more of the target compound. Next, we will develop a selection cassette for the evolution of new transcriptional regulators with desired properties. Finally, we will use the selection cassette to evolve new transcriptional regulators to detect specific target molecules. Accordingly, the specific tasks of this proposal are (1) to use the endogenous transcriptional activator PrpR to direct the evolution of a propionate production pathway, (2) to build a reporter/selection (RS) cassette for the evolution of new biosensors, and (3) to evolve new DitR-based biosensors that sense different target compounds.

0439124
Keasling

The proposed research will examine the mechanisms regulating mRNA stability, translation, and protein synthesis in multi-gene operons. It will develop a new method for balancing the expression of multiple genes in operons.

The PI has developed expression systems that allow for the introduction of secondary structures between the transcription and translation sites of heterologous genes to protect the mRNA from endonucleolytic cleavage resulting in increases in both mRNA and protein levels. There are many unknowns in mRNA degradation and a better understanding of the control of mRNA stability in multi-gene operons is a worthy objective that may result in the discovery of new mRNA degradation mechanisms.. This work will use laboratory techniques to evolve the desired levels of expression of multiple genes in an operon and/or new stability determinants for mRNA.

This technology may find use for the production of multi subunit proteins such as used for vaccines.

Project Summary
Synthetic biology is the design and construction of new biological entities such as enzymes, genetic circuits, and cells or the redesign of existing biological systems. Synthetic biology builds on advances in molecular, cell, and systems biology and seeks to transform biology in the same way that synthesis transformed chemistry and integrated circuit design transformed computing. Just as engineers now design integrated circuits based on known physical properties of materials and then fabricate functioning circuits and entire processors (with relatively high reliability), synthetic biologists will soon design and fabricate biological entities to accomplish a particular task. To make this happen, biological materials properties (gene sequences, protein properties, natural genetic circuit design) must be formulated into a set of design rules that can then be used to engineer new biological entities.
The Synthetic Biology Engineering Research Center (SynBERC) will lay the foundation for synthetic biology. The research program will develop the foundational understanding and technologies to build biological components and assemble them into an integrated system to accomplish a particular task. The Center's specific aims are 1) to develop a conceptual framework for designing small biological components (parts) that can be assembled into devices that will perform a well-characterized function under specified conditions, 2) to develop a small number of chasses (stable, robust bacterial hosts with known responses) to host the engineered devices and to assemble several devices to accomplish a larger vision or goal, 3) to develop a set of standards for the interactions of the parts and devices so that the devices can be built more readily and reproducibly, 4) to offer the parts, devices, and chasses as open source to other researchers and companies and 5) understand the biological and societal risks associated with synthetic biology and integrate these into the design process. These objectives will be achieved through four thrust areas in 1) Parts and Part Composition, 2) Devices and Device Composition, 3) Chassis Design, Construction, and Characterization, and 4) Societal Issues in Synthetic Biology. Two testbed applications will drive development of the thrusts. The resulting parts, devices, and chasses will be managed through a distributed web of Registries of Standard Biological Parts.
SynBERC will offer a complementary education program that will 1) educate a new cadre of biological engineers capable of designing biological components, just as electrical engineers design and build integrated circuits, 2) educate the general public about the benefits and possible risks of synthetic biology, 3) educate public policy students and policy-makers about the benefits and real threats of synthetic biology, and 4) educate K-12 students about the opportunities offered by careers in science, engineering, and synthetic biology. Coupled with a strong outreach program to minority institutions and local community colleges and high schools, SynBERC will increase the participation of minority students in this emerging area and encourage high-school students to enter this exciting new area of engineering.
The new discipline, synthetic biology, will transform the biotechnology, high-technology, pharmaceutical, and chemical industries as well as suppliers of genetic tools and custom DNA synthesis companies. SynBERC will partner with key companies in these sectors to invite applications for and advice on our research program, to provide internships for undergraduate and graduate students, to speed technology transfer, and to develop SynBERC funding.
Intellectual merit. Synthetic Biology will transform the field of biology into an engineering discipline by introducing into biology the concepts developed in other fields of engineering: ready access to off-the-shelf parts and devices with standard connections; a substrate onto which one can assemble the parts and devices and a power supply for the devices; standards for the basic components to enable their ready integration into a larger functional system; and open-source availability of parts, devices, and chasses. These developments will make the engineering of biology easier and more predictable. SynBERC brings together many of the pioneers (biologists and engineers from world-class institutions) of synthetic biology to work together to lay the foundation for this nascent field.
Broader impacts. Synthetic biology (catalyzed by SynBERC) will transform the biotechnology, high-technology, pharmaceutical, and chemical industries, as well as suppliers of genetic tools and custom DNA synthesis companies. SynBERC will educate a new cadre of synthetic biologists and biological engineers capable of designing biological parts and useful biological systems. Finally, SynBERC's education program will provide general information on synthetic biology for the general public, in-depth offerings for public policy professionals, and motivational information on opportunities in higher education for K-12 students.

DESCRIPTION (provided by applicant): Enzyme-Mediated Synthesis of Functionalized Terpene Structures Project Summary/Abstract This application addresses broad challenge area (06), Enabling Technologies, and specific Challenge Topic 06-GM-109: Green chemistry and engineering for drug discovery, development, and production. Terpenes, constituting one of the most diverse groups of compounds synthesized by biological systems, have been used as antibacterial, antifungal, and anticancer agents in the treatment of human disease. Despite the high value of many terpenes as therapeutic targets, chemical synthesis routes have proven elusive due to the presence of several chiral centers in the terpene core and the need for differential protection and deprotection of functional groups. In the last ten years there has been tremendous progress in the chemical synthesis of complex functionalized terpenes using metal-based catalysts to execute regio and stereospecific reactions, and having access to small quantities of terpenes has been sufficient to confirm the therapeutic value of a number of drug candidates. However, going forward we will need cost-effective and green synthetic methods to produce quantities of therapeutic terpenes sufficient for drug development and commercialization. To address this problem, we propose a green chemistry platform for the synthesis of terpene olefins and functionalized terpenes of therapeutic value. First, through expression of selected known and novel terpene synthases in engineered microbial hosts, we will generate terpene olefins that constitute core structures of therapeutic compounds. Second, we will functionalize these terpene olefins using mutants of a highly efficient bacterial cytochrome P450 monooxygenase. Finally, we will optimize expression of these enzymes in microbial strains that we have engineered to overproduce universal terpene precursors in order to achieve high-level production of terpene olefins and oxidized terpenes of therapeutic value. Successful completion of this proposal will provide a microbial platform for the economical and clean synthesis of terpene olefins and oxidized terpenes of therapeutic value. This work has three significant impacts on human health. First, the microbial terpenes can then be used as late intermediates in the synthesis of complete therapeutic terpenes and terpene analogs, thus accelerating the discovery of terpene-based therapeutics. Second, microbial production of terpenes is more cost-effective and environment-friendly when compared to traditional organic synthesis methods that rely on expensive starting materials and toxic solvents and catalysts. Finally, the generality of the proposed platform will streamline the synthesis of other functionalized terpenes via the introduction and mutagenesis of new enzymes in our host strains. PUBLIC HEALTH RELEVANCE: There are many natural products in the terpene family with promising or proven therapeutic characteristics. As yields from natural sources are frequently low, and typical chemical synthesis routes are often challenging and expensive, we aim to facilitate economical and clean enzyme-mediated synthesis of a number of these products. In addition, our plan enables green synthesis of product analogs for drug discovery and improvement programs though the use of mutant enzymes.

The Synthetic Biology Engineering Research Center (SynBERC) at the University of California-Berkeley is designing an interactive web portal and public forum, Ars Synthetica, to communicate SynBERC's research on synthetic biology, an emerging field that builds on advances in molecular, cell, and systems biology. Synthetic biologists will soon design and fabricate biological entities to accomplish particular tasks. Biological materials properties (gene sequences, protein properties, natural genetic circuit design) must be formulated into a set of design rules that can be used to engineer new biological entities. These design issues raise ethical concerns among the public and will form the core focus of Ars Synthetica. The forum will use Omeka, an open source software platform that will afford research scientists and the public with the ability to engage in community-wide discussions and debates, and will be customized to utilize a variety of media, such as articles, photo essays, and video vignettes addressing key questions surrounding synthetic biology. The forum will provide numerous links to research articles, data, and relevant websites, including SynBERC's synthetic biology website and the Anthropology of the Contemporary websites. Video webcasts will include guest lecturers with diverse perspectives on synthetic biology aimed at both scientists and the public, which will be disseminated on social networks, such as YouTube, Flickr, and WordPress blogs. The forum has the potential to make a significant impact on public discourse on science, as well as provide an important new exemplar for public engagement in research that can be replicated in other fields of science and informal science education.

ABSTRACT

0946510, Arkin, University of California-Berkeley

The primary purpose of this bio-fabrication facility (BioFab) is to ensure that all of the driving testbed applications of the Synthetic Biology Engineering Research Center (SynBERC) that are both existing and future have rapid access to high-quality standard biological parts as well as DNA synthesis and part assembly services. Two critical secondary purposes are: (1) the development and testing of the best organizational architecture for operating and scaling a production facility that is capable of producing many standard biological parts and that supports broader community participation across both academia and industry, and (2) ensuring that all foundational SynBERC-developed technologies, from rapid genome-scale reengineering of chassis to function composition standards for devices are broadly accessible and rapidly translated into SynBERC testbeds and to industry. Practically, the facility will pursue three specific projects at launch. Specifically, the BioFab staff will 1) rapidly prototype SynBERC-specified engineered genetic systems to achieve testbed-driven objectives, 2) design, construct, and test a collection of 6,000 new BioBrick parts for controlling replication, transcription, RNA processing and degradation, translation, and protein degradation in E.coli and S. cerevisiae, and 3) work in partnership with industrial and academic partners to develop improved tools for supporting the design, construction, and characterization/testing of engineered genetic systems assembled from standard biological parts.

Intellectual merit: The successful development, launch, and operation of the world?s first design and build facility for making high-quality standard biological parts will mark an important transition in the development of biology as a substrate for engineering. Within academia and industry, the BioFab will mark a transition from individual ?crafts? based production of genetic reagents to a more mature engineering technology platform. In turn, this will make the engineering of biology easier and more predictable, with potential applications in energy, human health, information processing, and more.

Broader impacts: This first-of-its-kind facility has significant broader impacts for academic research, education, industry, and globally. The BioFab facility will accelerate the adoption and development of biological standards while extending the SynBERC?s cutting-edge research to the broader industrial and academic communities. This professionally staffed and operated parts engineering facility will serve as an important resource for industry and academia for defining and promulgating standards, from technical to professional practice. Within education, engineering students will have access for the first time to improved collections of biological parts for controlling basic but essential cellular processes, allowing them to focus less on technical issues and more on identifying ways to engineer biology to solve the world?s problems.

Synthetic biology is emerging scientific and engineering discipline that seeks to make it possible to build new biological systems (using building blocks gleaned from the natural world) that can be customized to meet pressing needs in areas such as renewable energy, specialty chemical production, and various areas of biotechnology. A grand challenge in this field is the need for technologies that enable the construction of novel complex functions in biological systems. When these functions involve the expression and coordination of multiple genes, building them becomes increasingly difficult. Assembling multigenic functions in an organism by an iterative approach is both laborious and difficult, since the engineered genes and their products often interact strongly with both one another and with the pre-existing native functions in the organism. For example, such complications have presented major challenges to efforts to engineer metabolism in microbes and plants. Moreover, many desirable applications of synthetic biology comprise complex novel functions and great genetic diversity, such as the assembly of genes from a metagenomic library in order to synthesize novel small molecules. In these cases, one might not know a priori which genetic elements need to be included in such a synthetic assembly, much less how they should be regulated in order to maximize the performance of a particular function. While these properties make linear engineering inefficient and difficult, sometimes prohibitively so, Nature has evolved mechanisms to deal with such complexity. This research project will develop a synthetic system that harnesses the power of these natural mechanisms to enable synthetic biologists to generate, diversify, and refine complex multigenic functions. The core of this technology will be based on a bacterial innovation called integrons, which are natural cloning and expression systems that assemble multiple open reading frames, in the form of gene cassettes, by using site-specific recombination and conversion to functional genes by expression from an internal promoter. The ability to capture disparate individual genes and physically link them in arrays suitable for co-expression is a trait unique to these genetic elements. The result is an assembly of functionally coordinated genes theoretically facilitating the rapid evolution of new phenotypes. This project will generate a novel technology platform based on synthetic integrons (syntegrons), including computational optimization and analysis tools, that will enable the engineering of complex multigenic functions (such as the biosynthesis of plant-derived small molecules like taxol) through continuous directed evolution.

Broader impacts
This project will generate a robust technology enabling the engineering of biological systems, including both microbes and plants, for myriad useful purposes. Notable examples include the production of renewable bio-fuels and biomaterials, the synthesis of small biomolecules for applications in specialty chemicals, bioremediation, and improvement of crops for agriculture. This project will also provide a scientific tool for probing genome organization and dynamics in processes such as the emergence of microbial resistance to small-molecules and metabolic pathway evolution. In addition, this project will introduce students at both graduate and undergraduate levels to the potential of synthetic biology, including exposure through the annual International Genetically Engineered Machine (iGEM) competition. Finally, this project will engage the broader community (outside the university setting) through the Science, Art and Writing (SAW) initiative - a cross-curricular science education program that is particularly targeted towards school-age children (www.sawtrust.org). This initiative uses themes and images from science as the starting point for scientific experimentation, art and creative writing, and in doing so stimulates creativity and scientific curiosity.

This award provides funding for a three year continuing award to support a Research Experiences for Teachers (RET) in Engineering and Computer Science Site program at the University of California Berkeley entitled, "Berkeley Engineering Research Experiences for In-Service and Pre-Service Teachers (BERET)", under the direction of Dr. George C. Johnson.

This program will focus on the intersection between engineering, science, and mathematics through the interdisciplinary lens of synthetic biology. This program will place a total of 52 middle and high school STEM teachers (26 in-service teachers from the Berkeley Unified School District and the Oakland Unified School District and 26 UC Berkeley pre-service teachers enrolled in Cal Teach) within a variety of engineering-related research laboratories associated with UC Berkeley and will guide them to develop curricula based on their research that is relevant to the subjects that they teach in school and aligned with the educational standards for the subject addressed. BERET will be run jointly by the Cal Teach science and mathematics teacher education program and the NSF Synthetic Biology Engineering Research Center (SynBERC).

BERET will increase the understanding of math, science and engineering concepts, skills and careers for K-12 students in high need urban schools, and in so doing, contribute to increasing the number of women and under-represented minority students in the STEM pipeline, particularly in engineering fields. The rigorous K-12 curricula developed through BERET will be reviewed, tested and disseminated broadly through web-based resources such as the Industry Initiatives for Science and Math Education (IISME) Community website and the TeachEngineering digital library. The program will also help to build a vibrant network of in-service teachers, pre-service teachers, and faculty, postdoctoral, and graduate student researchers that will produce long-term collaborative partnerships among individuals and institutions.

Engineered microorganisms may serve as factories to convert renewable starting materials, such as sugars and plant-derived biomass, into valuable products including chemicals, fuels, and medicines. Although the feasibility and utility of this approach is now well established, engineering microorganisms and plants to produce novel products through the introduction of specific genes remains technically challenging and resource-intensive. Consequently, these barriers to bringing new biological synthesis platforms to commercial feasibility and efficiency limit the rate at which new technologies create public benefits and economic impacts. Moreover, it is not yet possible to efficiently harness sources of vast biodiversity, such as libraries of genetic information from plants and communities of organisms, because identifying and utilizing genetic "diamonds in the rough" remains too costly and technically intensive to conduct in most laboratories. To meet these needs, this project will develop a technology suite that enables researchers to efficiently assemble, evaluate, and optimize novel biological synthesis systems by harnessing evolutionary mechanisms to both generate and select for functional assemblies of genetic parts. This work includes novel approaches for identifying promising genes from plant genomes, biological sensors that enable engineers to monitor and control biomanufacturing within cells, and computational design tools that will enable the broader research community to apply these capabilities to a broad range of applications.

Broader Impacts: Together, this work will create tools that catalyze the development of sustainable biological manufacturing platforms and enable the production of new medically and industrially useful molecules, thus contributing significantly to the Nation's economy. The project will also provide multi-disciplinary training of students and postdoctoral researchers in a rapidly emerging field.

Intellectual merit: Saccharomyces cerevisiae (yeast) is one of the most widely used microorganisms for production of commodity chemicals and fuels, and proteins. This research will develop genetic, computer aided design, and strain engineering tools for yeast and will demonstrate their use in expressing multi-component polyketide synthases for the production of important chemicals. The choice to engineer yeast for polyketide synthesis is derived from the fact that the most useful natural products are polyketides and the biosynthetic enzymes involved can be engineered to produce a broad range of pharmaceuticals, commodity and specialty chemicals, and fuels. The expression of functional polyketide synthases and the production of the necessary precursors to supply the biosynthetic machinery is a challenging problem that requires a better biological host and the appropriate gene expression tools. The specific aims of the project are to 1) develop generalized methods for the rapid modification of the S. cerevisiae genome to create modified versions of yeast strains that are capable of growing and/or surviving extreme conditions; 2) create a suite of gene expression control devices and demonstrate their utility for producing polyketide synthase complexes for the production of specific commodity chemicals; 3) construct pathways for the synthesis of polyketide precursors; 4) create libraries of polyketide synthases for production of diverse chemicals; and 5) develop computer aided design (CAD) software that will enable the rapid construction of yeasts.

Broader impacts: The tools developed for the synthesis of polyketides will be broadly applicable for the engineering of yeast to produce a variety of products as well as exploring fundamental yeast biology. This translational research effort will bring state-of-the-art synthetic biology advances to an industrially-relevant organism. The major potential benefits to society will be replacement of existing, petroleum-based polymer monomers with monomers derived from renewable resources; production of novel monomers for new materials that cannot be made; biological components for engineering yeast for scientific projects and applications; and computer-aided design tools for biology. The investigators will use relationships developed through the Synthetic Biology Engineering Research Center to help small and large companies grow the bioeconomy. The investigators will use the research findings to teach and train a cutting-edge workforce of post-docs, graduate students, and undergraduate students in this new area of research. The project will support underrepresented undergraduate students through established REU programs at UC Berkeley and MIT, and underprivileged high school students through a novel high school program developed by post-doctoral fellows in SynBERC.

Proposed Title: Characterization of specific polyketide synthases and application toward the biological production of highly-desirable, short-chain olefins.



Recent concerns as to the supply and cost of petroleum-based fuels and commodity chemicals have sparked an interest in creating alternative routes to these important hydrocarbon compounds. In particular the alpha-olefins, a class of compounds currently derived from petroleum, serve as precursors to many industrially produced plastics, short chain fatty acids, surfactants and recently have been touted as potential fuel alternatives. One potential route for synthesizing commodity and specialty chemicals is to engineer microorganisms to transform inexpensive, renewable sugars to desired products. Unfortunately, the scope of biological transformations available to generate differing chemical functional groups is lacking, thus limiting our ability to replace all petroleum-derived commodity chemicals. In an effort to produce this important set of chemicals, scientists at the University of California, Berkeley have identified enzyme components comprising a class of polyketide synthase (PKS) terminal modules known to generate alpha-olefins by way of a unique structural feature. Understanding the mechanism of this enigmatic reaction will pave the way for future enzyme biocatalyst design, in particular, new designer PKS assemblies for specific terminal alkene production. Studies and engineering efforts concerning the PKS modules will be executed under the supervision of the awardee, Prof. Jay D. Keasling (UC, Berkeley), by a diverse group of students from a wide range of socioeconomic and educational backgrounds. In particular, through the iCLEM program and partnership with the University of California, Berkeley Extension Program, Keasling and his group aim to involve high school and undergraduate students pursuing education outside the formal university requirements (e.g. through Associate Degrees and certificate programs) to gain valuable hands-on experience.

The goal of this proposal is to understand how these unique, conserved terminal sulfotransferase-thioesterase (ST-TE) didomains of PKS function to produce alpha-olefins. The specific project aims include 1) to characterize the overall substrate tolerance and fundamental kinetic parameters of four terminal olefin-forming PKS modules; 2) to perform an in-depth mechanistic analysis of the CurM TE to better understand this pivotal transformation; and 3) to use this information to guide the generation of two chimeric synthases for the sustainable production of C5 (pentene) and C6 (hexene) terminal olefins, as well as the related aromatic alkene, styrene. PKS proteins will be expressed in Escherichia coli or a native olefin producer, such as cyanobacteria Synechococcus PCC 7002. Aggregate results will be leveraged to design highly active catalysts specific for producing these olefinic compounds. Chimeric PKSs will be evaluated in both E. coli and Synechococcus PCC 7002 to better explore PKS activity and olefin formation in both natural and unnatural hosts. Definition of this this unique termination mechanism and application of the findings to create novel enzymes will provide a biological route to terminal alkenes, which may ultimately contain novel functional groups (e.g., halogens, amines, etc.) that can be used to create new cross-linkable polymers with altered properties, or compounds with improved fuel characteristics, all from low-cost renewable resources.

This project, funded jointly by the Systems and Synthetic Biology Program in MCB and the Biotechnology, Biochemical and Biomass Engineering Program in the CBET, aims is to contribute a synthetic biology workflow for the efficient production of non-natural molecules in living cells. The specific aim is to produce novel biofuels that match the performance of existing fuels, while overcoming their limitations with respect to soot and nitrogen oxide formation. The outcomes will propel the development of Synthetic Biology in Europe and the US, as it contributes significantly to the development of a bio-refinery approach in the chemical industry. On the educational side, the investigator is involved in graduate education and research training. The project will also contribute to established REU programs at the University of California, Berkeley. Results will be disseminated broadly via publications in interdisciplinary international journals. Long-term, the major potential benefits of the proposed activity to society will be replacement of existing, petroleum-based fuels with fuels derived from renewable resources.

The multinational SynPath project follows a classic engineering approach of design, synthesis and analysis to explore metabolic pathways in microorganisms for production of drop-in biofuels. The focus will be on developing novel metabolic pathways for production of hydrocarbons and identify rate-limiting steps in biofuel production so that sugars can be efficiently converted to advanced biofuels. The proposed work will also create devices and modules that can be used to regulate and optimize biofuel production pathways. The success of this project relies on the integration of expertise in thermodynamics, chemical kinetics, microbial physiology, metabolic pathway engineering, and enzyme engineering.

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Keasling, Jay D.

The goal of the workshop is to convene experts in synthetic biology and related fields to identify broad principles and methods to translate fundamental discoveries from synthetic biology into useful bio manufacturing platforms. It is anticipated that the workshop will generate new ideas towards the next steps of leveraging the potential of synthetic biology for new technologies.

The workshop will survey a broad array of topics, including protein folding, metabolic pathways, cellular and tissue engineering, and evolutionary forces, to identify new research and educational directions that can capitalize on advances in synthetic biology and lead to innovative manufacturing methods and products.

The workshop is co-sponsored by the Biotechnology and Biochemical Engineering Program of CBET, the Office of Emerging Frontiers and Multidisciplinary Activities, the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biosciences, the Chemistry of Life Processes Program of the Division of Chemistry and by the Physics of Living Systems Program of the Division of Physics.

This award, funded jointly the Biological Sciences Division of Molecular and Cellular Biosciences and the Engineering Division of Chemical, Biological, Environmental, and Transport Systems Division, will bring together leading scientists and bioengineers from the US and India to develop an optimize cellular systems for the productions of industrially relevant chemicals and products using approaches derived from theoretical principles of biology and engineering. One of the anticipated outcomes will be the development of parallel strategic partnerships for research collaborations and student and postdoctoral exchange opportunities.

The workshop will be held March 18-20, 2016 at the India Institute of Technology in Mumbai, India. Approximately 30 senior researchers with expertise in the fields of systems and synthetic biology from the US and India will be in attendance in addition to researchers from select Asian countries. The outcomes of this workshop is to develop strategic collaborative and educational opportunities in addition to creating an infrastructure that will be necessary to realize the overarching goals of this workshop.

The Engineering Biology Research Consortium (EBRC) aims to bringing together the US synthetic biology community to develop the future vision for synthetic biology, catalyze leading-edge research and education programs, and promote dialogue about synthetic biology among policy-makers and public stakeholders. Biology is emerging as an important manufacturing platform for the 21st century; the market for genetically engineered products already exceeds the global semiconductor market. EBRC will conduct meetings, programs and workshops that strengthen the research and education communities; articulate compelling research visions and goals aligned with pressing national and global challenges; and engage the research and education communities, and government, industry and public stakeholders to develop and communicate ideas. EBRC will establish a forward-thinking forum at the nexus of these groups through a new non-profit organization and take on a new set of leadership challenges as synthetic biology moves from concept to reality.

NSF funding during EBRC's initial two years will support: (1) the development of two consecutive annual engineering biology community roadmaps; (2) the development of key white papers addressing needs outlined in the recent National Academies' Industrialization of Biology report; and (3) communication activities coordinated with NSF and other US government agencies to communicate the vision and content of the roadmap and white papers.

The chemical energy that cells use for growth is generated using respiration, a universal set of reactions that occur partly within lipid membranes. Lipid composition is used by cells to modulate the properties of their membranes, which could act as important physical structures for reactions that occur during respiration. In this project, novel synthetic biology approaches will be used to investigate biophysical roles for lipid chemistry and membrane structure in respiratory activity. This research will provide mechanistic insight into respiratory function and cell physiology that is applicable to a wide variety of organisms and is relevant to understanding the interplay of diet and disease in human health. In addition to the research activities, this grant will support education and outreach efforts that focus on scientific training for underprivileged high school and undergraduate students. These efforts will teach students fundamental concepts in metabolism and provide hands-on experience with emerging biotechnology research projects in order to support their future careers in science and technology.

Efforts to engineer fatty acid composition in bacteria have led to the observation that fluidizing lipid components, such as unsaturated lipids, act as a general regulator of respiratory metabolism. The project will test the hypothesis that respiratory flux by is controlled by the lipid-mediated diffusion rates of electron transport chain components. This will be done with a combination of biophysical measurements, physiological experiments, and physical modeling of bacterial respiratory chains. Lipid composition varies tremendously among prokaryotes and this could partly be understood through differences in their metabolic requirements. The project will therefore use synthetic biology approaches to assess the metabolic effects of isoprenoid-based membrane components that modulate membrane structure in a heterologous bacterial host. Lipid composition is also tightly regulated in mitochondrial membranes, which carry out respiration in all eukaryotic cells. The project will use engineered yeast strains to systematically investigate the role of fatty acid composition in mitochondrial structure and respiratory activity. These systems will serve as models for understanding how lipid composition can affect metabolic functions and may be broadly applicable to many important processes.

PROJECT SUMMARY/ABSTRACT Plant terpenes are a critical source of clinically approved drugs and clinical candidates, yet very few complete biosynthetic pathways have been characterized. Due to the lack of efficient chemical synthesis routes, many complex plant natural product scaffolds including terpenes are currently still isolated from the producing plant or plant cell culture and then converted to a clinically-used drug by semisynthetic routes (e.g. digoxin and taxol on the 2015 WHO list of essential medicines). Lack of information regarding terpene biosynthetic pathways severely limits the use of promising new approaches to produce plant molecules in heterologous hosts (e.g. yeast strains that make artemisinin), as well as the intriguing possibility of engineering the biosynthetic pathways to access analogs and non-natural derivatives with greater efficacy. Given the critical role of medicinal plant terpenes in human health and utility of biosynthetic genes, we propose here systematic discovery of key medicinal plant terpene biosynthesis pathways, taxol and cyclopamine, and the engineering of yeast strains for scalable production. Classically, the discovery of plant pathways has been slower and more painstaking than bacterial pathways; however, our team has demonstrated two approaches that greatly accelerates identification of complete biosynthetic routes: (1) rapid combinatorial testing of enzymes in a N. benthamiana heterologous host, and (2) transcriptional profiling and co-expression analysis to identify pathway genes. This approach enabled the discovery of six enzymes that complete the pathway to the etoposide aglycone from the unsequenced medicinal plant Podophyllum in a matter of months and has also led to the discovery of numerous plant terpene pathways including a novel class of sesterterpenes. In this proposal, we have prioritized pathways for valuable medicinal plant terpenes that are notoriously difficult to access: the clinically used anticancer agent taxol and the clinical candidate cyclopamine. These compounds are representative medicinal plant terpenes that will be used to demonstrate the broad utility of our discovery and yeast engineering approach that can be applied for accessing many of the other >100,000 different plant terpenes in nature. In addition to yeast strains that produce these highly valuable plant terpenes, a major outcome of this work will be broadly applicable yeast synthetic biology tools for efficient production of multiple cytochromes P450s in series which represents a major bottleneck for efficient transfer of plant pathways to yeast heterologous hosts.

As products change over time, so do the processes that manufacture them. At the beginning of the twentieth century, most consumer goods were made from natural fibers, wood, ceramics, and metal. As the century progressed, petroleum-based plastics became the preferred materials for most consumer products. Replacing petroleum-based plastics in the economy is a serious challenge, and converting renewable raw materials to recyclable products is critical. One goal of this project is to synthesize monomers (the building blocks of polymers) from biomass sugars. Another goal is to design monomers that make polymers that are easy to recycle. The third goal is to demonstrate a digital light manufacturing (DLM) process that produces high-quality 3D-printed parts using those monomers. The ultimate objective is to cycle the monomers through the product and back to monomers, creating a circular path for the material, thereby reducing waste. Also, interactive activities will be developed for K?12 and public audiences to demonstrate the circular material flow.

Chemically recyclable photopolymerizable cycloolefin resins with properties tailored for DLM will be designed. The ability to form reversible polymer bonds will guide monomer design. Enzymes and microbial cells for biomanufacturing DLM monomers from renewable feedstocks will be developed based on polyketide synthases (PKSs). DLM processes for photo-polymerization of cycloolefin resins will be developed and improved. Photocatalyst systems, resin rheology, and instrumentation will be co-developed to digitally manufacture precision parts from circular cycloolefin resins. An extensive suite of mechanical tests will be carried out on structures printed from candidate resin formulations for both hard and elastomeric 3D-printed products, based on volumetric 3D printing via tomographic reconstruction. The rigidity, strength, and fracture toughness of 3D-printed cycloolefin resins will be benchmarked against leading conventional photopolymer resins, to guide material and process selection and maximize their impact on future manufacturing.

This project is jointly supported by the Cellular and Biochemical Engineering Program (ENG/CBET/CBE), the Synstems and Synthetic Biology Program (BIO/MCB/SSB) and the Chemical Catalysis Program (MPS/CHE/CAT).

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Microbes can make organic molecules with complex structures. Many of these molecules or their analogs are central ingredients in a variety of products, but the diversity of these molecules is limited by the range of the reactions catalyzed by natural enzymes. Engineering cells to produce artificial metalloenzymes (AMEs) will expand the range of possible reactions. Novel biosynthetic pathways will be created by complementing natural enzymes with AMEs. Graduate students and postdoctoral associates will be trained in this convergent field of synthetic chemistry and synthetic biology. Outreach activities to encompass topics related to artificial biosynthesis will be offered to underserved high school students and teachers, as well as to K-8 students.


This project will build upon the general concept of creating artificial biosynthetic pathways containing artificial metalloenzymes and preliminary results showing the feasibility of creating these pathways in bacteria. We will increase the numbers and types of microorganisms that can host the chemistry catalyzed by artificial metalloenzymes to expand the range of natural products that react with AMEs; expand the types of metallo-cofactors that are incorporated intracellularly into AMEs to increase the scope of unnatural reactions in these pathways; and combine the abiotic chemistry with natural biosynthesis in varying sequences. Specifically, we will 1) introduce AMEs into Streptomyces strains and test activity on heterologously produced terpenes and polyketides; 2) incorporate new cofactors into AMEs expressed in E. coli and Streptomyces; 3) broaden the scope of transformations catalyzed by AMEs in the artificial biosynthetic pathways to encompass abiotic C-H bond functionalizations; 4) create pathways in which the unnatural chemistry occurs in the middle of the artificial biosynthesis; and 5) elucidate the pathways for diazo-containing small molecules. By doing so, we will generate the fundamental knowledge and demonstrate guiding principles to create artificial biosynthetic pathways that convert simple carbon sources to valuable unnatural products in whole microorganisms.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.