Presentation Abstracts

Opening Keynote

Christina Smolke
Stanford University

Biosynthesis of complex plant-derived natural products

Plants are a rich source of unique molecules, including 25% of natural-product-derived drugs. However, the discovery, synthesis, and overall material supply chains for sourcing plant-based medicines remain ad hoc, biased, and tedious. While microbial biosynthesis presents compelling alternatives to traditional approaches based on extraction from natural plant hosts, many challenges exist in the reconstruction of plant specialized metabolic pathways in microbial hosts. We have developed approaches to address the challenges that arise in the reconstruction of complex plant biosynthetic pathways in microbial hosts. We have utilized these strategies to develop yeast production platforms for an important class of plant alkaloids, which include the medicinal opioids and noscapinoids. The intersection of synthetic biology, genomics, and informatics will lead to transformative advances in how we make and discover essential medicines.

Session 1: Natural Product Discovery and Engineering

Ikuro Abe
The University of Tokyo

Engineered Biosynthesis of Medicinal Natural Products

Meroterpenoids are hybrid natural products that are partially derived from terpenoids, and those from fungi exhibit extremely diverse structures and biological activities. Recent progress in the research of fungal meroterpenoid biosynthesis has revealed several unusual enzyme reactions, including post-cyclization modification reactions by oxygenases, such as FAD-dependent monooxygenases, cytochrome P450 monooxygenases, and non-heme iron-dependent dioxygenases. These oxidative processes build molecular complexity and contribute to the structural diversification of fungal meroterpenoids. In this presentation, our most recent structure-function studies of these unique enzymes will be discussed.

Yan Feng
Shanghai Jiao Tong University

Enzyme evolution for efficient biosynthesis of natural products

Exploring the novel functions of enzymes and pathways used in green chemistry and pharmacy are important goals for synthetic biologists. Protein engineering has provided crucial strategies to create better biocatalysts to be more stable, to accept unnatural substrates and even to catalyze unnatural reactions. Here, we gave some examples for the molecular evolution and optimization of enzymes to exhibit new catalytic abilities. More specifically, the lecture will focus on the engineered enzymes are applied to de novo biosynthesis of products.

Zhao-Xun Liang
Nanyang Technological University

Microbial natural product discovery by genome tinkering

With the aim of uncovering novel microbial secondary metabolites, our lab recently isolated several hundred microbial strains from the aquatic environment of Southeast Asia. By metabolite profiling and genome sequencing, we identified a number of talented strains that contain unique cryptic biosynthetic gene clusters (BGCs) predicted to produce structurally novel compounds. We are currently targeting the cryptic BGCs for the discovery of new biosynthetic pathways and natural products. We often manipulate the microbial genomes to relieve the repression of gene expression and to probe the biosynthetic mechanism. I will present some of the results obtained from our recent research work.

Session 2: Biocatalyst Discovery, Design and Engineering

Chan Beum Park

Shedding Light on Biocatalysis: Photobiocatalytic Platforms for Solar Production of Fuels and Value-Added Chemicals

The idea of solar energy utilization in chemical synthesis through the combination of photocatalysis and biocatalysis provides an opportunity to make the green process greener. Recent progress indicates that photoinduced electron transfer using organic or inorganic photosensitizing materials can activate a wide spectrum of redox enzymes to catalyze fuel-forming reactions (e.g., H2 evolution, CO2 reduction) and synthetically useful reductions. This talk will provide a conceptual description of photobiocatalysis that couple enzymatic reduction reactions with photochemical water oxidation towards mimicking natural photosynthesis. Our research works in the light-driven, enzyme-based production of fuels and value-added chemicals will be presented according to the methods of cofactor regeneration and the hybridization of oxidoreductases and photosensitizing materials. For more efficient utilization of solar energy, we have recently developed Z-scheme-based biocatalytic photoelectrochemical cells (e.g., a tandem platform integrated with an enzyme-cascade system for sequential reduction of CO2 to formate, formaldehyde, and methanol), involving dual illumination on both anode and cathode through mimicking the two-step photoexcitation scheme of natural photosystems. I will discuss future perspectives to take yet-to-be-expected next steps in the vibrant field of biocatalyzed artificial photosynthesis.

Isabelle AndrŽ
INSA, CNRS, INRA, University of Toulouse

Computer-aided engineering of enzymes for in vitro and in vivo production of novel precursors

Development of enzyme-based synthetic processes is often hampered by the lack of natural enzymes with requisite properties or specificities. With the potential offered nowadays by computer-aided molecular design and enzyme engineering techniques, we have seen in recent years numerous examples of successful enzyme designs that enabled tremendous improvements of catalytic properties for various applications, including catalysis of novel synthetic reactions. Nonetheless, progress in this field, in particular with computational techniques, is still required in order to fasten enzyme design and accelerate the generation of efficient biocatalysts.
This lecture will report and discuss recent developments and specific research projects of our laboratory. Special emphasis will be placed on the contribution of computational methods in our strategies to engineer enzymes for the production of novel chemical precursors. Two examples will be presented: (i) computer-aided engineering of carbohydrate-active enzymes to conceive catalysts acting on non-natural substrates, to enter programmed chemo-enzymatic cascades, and ultimately produce antigenic oligosaccharide precursors [1]; (ii) structure-based engineering of enzymes to conceive an artificial metabolic pathway dedicated to in vivo production of non-natural chemical precursors [2].

This work was partially funded by the French National Research Agency (PROTICAD, ANR-12-MONU-0015-03; GLUCODESIGN ANR-08-PCVI-0002-02; CarbUniVax, ANR-15-CE07-0019-01; SYNTHACS ANR-10-BTBR-05-01).

[1] VergŽs A. et al. Computer-aided engineering of a transglucosylase for the glucosylation of an unnatural disaccharide of relevance for bacterial antigen synthesis.ACS Catalysis. 2015, 5(2), 1186
[2] Walther T. et al.. Construction of a synthetic methabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid. Nature Comm. 2017, (8),15828

Yifeng Wei
Institute of Chemical and Engineering Sciences

Radical chemistry in anaerobic bacteria

The anaerobic biosphere constitutes a significant fraction of our environment and bodies, and has great relevance to human health and industry. However, the diversity of anaerobic biochemical transformations is poorly understood. Here we describe the bioinformatics-aided discovery and biochemical characterization of two new oxygen-sensitive enzymes from anaerobic bacteria. 1) Indoleacetate decarboxylase catalyzes the formation of 3-methylindole (skatole) as an end-product of tryptophan catabolism in certain fermenting bacteria. Skatole is notorious as the characteristic odorant of animal feces, and is of great concern to the livestock industry. 2) Isethionate C-S lyase catalyzes the cleavage of hydroxyethylsulfonate (isethionate), to give acetaldehyde and sulfite. It is present in many sulfate- and sulfite-reducing bacteria, including human gut bacteria, which use sulfite as a terminal electron acceptor to produce toxic hydrogen sulfide. Both enzymes carry out the chemically challenging transformations through free radical chemistry enabled by a protein-based glycyl radical cofactor.

Session 3: Novel Biomolecules

Steven Benner
Foundation for Applied Molecular Evolution

Re-inventing Darwinism from the Ground Up

By dragging scientists across uncharted terrain where they are forced to answer unscripted questions, "Grand Challenge" synthesis can drive discovery and paradigm change in ways that hypothesis-directed research cannot. Here, our grand challenge in synthetic biology seeks to reproduce the Darwinism displayed by terran biology, but on a molecular platform different from standard DNA; access to Darwinism is believed by many to distinguish the living state from the non-living state. This recognizes that alien Darwinism, with a natural history (including origins) independent of terran Darwinism, might support Darwinism on a different biopolymer. Here, we explore the possibility that Darwinism can be universally supported by any biopolymer that has just two structural features, (a) an ability to fit into a Schršdingerian "aperiodic crystal", lattice, and (b) an "polyelectrolyte" backbone.

Ichiro Hirao
Institute of Bioengineering and Nanotechnology

Xenobiology applications by genetic alphabet expansion

Artificial extra base pairs (unnatural base pairs) that function as a third base pair in replication, transcription, and/or translation expand the genetic alphabet of DNA, creating a new research area, xenobiology, for novel biology systems. Through the unnatural base pairs, additional functional components can be incorporated into DNA, RNA, and protein. Unnatural base pairs can be used for new qPCR, PCR visualization, and multiplex PCR methods. Hydrophobic unnatural bases greatly augment the affinities of DNA aptamers that specifically bind to target molecules. Semi-synthetic organisms with six-letter DNA can produce new proteins containing unnatural amino acids. In this talk, I will introduce xenobiology using unnatural base pair systems and a wide variety of their applications.

Charles Johannes
p53 Lab

A*STAR Peptide Engineering Platform (PEP): Enabling Naturally Inspired Modalities and Technologies

Peptides are a cornerstone of natureÕs diversity and function. As part of the Peptide Engineering Programme we have been inspired by the simplicity of how peptides are assembled and the seemingly infinite properties and function that can be derived. With innovations in technologies such as deep sequencing and microwave synthesizers we can now begin to harness natureÕs power and evolve our contemporary chemical and biological tools to develop new modalities. I will highlight our approach and share our preliminary results.

Session 4: Therapeutics

Matthew Chang
National University of Singapore

Engineering microbes to rewire host-microbiome interactions

The wealth of knowledge on the human microbiota composition and its role in health and disease has recently spurred the development of novel therapeutic strategies. Furthermore, with an array of genetic tools that are readily available, we can design and build genetic circuits, edit and rewrite genomes and reprogram cells to foster novel microbiota-based interventions. Herein, we present our work in engineering gut-resident microbes as a versatile platform equipped with clinically relevant functionalities for various diseases. This conceptual approach of transforming gut microbes as live biotherapeutics with prophylactic and therapeutic efficacy has been demonstrated against pathogenic infections and chronic metabolic diseases such as cancer. This work provides a strong foundation in the application of engineered microbes for modulating microbiota-host metabolism and interactions and supports the use of functional microbes as a viable strategy for clinical intervention.

Wei Leong Chew
Genome Institute of Singapore

DNA engineering from molecule to animal

We now find ourselves stepping into an exciting new age, brought here by DNA technologies that enables redesign of the blueprint of life. Today I will share how we launched two research programmes in Singapore to develop technologies that enable new ways of writing DNA, from the single-molecular level to that within the human subject.

James Tam
Nanyang Technological University

Superglue for Precision Biomanufacturing

Site-specific ligation is enormously useful to enable selective bonding of chemicals, polymers, peptides and proteins to form new compounds. Recent advances in chemistry, biochemistry and molecular biology have provided novel methods to staple peptide and non-peptide bonds under physiological conditions, using superglue derived from nature. An example is the family of linkage-specific, peptide asparagus-specific ligases (PAL). A prototypic member of PAL is Butelase 1 isolated from butterfly pea (Bunga Telang). Butelase 1, catalyzing the reverse reaction of an asparaginyl endopeptidase, is a stand-alone and ATP-independent ligase. Importantly, butelase 1 is C-terminus-specific for Asn/Asp (Asx), traceless, and accepts a tripeptide Asx-His-Val with the dipeptide His-Val as the leaving group. Butelase 1 accepts most N-terminal amino acids with D- or L-configuration. It exhibits unmatched kinetics with catalytic efficiencies of up to 1,340,000 M-1 s-1 and >10,000 times faster than other known ligases. Our recently published work showed that butelase 1 is useful for both intra- and intermolecular ligation, cyclizing or ligating efficiently various peptides and proteins ranging in size from 8 to >300 amino acids. Thus, the high catalytic efficiency and broad substrate specificity of butelase 1 could augment new applications, both in vitro and in vivo systems for basic and translational research. Here, I will present our latest results on Asx-specific ligases, their applications and live-cell labeling to explore new frontiers in biochemical, medical, material sciences, and precision biomanufacturing.

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Michael Winther
Engine Biosciences

Building an Engine for Drug Discovery with AI and Synthetic Biology

Synthetic biology and metabolic engineering technologies provide powerful new tools for developing novel products and services and advancing understanding of complex biological systems. At Engine Biosciences we are building a predictive engine for drug discovery using cellular maps, high-throughput, massively parallel biological experimentation and high-throughput computation for drug discovery. This is achieved by building an integrated team spanning multiple disciplines including systems and synthetic biology, genome engineering, and data science. Our platform, which combines artificial intelligence and genetic perturbation, is being used to uncover the gene interactions and biological networks underlying diseases to identify new drug therapies faster and at lower cost. This alliance of technologies is already helping make important analyses and predictions for precision medicine applications.

Session 5: Foundational Technology I

Huimin Zhao
University of Illinois at Urbana-Champaign

Biosystems Design via Directed Evolution

By mimicking the Darwinian evolution in the test tube, directed evolution has become a powerful and indispensable tool for the design and engineering of biological systems including proteins, pathways, and genomes for fundamental research and biotechnological applications. In this talk, I will give a historical account of the directed evolution field and discuss the challenges and opportunities in directed evolution. I will highlight our recent work on the development and application of novel directed evolution tools for genome engineering. Specifically, I will introduce three genome evolution strategies including RNA interference assisted genome evolution (RAGE), a CRISPR transcriptional activation, interference, and gene deletion (CRISPR-AID) based genome evolution method, and a CRISPRÐCas9- and homology directed-repair (HDR)-assisted genome-scale engineering (CHAnGE) method that enables rapid engineering of Saccharomyces cerevisiae on a genome scale with precise and trackable edits. In addition, I will introduce a data-driven directed evolution approach for biosystems design.

Yuansheng Yang
Bioprocessing Technology Institute

Precise engineering of glycosylation pathway in CHO cells for producing monoclonal antibodies with improved efficacies

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are the best-selling class of therapeutic proteins and their demand is still rapidly growing. Chinese hamster ovary (CHO) cells are the dominant host for producing mAbs due to their capacity to perform proper folding and assembly of complex proteins and human-compatible glycosylation. Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic mAbs. Cell engineering can be performed by knockout, knockdown and overexpression of one or more critical genes in the glycosylation pathway for obtaining desired glycosylation on a mAb product. The effectiveness of traditional cell engineering technologies could be impeded by inability to control the modified genes at optimal expression levels due to random nature of plasmid integration and the large variations in the activity of the numerous possible integration sites. At Bioprocessing Technology Institute, we have developed a FLP/FRT recombinase mediated cassette exchange (RMCE)-based targeted integration platform to avoid random integration of plasmid vectors. Together with one set of internal ribosome entry site (IRES) mutants with different strength, we are able to accurately control the expression level of multiple genes in CHO cells. We have used these tools to produce anti-CD20 IgG1 rituximab with different glycan structures. Cell based functional assays indicate low fucose and high galactose enhances the ADCC and CDC of rituximab respectively, while enhanced sialic acids decreases both ADCC and CDC. Our technology provides an approach for efficient manufacturing of glycoengineered mAbs with improved efficacies.

Ann Koay
Biotransformation Innovation Platform

Biotrans Taste Receptor Platform for Novel Taste and Sensate Compounds

Food companies are driven to increase product innovation and differentiation to meet the demands of the modern-day consumer market. This has resulted in an increased search for natural and novel ingredients for tastier and more nutritious products. At the taste receptor platform in BioTrans we have developed in vitro cell systems that mimic individual human taste responses and sensations enabling us to quickly evaluate thousands of molecules for any taste-modulating or sensate properties. These approaches provide opportunities to discover and develop new compounds that will add to the pipieline of molecules with potential for commercialization in the food and consumer care industry. Some of the key technology factors involved in developing this taste receptor platform will be presented.

Session 6: Foundational Technology II

Zhongjun Qin
Shanghai Institute of Plant Physiology and Ecology

Creating a functional single chromosome yeast

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single chromosome yeast from a Saccharomyces cerevisae haploid cell containing sixteen linear chromosomes through successive chromosome end-to-end fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome result in dramatic changes in the global chromosomal three-dimensional structure due to the loss of all centromere-, most of the telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, but displays less fitness in several aspects such as growth across environments and competitiveness, gametes production and viability. This synthetic biology study paves a new path for exploring the eukaryote evolution with respect to chromosome structure and function.

Fayza Daboussi
French National Institute for Agricultural Research

An innovative DNA-free genome editing strategy for the one-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum

Diatoms are highly prized in industrial biotechnology, due to both their richness in natural lipids and carotenoids and their ability to produce recombinant proteins of considerable value in diverse markets. Studies of their metabolism will provide insight into their adaptation capacity, a prerequisite for metabolic engineering. Several years of investigation have led to the development of genome engineering tools for exploring and exploiting the metabolism of these organisms. Thus, custom molecular scissors have recently emerged as useful tools to inactivate single target genes in Phaeodactylum, either for functional analysis or to redirect natural metabolism towards increased neutral lipid biosynthesis (Daboussi et al., Nature Communications, 2014). Until now, it has been mediated by introducing plasmids encoding a nuclease and an antibiotic resistance cassette into the cells, both then stably integrated at random sites within the nuclear genome. Disadvantages of this approach include: the random integration of all or part of the plasmid DNA into the genome can lead to undesired gene disruptions or uncontrolled effects on gene expression near the integration site(s); the long-term expression of the nuclease can potentially induce off-target cleavage; and the impossibility to eliminate background mutations or integrated transgenes through outcrossing in Phaeodactylum, because this is a diploid organism with no known sexual reproduction.
Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of the CRISPR/Cas9 molecular scissors in the protein form (RNPs) coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. The power of this methodology was confirmed by creating strains in which three genes were simultaneously inactivated (triple knock-out) without introducing any selection marker or DNA into the cells (Serif et al., Nature Communications, 2018). This remarkable result allows microalgae to catch up with other industrial chassis (yeasts, bacteria).
Another attractive perspective relies on the fact that the counter-selectable markers are well conserved within the microalgae phylogenetic tree and among other eukaryotic groups, making this strategy extendable to other organisms. This is particularly important for hard-to-transfect species or those for which no biobricks (promoters, terminators) are available.

Jihyun F. Kim
Yonsei University

Genomics and Systems/Synthetic Biology of Microbes and Microbiomes

Powered by high-speed high-throughput next-generation genomic technologies, life science and biotechnology are being transformed. In our laboratory, we apply genomic and metagenomic tools to study model microbes and microbial communities. Multi-omics systems-level understanding of the Escherichia coli cell factory may open the door to synthetic biology and next-generation biotechnology. Analysis of genomes sampled from a long-term evolution experiment revealed that the coupling between genomic and adaptive evolution is complex and can be counterintuitive even in a constant environment [1]. The microbiome, comprised of the microbiota and its collective genomes called the metagenome, is an integral part of our body and the ecosystem. Systems understanding of host physiology can be possible only if the microbial counterparts that reside in are fully appreciated and both are considered as a unit, i.e. holobiont. Recent analyses reveal that a myriad of microbial members, mutualistic, commensal, or pathogenic to the host, play pivotal roles in health and disease by producing diverse macromolecules and metabolites. Host-microbiota relationships in the plant rhizosphere [2] and the human gastrointestinal tract, as well as the dynamics of microbial communities, will be presented as examples. In the talk, efforts to develop probiotics or more preferably pharmabiotics for the prevention or treatment of gastrointestinal cancers will also be presented. Synthetic biology concepts and toolkits enable us to modulate the microbiome to maintain (eubiosis) or regain (rebiosis) homeostasis, and even to transform it to become preventive or curative.

1. Barrick JE, Yu DS, Yoon SH, Jeong H, Oh TK, Schneider D, Lenski RE, Kim JF. (2009) Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461:1243-1247.
2. Kwak MJ, Kong HG, Choi K, Kwon SK, Song JY, Lee J, Lee PA, Choi SY, Seo M, Lee HJ, Jung EJ, Park H, Roy N, Kim H, Lee MM, Rubin EM, Lee SW, Kim JF. (2018) Rhizosphere microbiome structure alters to enable wilt resistance in tomato. Nat Biotechnol 36:1100-1109.

Closing Keynote

Akihiko Kondo
Kobe University

Genome editing and synthesis platforms which facilitate the construction of cell factories

We have developed the platform technologies such as genome editing and a large gene cluster synthesis systems and are going to integrate to set up the automated systems for efficient construction of microbial cell factories.
By tethering the DNA deaminase activity to nuclease-deficient CRISPR/Cas9 system, we have developed a genome editing tool that enables targeted point mutagenesis. An AID orthologue PmCDA1 was attached to nuclease-deficient mutant of Cas9 (D10A and H840A) to perform highly efficient and target-specific nucleotide editing. This hybrid system, termed Target-AID, induced cytosine point mutation in 3-5 bases range at the distal site within target sequence. Use of nickase Cas9 (D10A), which retains single-strand cleaving activity, greatly increase the efficiency, although it also occasionally induce insertion/deletion (indel) in higher eukaryotes. Uracil-DNA glycosylase inhibitor further increase the efficiency and reduced the indel formation. The toxicity associated with Cas9 has been greatly diminished, enabling application of this technique to wider range of organisms including yeast, bacteria, animals and plants._In addition, by tethering Glycosilase activity to nuclease-deficient CRISPR/Cas9 system, we have developed a genome editing tool that enables targeted randam mutagenesis.
We have also developed an efficient DNA assembly method, namely, Ordered Gene Assembly in B. subtilis (OGAB) method. OGAB method can assemble more than 50 DNA fragments in one-step using B. subtilis [3]. Thanks to this high processability, even in construction of long DNA (~100 kb), material DNA fragments can be kept in chemical DNA synthesis-friendly and sequencing-friendly small size (< 2 kb). Since there is no in vitro DNA synthesis step that may cause unexpected mutation(s), long DNA by OGAB method using sequence-confirmed material DNA thus contains essentially no mutation. We are now constructing user friendly DNA synthesis system by integrating new automation system, such like a liquid handling robot that is specifically developed for OGAB method
These technologies might lead to new pipelines through which functional genomes are cleated with much faster speed to construct microbial cell factories to produce variety of biofuels and chemicals.