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2019 Genome Project-Write and 8Th Annual Sc2.0 Meeting ABSTRACT BOOKLET 2019 Genome Project-write and 8th Annual Sc2.0 Meeting New York, New York, United States November 11-14, 2019 ABSTRACT BOOKLET Hosted by the Institute for Systems Genetics at NYU Langone Health 1 Abstracts Selected for Posters (P1 – P28): P1. A “Marionette” S. cerevisiae strain to control metabolic pathways Marcelo Bassalo, Chen Ye, Joep Schmitz, Hans Roubos, Christopher Voigt Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States Optimizing metabolic networks often requires fine-tuning of gene expression levels to minimize buildup of toxic intermediates while maximizing productivity. Inducible promoters are a straight-forward strategy to systematically test different expression levels, providing levers to independently control targeted genes. However, the limited availability of orthogonal transcriptional sensors in the yeast, Saccharomyces cerevisiae, hinders their use to optimize an engineered biosynthetic pathway. In this work, we aim to expand the set of inducible promoters and develop a “Marionette” yeast strain, containing a genome integrated array of optimized sensors. We have taken steps towards this “Marionette” strain by constructing and testing an initial set of 4 orthogonal sensors, engineered by placing bacterial operator elements into yeast core promoters. We then demonstrate “Marionette” in yeast by tuning a toxic metabolic pathway to produce the monoterpene Linalool. Initially, a two-level factorial experiment was performed to uncover expression rules of the targeted genes. By incorporating these rules, we performed a second optimization round. Overall, this pilot test of expression profiles allowed us to explore the equivalent of ~300 kb of pathway variant constructs with a single genetic design. Finally, we also demonstrate staging order of operations on the controlled genes. The ability to establish a synthetic metabolic pathway control to independently tune component genes will accelerate metabolic engineering cycles in yeast, enabling rapid testing of multiple expression levels that ultimately can be used to train learning algorithms and uncover rules for optimal pathway flux. P2. Dissecting the α-globin super-enhancer with synthetic regulatory genomics Brendan R. Camellato, Leslie A. Mitchell, Helena Francis, Mira T. Kassouf, Matthew T. Maurano, Douglas R. Higgs, Jef D. Boeke Institute of Systems Genetics, NYU Langone Health, New York, New York, USA The α-globin locus has been a pioneering model to study gene regulation by distal enhancer elements, locus control regions, and super-enhancers. In the mouse, α-globin expression is regulated by a cluster of five enhancers located 50 kb away from the coding genes. It’s not clear, however, whether these enhancers function as a cluster of independent elements, or synergistically as a super-enhancer. Current techniques to study enhancer function are not suitable for simultaneously assessing the roles of multiple elements in their native genomic context. We are thus applying our approach of synthetic regulatory genomics to dissect the 2 α-globin super-enhancer and characterize the individual elements. Using our “Big DNA” synthesis technologies, we built a synthetic version of the mouse α-globin locus, as well as 20 different variants which feature combinatorial perturbations of specific enhancers and addition of various features to facilitate phenotypic readouts. These synthetic loci are assembled directly into BAC vectors allowing delivery into suitably engineered mouse embryonic stem cells (mESCs) and integration into the genome using a recombinase- mediated cassette exchange strategy, overwriting the endogenous locus. Following in vitro differentiation of the mESCs into erythrocytes, or establishment of mouse strains, phenotypes can be assessed relating to gene expression, transcription factor binding, and chromatin state. So far, three synthetic loci have been integrated, establishing three mESC cell lines and one mouse strain. Characterization of these locus variants has produced results that were unexpected based on previous work and support a model in which the α- globin enhancer cluster functions synergistically as a super-enhancer. P3. Development of platform technologies for metabolic engineering in Saccharomyces cerevisiae Alexander C. Carpenter 1,2, Thomas C. Williams 1,2, Isak S. Pretorius1 and Ian T. Paulsen1 1Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia 2CSIRO Synthetic Biology Future Science Platform, Canberra, ACT 2601, Australia Directed evolution is an attractive method for metabolic engineering that allows for the identification of non-obvious changes, which increase compound of interest production. Two major limitations in directed evolution experiments are the emergence of cheater cells within the population, and the difficulties involved in generating new biosensors. Two methods are currently being developed to mitigate both of these limitations in S. cerevisiae. Two cell directed evolution (2CDE) creates a synthetic co-dependency in which production of a compound of interest by one strain triggers the activation of a biosensor in a second strain. Genetic separation of biosensor from compound of interest production would greatly reduce the formation of cheating sub-populations. Underlying assumptions involved in 2CDE have successfully been tested using doxycycline induced amino acid cross-feeding, and PHBA biosensor activation from adjacent overproducing strains. Iterative simultaneous yeast display (ISYD) is a technique to create binding peptides for compounds of interest which can be modularly used in biosensor designs. The technique uses simultaneous screening of yeast display libraries in a non-competitive binding assay for a compound of interest. Furthermore, the construction uses intron mediated genetic assembly to allow iterative addition of new peptides to identified binding peptides, increasing binding affinity and specificity. Intron mediated genetic assembly is being tested in model systems using fluorescence activated cell sorting based outputs. 3 P4. Identification of optimal orientation and insertion loci for contextual gene expression in bacterial plasmid by in vitro transposition JungHwan Cho, Seungwoo Baek, In-Geol Choi Department of Biotechnology, Graduate School, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea The bacterial gene expression using a plasmid vector is mainly affected by various non- contextual factors such as ribosome binding site, codon usage, promoter intensity. However, the contextual effect of gene expression (e.g. orientation and location of genes) is also a critical factor for gene expression and its contribution to the gene expression has not been accessed thoroughly. Here, we employed the Tn5 transposase system, which makes a random insertion of reporter gene cassette into plasmid vectors, for examine the contextual effect of gene expression. The main objective is to find the ‘optimal orientation and insertion loci’ that are correlated with various expression levels. To do this, we performed in vitro transposition experiments to build a library of expression vectors having various orientation and insertion loci of gene cassette. Random and single insertion of a GFPuv gene cassette into pUC19 was examined by NGS sequencing of a pooled library (1,000 colonies). The insertion loci were screened by the fluorescent intensity in E. coli. Relating the insertion sequential position data to the expression level, we categorized contextual effects by GFPuv expression level. P5. Methanol assimilation in native and synthetic strains of Saccharomyces cerevisiae Monica I. Espinosa1,2, Kaspar Valgepea3,4, Ricardo A. Gonzalez-Garcia3, Colin Scott 2,5, Isak S. Pretorius1, Esteban Marcellin3, Ian T. Paulsen1* and Thomas C. Williams1,2* 1Department of Molecular Sciences, Macquarie University, Sydney, Australia 2Synthetic Biology Future Science Platform, CSIRO, Sydney, Australia 3Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Australia 4ERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, Estonia 5Biocatalysis and Synthetic Biology Team, CSIRO, Canberra, Australia Microbial fermentation for chemical production is becoming more broadly adopted as an alternative to petrochemical refining. Fermentation typically relies on sugar as a feed- stock. However, one-carbon compounds like methanol are a more sustainable alternative as they do not compete with arable land. This study focused on engineering the capacity for methylotrophy in the yeast Saccharomyces cerevisiae through a yeast xylulose monophosphate (XuMP) pathway, a ‘hybrid’ XuMP pathway, and a bacterial ribulose monophosphate (RuMP) pathway. Through methanol toxicity assays and 13C-methanol- growth phenotypic characterization, the bacterial RuMP pathway was identified as the most promising synthetic pathway for methanol assimilation. When testing higher 4 methanol concentrations, methanol assimilation was also observed in the wild-type strain, as 13C-ethanol was produced from 13C-methanol. These results demonstrate that S. cerevisiae has a previously undiscovered native capacity for methanol assimilation and pave the way for further development of both native and synthetic one-carbon assimilation pathways in S. cerevisiae. P6. Rewriting the firmware of RNA-guided nucleases via directed evolution Gregory W. Goldberg, Brendan Camellato,
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