Functional and nutritious food crops are beneficial to human health. Refined grains are mainly starch of endosperm and lack nutrients. Therefore, using plant design and synthetic biology methods to synthesize nutrients and bioactive components in crops has important research significance for the nutrition and functionalization of crops. To this end, we have developed efficient DNA assembly methods, TransGene Stacking system, and CRISPR/Cas9 editing system tools etc. for plant design and synthetic biology applications. Using these tools and rice as a chassis, a number of phytonutrients and functional active substances were synthesized de novo in the endosperm of rice, realizing the biosynthesis and accumulation of three types of plant pigments in rice endosperm, and creating a number of new functional rice germplasms, such as the first endosperm anthocyanin-rich Purple Endosperm Rice (PER, Zi Jingmi in Chinese) and the first endosperm astaxanthin-rich Astaxanthin Rice (AR, Ci Jing mi in Chinese).
Dr. Qinlong Zhu is a professor at South China Agricultural University and State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources. He also was a visiting associate professor at Cornell University (2018-2019). His research mainly focuses on plant synthetic biology tool development and germplasm innovation of functional food crops for addressing the challenges in human health, including DNA assembly methods, TransGene Stacking system, and CRISPR/Cas9 genome and base editing tools, as well as phytonutrient biosynthesis, biofortification of crops and synthesis of bioactive substances. He supervised the students to win the first Best Plant Synthetic Biology Award of iGEM in 2016. He won the Best Youth Outstanding Paper Award of CSBT in 2019.
Methanol is an ideal feedstock for bio-manufacturing that can be beneficial for global carbon neutrality. However, the toxicity of methanol limits the efficiency of methanol metabolism toward biochemical production and it is still challenging in engineering this non-conventional yeast due to serious lack of genetic editing tools. In this presentation, we will show our recent progress in establishing CRISPR-Cas9 based genome editing tools and enhancing the homologous recombinations in methylotrophic yeast Ogataea polymorpha. With this genetine platform, we tried to engineering cellular metabolism for fatty acid production from methanol. We found that engineering overproduction of free fatty acids (FFA) from sole methanol resulted cell death with a decreased cellular phospholipid in O. polymorpha, and the cell growth was restored by adaptive laboratory evolution (ALE). Whole genome sequencing of the adapted strains reveals that inactivation of LPL1 (encoding a putative lipase) and IZH3 (encoding a membrane protein related to zinc metabolism) preserve cell survival by restoring phospholipid metabolism. Engineering the pentose phosphate pathway and gluconeogenesis enabled high-level production of FFA (15.9 g/L) from sole methanol. Preventing methanol-associated toxicity underscored the link between lipid metabolism and methanol tolerance, which should contribute to enhancing significantly methanol-based bio-manufacturing.
Yongjin Zhou is a Chair Professor at Dalian Institute of Chemical Physics, Chinese Academy of Sciences. His research areas include: Synthetic microbiology for cell factory construction, yeast genetics and metabolic engineering. His lab mainly focus on engineering yeast cell factories for biosynthesis of natural products and establishing methanol biotransformation process for chemical production. He co-authored more than 80 peer reviewed papers on prestigious journal such as Cell, Nature Energy, Nature Metabolism, Nature Chemical Biology, JACS, PNAS, Nature Communications with >3900 citations and hold 8 patents. He was honored several awards includes “Lun Shiyi” Outstanding Young Scientists (2018), and The first prize of Chinese Pharmaceutical Association (2015) etc.
Glycosylation by uridine diphosphate-dependent glycosyltransferases (UGTs) in plants contributes to the complexity and diversity of secondary metabolites. UGTs are generally promiscuous in their use of acceptors, making it challenging to reveal the function of UGTs in vivo. Here, we described an approach that combined glycoside-specific metabolomics and precursor isotopic labeling analysis to characterize UGTs in Arabidopsis. We revisited the UGT72E cluster, which has been reported to catalyze the glycosylation of monolignols. Glycoside-specific metabolomics analysis reduced the number of differentially accumulated metabolites in the ugt72e1e2e3 mutant by at least 90% compared with that from traditional untargeted metabolomics analysis. In addition to the two previously reported monolignol glycosides, a total of 62 glycosides showed reduced accumulation in the ugt72e1e2e3 mutant, 22 of which were phenylalanine-derived glycosides, including 5-OH coniferyl alcohol-derived and lignan-derived glycosides, as confirmed by isotopic tracing of [13C6]-phenylalanine precursor. Our method revealed that UGT72Es could use coumarins as substrates, and genetic evidence showed that UGT72Es endowed plants with enhanced tolerance to low iron availability under alkaline conditions. Using the newly developed method, the function of UGT78D2 was also evaluated. These case studies suggest that this method can substantially contribute to the characterization of UGTs and efficiently investigate glycosylation processes, the complexity of which have been highly underestimated.
Qiao Zhao is a professor in the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. He received his Ph.D. training in the department of plant molecular and cellular at the Ohio State University in United States in 2008. He started his independent research career at Tsinghua University in 2014 and moved to the Shenzhen Institutes of Advanced Technology in 2019.
For precision engineering of microbial cell factories, developing high throughput and automated (HTA) biobreeding technology as well as the novel instruments are of importance. We have systematically developed serial enabling novel HTA breeding instruments, including 1) a rapid and powerful ARTP (atmospheric and room temperature plasma) mutagenesis, high throughput and automated microdroplet-based microbial culture (MMC) and screening (Drem cell，MISS cell ) systems, and demonstrated their wide application in adaptive evolution and genome-phenotype association of microbial cell factories including various prokaryotic and eukaryotic microbes.
Prof. Xinhui Xing received his Ph.D. from Tokyo Institute of Technology in 1992. He had been the Assistant Professor at Tokyo Institute of Technology from 1992 to 1998, and Associate Professor at Yokohama National University from 1998 to2001. He was selected as a full professor by the 100-Telent Scholar Program of Tsinghua University in 2000 and joined Department of Chemical Engineering since then. He has served the director of Institute of Biochemical Engineering from 2002 to 2022. He had been the vice department chairman from 2009 to 2018. From 2019, he serves the Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School as the Deputy Director. His research field covers enzyme engineering, biobreeding technology and instrumentation, synthetic biotechnology, health-care product engineering and glycodrug technology, bioactive peptide mining and production technology. He serves as associate editor of Biochemical Engineering Journal, and editorial board of several domestic and international journals. He has published more than 300 papers in international and domestic academic journals, co-authored 8 books, translated 2 teaching materials, 1 English monograph, and obtained more than 100 invention patents, and has cofounded 3 venture companies.
Professor of Biomedical Pioneering Innovation Center, Peking-Tsinghua Center for Life Sciences, and the School of Life Sciences at Peking University, and the director of Peking University Genome Editing Research Center. The research of Wei group is mainly focused on the development of eukaryotic gene editing tools with the emphasis on high-throughput functional genomics, gene and cell therapy, and the novel vaccination approach based on circular RNAs.
Saccharomyces cerevisiae is far from being the only yeast of potential scientific and economic importance. Many of the 2000 other known yeast species have highly unusual metabolic, biosynthetic, and physiological capacities. However, significant hurdles lay in front due to the missing radical technologies to effectively engineer nonconventional microorganisms. Our research is geared towards illustrating how to design systematic rules and enable generalizable technologies that can be effectively applied from one species to another. A common technology hurdle for engineering nonconventional species is that majority of them preferentially employ non-homologous end joining (NHEJ) to repair DNA double-strand breaks (DSB), which prohibits precise CRISPR/Cas9 genome editing. Most recently, we identified the shortcomings of the conventional genome editing strategies and developed a new CRISPR platform, named Lowered Indel Nuclease system Enabling Accurate Repair (LINEAR), which enabled precision editing without NHEJ disruption with efficiencies of 67-100% in four industrially relevant yeasts. With NHEJ preserved, we demonstrated its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yielded favorable expression dynamics for heterologous pathways. A case study will be presented to demonstrate how to leverage an antagonizing pair of DNA DSB repair mechanisms to rapidly engineer a microbial factory to produce (S)-norcoclaurine, an entrance molecule of the large group of benzylisoquinoline alkaloids with medicinal applications. Taken together, the development of radical technologies pushes the frontier of synthetic biology to explore novel species and accelerates strain engineering for generating interesting products.
Zengyi Shao received her Ph.D. in Chemical and Biomolecular Engineering from the University of Illinois, Urbana-Champaign in 2009. She is now a Vernon Guse Associate Professor in the Department of Chemical and Biological Engineering at the Iowa State University and affiliated with the NSF Center for Biorenewable Chemicals (CBiRC) and the DOE Center for Advanced Bioenergy and Bioproducts Innovation (CABBI). Her research group strives to develop platform strain-engineering technologies to explore the biochemical and biomedical potentials of high-performance microbes. She is the awardee of the National Academies Keck Futures Initiative Award (2010), the Iowa Energy Center Impact Award (2016), the NSF CAREER Award (2018), the Bailey Research Career Development Award (2021), and the NIH Maximizing Investigators' Research Award (2021).
Epigenetic regulation allows genetically identical cells to generate distinct gene expressions and phenotypic states that persist during cell division. The repurposing of epigenetic regulation offers great opportunities for the development of next-generation cellular control. While the engineering of epigenetic regulation is promising, it remains challenging due to the lack of identifiable parts and components sufficient for epigenetic behaviors.
We apply a bottom-up synthetic biology approach to develop a minimal synthetic epigenetic system based on the principle of classic “read-write” motif. We exploit DNA adenine methylation (6mA), a DNA modification that is rarely found in metazoan genomes, to create a fully synthetic chromatin system in human cells. Together with a quantitative model of chromatin dynamics, we show these circuits mediate the spreading of the modification to regulate genes at a distance and the establishment of long-term epigenetic memory, demonstrating the sufficiency of the read-write mechanism for epigenetic memory. We also explore additional motifs governing epigenetic regulation, one of which is the 3D chromatin structure. We combine user-defined synthetic control of epigenetic silencing and super-resolution 3D chromatin imaging to quantitatively delineate 3D chromatin organization and epigenetic memory mechanisms at single-nucleus resolution.
Minhee Park received her Ph.D. in Biomedical Engineering from Boston University in the lab of Prof. Ahmad Khalil in 2019. In her thesis work, she developed an artificial epigenetic regulatory system from bottom-up, taking a synthetic biology approach to study and control epigenetic regulation in mammalian cells. She then moved to Stanford University for her post-doc training as a Damon Runyon Postdoctoral Fellow in the lab of Prof. Alistair Boettiger. Leveraging her expertise at the intersection of synthetic biology and chromatin/epigenetics, she worked on studying mechanisms governing spatial chromosome organization across genomic scales and their implications on gene regulation using super-resolution single cell 3D chromatin imaging technique developed in the Boettiger lab. After post-doctoral training, she moved to KAIST in 2021 as an Assistant Professor in the Department of Biological Sciences. Her group focuses on studying the basic grammar rules of chromatin regulation for better mammalian epigenome engineering.
Terpenoids are a class of high-value natural products with wide application in both industrial and human health spaces, but sourcing them from nature or deriving from petrochemicals is no longer sustainable. Microbial biosynthesis of terpenoids has emerged as the most commercially viable option for their large-scale production. However, available efficient microbial cell factories are often hampered by unknown metabolic pathways and/or frustrated performance of heterologous pathways in host. Here, we will show some works and experience how to uncover the metabolic pathways of terpenoids in plants and their efficient production in microbial cell factories.
Dr. Ma obtained his PhD degree at Chinese Academy of Agricultural Sciences at 2017. He is performing his postdoctoral research at MIT under the supervisor of Prof. Gregory Stephanopoulos since 2018. His interests mainly focus on synthetic biology of plant natural products. On the one hand, he established the multi-omics-based research system for plant secondary metabolism, and systematically uncovered the metabolic pathways, regulation and transport mechanisms of many plant natural products. On the other hand, He designed and reconstituted the microbial cell factories for efficient synthesis of natural products by synthetic biology and metabolic engineering approaches. He aims to combine the multi-omics, synthetic biology, metabolic engineering, protein engineering and artificial intelligence, and to explore the alternatives for changing the access to raw materials instead of traditional ways. His works were published in Science, Nature Plants, Nature Communications, PNAS, etc.
The genome of an organism is inherited from its ancestor and keeps evolving over time, however, how much the current version could be altered remains unknown. Here, we used the left arm of chromosome XII (chrXIIL) as an example to probe the genome plasticity in Saccharomyces cerevisiae. A neochromosome was designed to harbor originally dispersed essential genes in chrXIIL. Using a synthetic version of chromosome XIIL, we systematically probed the essentiality of sequences in chrXIIL by targeted DNA removal, chromosome truncation and random deletion. We found that a combination of 12 genes on this arm is sufficient for cell viability. However, 25 genes are required to retain near wild type fitness. Next, we demonstrated most of these genes could be reconstructed using completely synthetic promoters and terminators, and recoded open-reading frame with “one-amino-acid-one-codon” strategy. Finally, by arranging these reconstructed genes together, we designed and built a neochromsome, which could substitute for chrXIIL for cell viability. Our work not only directly demonstrates the high plasticity of yeast genome, but also illustrates the possibility of making completely artificial chromosomes to replace the native genome.
Zhouqing Luo is now a professor at Xiamen University, a member of the Youth Innovation Promotion Association of Chinese Academy of Sciences, and awarded the Young Elite Scientists Sponsorship Program by China Association for Science and Technology. He graduated from Xiamen University with a bachelor's degree in 2017 and from Tsinghua University with a doctor's degree in 2017. His main research interests include synthetic biology and epigenetics. Up to now, he has published papers in journals including Science, PNAS, Cell Research and Nature Communications, revealing new regulatory mechanisms of histone modification and realizing the construction, minimization and application of yeast synthetic chromosomes.
Corynebacterium glutamicum, a Gram-positive bacterium, is an important industrial workhorse. However, its genome synthesis is impeded by the low efficiencies in DNA delivery and in genomic recombination/replacement. Recently, we described a genomic iterative replacement system based on RecET recombination for C. glutamicum, involving the successive integration of up to 10 kb DNA fragments obtained in vitro, and the transformants were selected by the alternative use of kanR and speR selectable markers. As a proof of concept, we systematically redesigned and replaced a 54.3 kb wild-type sequence of C. glutamicum ATCC13032 with its 55.1 kb synthetic counterpart with several novel features, including decoupled genes, the standard PCRTags, and 20 loxPsym sites, which was for the first time incorporated into a bacterial genome. The resulting strain semi-synCG-A1 had a phenotype and fitness similar to the wild-type strain under various stress conditions. The stability of the synthetic genome region faithfully maintained over 100 generations of nonselective growth. Upon induction of synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE), genomic deletions, inversions, and translocations occurred in the synthetic genome region, revealing potential genetic flexibility for C. glutamicum. To date, we have accomplished >5% (~160kb) of the genomic replacement. To scale up the synthesis and editing of the C. glutamicum genome, we have been testing a BAC-like vector for C. glutamicum. These strategies can be used for the synthesis of a larger region of the genome and facilitate metabolic engineering of C. glutamicum.
Zhanglin Lin is a professor at the Shenzhen Institute of Advanced Technology. He received an PhD in molecular biology from the University of Maryland, College Park, and completed his postdoctoral training in Frances Arnold Lab at the California Institute of Technology. He was a professor at Tsinghua University for 15 years, and a professor at the South China University of Technology for 6 years. In 2022, he joined at the Shenzhen Institute of Advanced Technology as a professor. His research interests include protein engineering, protein synthesis, and synthetic biology.
Plant synthetic biology is an emerging research field, which has been exhibiting great promise to introduce new traits into plant. However, it is still in its infancy, with many knowledge and technology gaps that remain to be addressed. For example, the unpredictable performance of introduced proteins and the lack of well-established genetic parts, severely impede the accurate control of synthetic pathways modulation, metabolic flux regulation, and new traits introduction. In this meeting report, we gave two examples of pathway engineering in a plant-based chassis-N. benthamiana, by using different strategies. In one study, we optimized the taxane biosynthesis using chloroplast metabolic engineering. In another study, we quantitatively characterized a library of plant-based promoter and terminators, and further optimized the synthesis of betalian by rationally selecting those well-established parts.
Jianhua Li, associate professor. Dr. Li received his PhD in pharmacognosy from Peking Union Medical College-Institute of Materia Medica in 2014. In the same year, he joined in the biosynthesis of natural product research group, leaded by Prof. Yong Wang, in CAS Center for Excellence in Molecular Plant Sciences. His main research interests are focused on deciphering the synthetic mechanism of natural products, and further re-constructing and bio-engineering their synthetic pathway in a heterologous chassis. He also works on the plant-based biotechnologies, such as DNA fragment assembly, DNA transmission, plant genome editing and metabolic pathway regulation.
One of the goals of synthetic biology is to design and assemble organisms from the ground up. Rapid advances in DNA synthesis technology have enabled the assembly of complete chromosomes in bacteria and yeast. However, it remains a challenge to apply synthetic genomics technologies in multicellular organisms. Here I present a pilot experiment that assembles chromosomal arm-level fragments in the model moss Physcomitrium patens. A ~150 kb region on chromosome 18L has been replaced with a 75 kb synthetic fragment. In addition to removal of destabilizing elements, systemic modifications are included. 3D genome organization analysis suggests changes of genome organization. I will also discuss effects to overcome obstacles hindering genome assembly in seeds plants. Elucidating the principles of single cell regeneration holds promise to increase regeneration efficiency and pave the way for genome assembly.
Yuling Jiao earned his B.S. degree in Biochemistry and Molecular Biology at Peking University and his M.S. and Ph.D. in Molecular, Cellular and Developmental Biology at Yale University. After postdoctoral research at the California Institute of Technology in the laboratory of Prof. Elliot Meyerowitz, he started his own research group at the Institute of Genetics and Developmental Biology in 2010. He has been a professor at the School of Life Sciences, Peking University since 2021. His group combines multidisciplinary approaches to study plant development. More recently, his group has spearheaded plant synthetic genomics research, and aims at establishing the first synthetic plant chromosome.
Synthetic biology tools have the potential to support wider advanced applications of cell and gene-based therapies through introduction of new modalities of regulating mammalian cells. Advancing beyond the traditional transcriptional regulation by ectopic proteins harvested from other organisms we introduced sensors originating from endogenous proteins (1), faster and more potent response to the chemical signals (2-4) and allosteric regulation of protein function through coiled-coil peptides (5). Translation to therapeutic applications has been limited due to unfavorable ligand characteristics or an immune response in vivo to non-human protein domains. A strategy for engineering inducible split protein regulators based on human proteins (INSPIRE) was devised, with physiological ligands or clinically approved drug as the regulators. The INSPIRE platform can be used for the dynamic, orthogonal, and multiplex control of gene expression in mammalian cells and we demonstrated glucocorticoid sensing and response in vivo (1). While transcriptional regulation is widely used, faster response is required for some physiological processes. This could be accomplished using chemicaly regulated proteases that can rapidly activate the selected cellular processes or release of the synthesized proteins to the plasma membrane or for secretion (2,4). Designable coiled-coil dimers have demonstrated great potential to drive diverse processes within mammalian cells (2,3). Finally, a protein function regulation INSRTR (inserted peptide structure regulator) is based on the insertion of a coiled-coil forming peptide into the selected protein. Formation of a heterodimer disrupts the protein function. This platform enables the construction of ON/OFF protein switches, regulation by small molecules, and logic functions with rapid response in mammalian cells (5). INSRTR was demonstrated on ten different proteins with diverse functions including enzymes, signaling mediators, DNA binders/transcriptional regulators, fluorescent protein, and antibodies implemented as a sensing domain of anticancer chimeric antigen receptors on T cells, offering extraordinary potentials for regulation of biological systems and therapeutic applications.
1. Rihtar et al., Nat.Chem. Biol. (2022) doi: 10.1038/s41589-022-01136-x
2. Fink et al. Nat.Chem. Biol. (2018) 15 (2), 115-122.
3. Lebar et al. Nat.Chem.Biol. (2020) 16 (5), 513-519.
4. Praznik et al., Nat.Commun. (2022) 1323.
5. Plaper et al. bioRxiv (2022) doi:10.1101/2022.06.03.494683.
Roman Jerala is head of the Department of synthetic biology and immunology at the National institute of chemistry in Ljubljana, Slovenia and professor at the University of Ljubljana. He obtained PhD in chemistry at the University of Ljubljana and was a postdoc at the University of Virginia USA. Upon return to Slovenia he moved to the National institute of chemistry and organized a strong group and established a Department of synthetic biology and immunology, working on diverse topics, including antimicrobial peptides, prion proteins and in the last two decades synthetic biology and molecular immunology. His group contributed to the advance of synthetic biology with innovative ideas in the area of mammalian synthetic biology and protein design, which includes the coiled-coil protein origami. Recently his group put strong emphasis on translational medicine including gene and cell therapy of cancer and genetic diseases using the arsenal of synbio and genome engineering tools.
He is member of the European Molecular Biology Organization (EMBO), member of the Slovenian Academy of Sciences and Arts, Academia Europaea and a recipient of the ERC Advanced Grant. He published his work in Nature Chemical Biology, Nature Biotechnology, Nature Communications, Science Advances, PNAS, Blood, JACS, Nucleic Acids Research and many other journals.
Plant specialized metabolites are bioactive molecules that have found wide applications in industry. However, they are usually produced by plants at very low contents and under specific conditions, posing significant challenges for their discovery and synthesis and further utilization. Uncovering their biosynthesis opens avenue towards accessing their structure, bioactivity and production. Nicotianna benthamiana has emerged to be an outstanding plant-based expression platform for eludidating functions of biosynthetic genes involved in plant specialized metabolite biosynthesis and engineering for their production, owing to its features including large biomass, fast-growing, rich endogenous metabolic bulding blocks, amenable to transient expression and scale-up infiltration. In this talk, I will present our recent discovery of plant specialized metabolites and their biosynthesis using N. benthamiana-based expression system and our efforts on engineering N. benthamiana for producing various types of plant specialized metabolites.
Dr. Ancheng Huang obtained his Ph.D. degree from The University of Adelaide, Australia, in 2014, receiving the ‘Dean's Commendation for Doctoral Thesis Excellence’. Dr Huang moved to the John Innes Centre, UK, working on natural product biosynthesis under the supervision of Professor Anne Osbourn (FRS) in 2015 with the support of a Marie Curie Individual Fellowship. He returned to China in 2019 and is now an Associate Professor in the Department of Biology of Southern University of Science and Technology. Dr Huang has published over 20 papers on international journals including Science, PNAS and Angew Chem. Int. Ed. His research is centered on the interface of plant chemistry and biology, with a particular focus on natural product biosynthesis. His team employs multi-disciplinary approaches ranging from chemistry and genetics to bioinformatics to address fundamental questions concerning how and why natural products are made in nature and mechanisms underpinning enzyme catalysis, with a view to developing production platforms for high-value natural products using synthetic biology.
With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the studies of and therapeutic interventions for specific cell types/states in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular and programmable design for live RNA sensing using adenosine deaminases acting on RNA (RADAR). We validated and then expanded our basic design, characterized its performance, and analyzed its compatibility with the human/mouse transcriptomes. We identified strategies to boost output levels and improve the dynamic range. We show that RADAR is programmable and modular, and uniquely enables compact AND logic. In addition to responding to the levels of transcripts, RADAR can potentially distinguish disease-relevant alterations of transcript identities, such as point mutations and fusions. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.
Dr. Xiaojing Gao is an Assistant Professor of Chemical Engineering from Stanford University. He received a B.S. in Biology from Peking University and a Ph.D. in Biology from Stanford University. He received his postdoctoral training from Biology and Biological Engineering at Caltech. His lab tackles fundamental engineering challenges across different levels of complexity, such as (1) protein components that minimize their crosstalk with human cells and immunogenicity, (2) biomolecular circuits that function robustly in different cells and are easy to deliver, (3) multicellular consortia that communicate through scalable channels, and (4) therapeutic modules that interface with physiological inputs/outputs. Their engineering targets include biomolecules, molecular circuits, viruses, and cells, and their approach combines quantitative experimental analysis with computational simulation. The molecular tools they build will be applied to diverse fields such as immunology, neurobiology, and cancer therapy.
The site-specific incorporation of the noncanonical amino acids (ncAAs) into proteins via genetic code expansion (GCE) has enabled the development of new and powerful ways to learn, regulate and evolve biological functions in vivo. Among several expression systems, yeast cell factories combine the advantages of being single cells, such as fast growth and easy genetic manipulation, as well as eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications. Here, I will present our recent progress in creating efficient genetic code expansion system in yeast cells by toolbox development and chassis engineering, as well as some applications of the GCE technology in yeast.
Dr. Xian Fu earned his Ph.D. from University of Florida mentored by Dr. Julie Maupin-Furlow in 2016. His Ph.D. thesis uncovered key components involved in targeted proteolysis and ubiquitin-like protein modification in archaea. He finished the postdoctoral training in Dr. Dieter Söll’s lab at Yale University to develop novel ways for biosynthesis of selenoprotein. He later joined the synthetic biology platform of BGI-Research at Shenzhen as a principal investigator. By combining synthetic genomics, genome mining, and advanced directed evolution methods, his main research interests focus on developing efficient genetic code expansion system in yeast to genetically encode a variety of “designer” noncanonical amino acids (ncAAs) for investigation of fundamental biology and industrial applications. So far, he has published 23 peer-reviewed articles. He also serves on the editorial board of Frontiers in Genetics.
The large intestine is the site of many human diseases yet is difficult to access with drugs due to digestion. At the same time, the gut provides a habitat for a numerous and diverse population of microbes that perform important, health-relevant chemistries. Our group is exploring the possibility of genetically engineering gut microbes to produce therapeutic molecules in the gut, ultimately improving drug delivery and reducing drug costs. In this talk, I will discuss our lab’s recent progress in optimizing both small molecule and peptide biosynthesis in the probiotic yeast Saccharomyces boulardii, as well as our efforts to enhance S. boulardii’s in vivo residence time. I will also discuss some new work from our lab focusing on the development of gut- and small-molecule inducible promoters for this organism.
Dr. Crook received his B.S. in Chemical Engineering from the California Institute of Technology in 2009, and his Ph.D. in Chemical Engineering from the University of Texas at Austin in 2014, studying under Dr. Hal Alper. He pursued postdoctoral studies in Pathology and Immunology at Washington University in Saint Louis School Medicine from 2014-2017 in the lab of Dr. Gautam Dantas. Dr. Crook joined the department of Chemical and Biomolecular Engineering at NCSU in January 2018, and his lab focuses on how to engineer microbial communities.
Artificial neural networks provide a powerful paradigm for information processing that has transformed diverse fields. Within living cells, genetically encoded synthetic molecular networks could, in principle, harness principles of neural computation to classify molecular signals. Here, we combine de novo designed protein heterodimers and engineered viral proteases to implement a synthetic protein circuit that performs winner-take-all neural network computation. This “perceptein” circuit includes modules that compute weighted sums of input protein concentrations through reversible binding interactions, and allow for self-activation and mutual inhibition of protein components using irreversible proteolytic cleavage reactions. Altogether, these interactions comprise a network of 310 chemical reactions stemming from 8 expressed protein species. The complete system achieves signal classification with tunable decision boundaries in mammalian cells. These results demonstrate how engineered protein-based networks can enable programmable signal classification in living cells.
The wealth of knowledge on the human microbiota composition and its roles in health and disease has recently spurred the development of novel therapeutic strategies. Moreover, with an array of genetic tools that are readily available, programmable genetic circuits can be designed, genomes can be edited and rewritten, and cells can be reprogrammed to foster novel microbiota-based interventions. In this talk, our recent work on engineering gut-resident microbes as versatile platforms equipped with clinically relevant functionalities will be presented. A particular emphasis will be placed on our efforts to transform gut microbes into live biotherapeutics with prophylactic and therapeutic efficacy against pathogenic infections and chronic metabolic diseases. This work provides a strong foundation for engineering microbes to modulate host-microbiome interactions and supports the use of live biotherapeutics as a viable strategy for clinical intervention.
Matthew Chang is Director of the Singapore Consortium for Synthetic Biology, Wilmar-NUS Corporate Laboratory, and NUS Synthetic Biology for Clinical and Technological Innovation, and Dean’s Chair in Medicine and Associate Professor of Biochemistry and Synthetic Biology at the Yong Loo Lin School of Medicine at the National University of Singapore. His research focuses on studying the engineering of biology to develop autonomous, programmable cells for biomedical and biomanufacturing applications. He co-founded the Global Biofoundry Alliance and the Asian Synthetic Biology Association and serves on the World Economic Forum’s Global Future Council on Synthetic Biology.
The tunability, reversibility, and orthogonality of light inputs make optogenetics a powerful strategy to dynamically control microbial metabolisms engineered for chemical production. My group has developed several transcriptional optogenetic circuits to dynamically control the expression of metabolic enzymes during the growth and production phases of microbial fermentations. These circuits can be designed to invert or amplify metabolic responses to light inputs to boost the production of various chemicals. The activation rates of these circuits can have a significant impact on chemical yields and titers, and on our strategies to use open- or closed-loop controls of metabolism. We have found ways to increase these activation rates by manipulating the protein stability of circuit components. However, to further increase the rates of response to light inputs beyond the limitations imposed by transcription, we have also developed post-translational optogenetic controls. Some of these controls are based on highly specific protein binders, such as nanobodies, that can be used to control enzymatic activity post-translationally, which can also be engineered to be light-switchable. I will discuss how these various optogenetic technologies can be integrated to optimize microbial fermentations for chemical production.
José Avalos is an Associate Professor in the department of Chemical and Biological Engineering, the Andlinger Center for Energy and the Environment, and the Bioengineering Initiative at Princeton University. He earned a B.E. in chemical engineering from Universidad Iberoamericana in Mexico City and an MSc in biochemical research from Imperial College in London. He then received a Ph.D. in biochemistry and biophysics from Johns Hopkins University. He conducted postdoctoral research at The Rockefeller University on molecular neuroscience, and then at MIT/Whitehead Institute, in the Department of Chemical Engineering on metabolic engineering and synthetic biology. He has been a faculty member at Princeton University since 2015, where he leads a research group focused on the use of biotechnology to address challenges in renewable energy, sustainable manufacturing, the environment, and human health. He has received several awards, including the Damon Runyon Cancer Research Fellowship, the NIH Ruth L. Kirschstein National Research Service Award, the Alfred P. Sloan Foundation Research Fellowship Award, the Pew scholarship, the NSF CAREER Award, the Camille Dreyfus Teacher-Scholar Award, the HHMI Gilliam award, and the ACS BIOT Young Investigator Award.
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