Shanghai Jiao Tong University
Expanding the Metabolic Power of the Mined Natural Products Pathways
In this talk, I will discuss the future directions towards the discovery and development of novel antibiotics NPs by pathway engineering, combinatorial biosynthesis and synthetic biology. Two classes of antibiotics will be the central theme for my presentation. First is an old class of aminoglycosides (AGs) drugs, which remains a valuable component of the antibiotic arsenal, despite their inherent toxicity and the global spread of bacterial resistance. Recent studies have continued to reveal the fascinating biochemistry of AG biosynthesis and the rich potential in their pathway engineering. In particular, parallel pathways have been shown to be common and widespread in AG biosynthesis, highlighting nature's ingenuity in accessing diverse natural products from a limited set of genes.
A second extensively investigated group of NPs is the structurally-related peptidyl nucleoside antibiotics. Their mechanistic insights representing very distinctive enzymatic reactions involved in the biosynthesis of their building blocks, nucleoside skeleton and peptidyl moieties were successfully used for the engineering of the industrial polyoxin producer as efficient cell factories for components optimization and successful production of the hybrid nucleoside antibiotics. As successful examples, introducing heterologous genes from one nucleoside antibiotics nikkomycin producer into an industrial polyoxin producer generated seven polyoxin-nikkomycin designer hybrid antibiotics (designated as nikkoxin A-G), some of which were significantly more potent against indicator human or plant fungal pathogens than both parental antibiotics.
Apparently, elucidation of the molecular mechanisms for aminoglycosides as well as for different nucleoside antibiotics biosynthesis would greatly expand the ways for rational designing and generation of natural artificial molecules with enhanced/selective bioactivity.
Seoul National University
Lactic acid production using glucose or methane
There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Currently, glucose and other biomass-derived sugars are used as major carbon sources for microbial fermentation, but other abundant and inexpensive carbon sources such as CO2, CO, and methane are considered promising alternatives to sugars. We engineered Saccharomyces cerevisiae and Methylomonas sp. DH-1, a methanotroph which can use methane as a sole carbon and energy source, to produced D-LA using glucose and methane, respectively. LA toxicity is one of the limiting factors for high-level production of LA. Therefore, Saccharomyces cerevisiae, having high acid tolerance, is a promising host for LA production. We generated S. cerevisiae strains producing D-LA with high efficiency under acidic fermentation conditions by introducing D form-specific lactate dehydrogenase, eliminating ethanol and glycerol production pathways, and adaptive laboratory evolution to increase lactate tolerance. In addition, mutated genes responsible for lactate tolerance in the evolved strains were identified and characterized. We also used adaptive laboratory evolution to increase lactate tolerance of Methylomonas sp. DH-1 and identified the genes responsible for the tolerance. When D form-specific lactate dehydrogenase was introduced into the genome, the evolved strain produced about 8-fold higher level of D-LA from methane compared to wild-type strain, suggesting that LA tolerance is a critical limiting factor for LA production in this host.
University of Illinois at Urbana-Champaign
Rapid Engineering of Yeast Cell Factories using Iterative Cycles of Design, Build, Test, and Learn on a Genome Scale
Advances in reading, writing and editing genomes have greatly expanded our ability to reprogram biological systems at the resolution of a single nucleotide and on the scale of a whole genome. Such capacity has drastically accelerated the cycle of design, build and test in synthetic biology to construct organisms as cell factories for synthesis of fuels and chemicals. In this presentation, I will introduce three new strategies for genome-scale engineering of Saccharomyces cerevisiae, a prominent industrial production host. These strategies include: (a) a CRISPR/Cas9 and homology-directed repair assisted genome-scale engineering (CHAnGE) method for rapid engineering of S. cerevisiae with single nucleotide resolution; (b) a tri-functional CRISPR-Cas system for simultaneous gene activation, interference, and deletion in S. cerevisiae either for a set of pre-selected targets or on a whole genome scale; and (c) design and construction of the RNA interference machinery for automated genome-scale engineering in S. cerevisiae. Coupled with our recently developed Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB), these strategies should greatly accelerate the metabolic engineering of S. cerevisiae for production of value-added products and provide new insights into cellular metabolism and physiology.
National University of Singapore
Programmable biological functionalities for autonomous cell therapeutics
Advances in sequencing technologies have greatly increased our knowledge of the composition of the human intestinal microbiota and its importance in health and disease. Various omics and molecular studies have also revealed further insights in host-microbiome interactions at the cellular and molecular level. In order to leverage the close associations between microbes and their host, the development of therapeutics targeting the microbiota has surged in recent years. Many advanced microbiota-targeting intervention strategies are being explored, ranging from the selection of novel probiotic strains and synthetic stool substitutes to maintaining the dynamics of metabolism by prebiotics and dietary interventions. Applying engineering biology to reprogramme gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics and deliver therapeutics. Herein, we present our work in exploiting commensal microbes to develop therapeutic microbes with programmable functionalities to prevent pathogenesis of various diseases. In particular, we have established and tested in vivo a combination of clinically relevant functionalities to effectively exert specific activities against opportunistic pathogens and chronic metabolic diseases such as cancer. This work provides a strong foundation for the use of engineered functional microbes that modulate microbiota-host metabolism and interactions as a viable clinical intervention.
National Institute of Advanced Industrial Science and Technology (AIST)
Bacterial cellular engineering by ribosome engineering
Bacterial ribosomes are structurally/functionally complex molecules that comprise 3 RNAs and more than 50 proteins with a molecular mass of ~2.3 MDa. Because of this complexity, ribosomal components, particularly the 16S and 23S ribosomal RNAs, have long been believed to be species-specific: no horizontal gene transfer should occur. Despite this general view, however, we recently found that the Escherichia coli ribosome is unexpectedly robust enough to accommodate foreign 16S rRNA in place of self 16S rRNA [1, 2]. We applied this RINSPEX (rRNA interspecies exchange) technique to bacterial cellular engineering. We assumed that the alteration in ribosomal function caused by RINSPEX directly affects the proteomic profile, which would in turn perturb cellular metabolism and phenotypes of the host strain. As a proof of concept, we attempted to evolve E. coli to a high temperature through rounds of interspecies exchange of 16S rRNA. After iterative rounds of substitution of the full-length and/or portion of the gene, we obtained a mutant strain, which showed enhanced growth at 45°C. We applied the same technique to a thermophilic bacterium, Thermus thermophiles, to obtain mutants that were capable of growing under wide range of temperatures, where the growth of the wild type strain was severely inhibited.
1. Kitahara K, Yasutake Y, and Miyazaki K (2012) Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli. PNAS 109(47):19220-19225
2. Tsukuda M, Kitahara K, Miyazaki K (2017) Comparative RNA functional analysis reveals high functional similarity between distantly related bacterial 16S rRNAs. Sci Rep 7:9993
Institute of Bioengineering and Nanotechnology
Synthetic Xenobiology using genetic alphabet expansion
A new research area, synthetic xenobiology, has rapidly advanced by the advent of artificial extra base pairs (unnatural base pairs) that function as a third base pair in replication, transcription, and/or translation. Unnatural base pairs can expand the genetic alphabet of biology systems in the central dogma. By applying unnatural base pairs to biotechnology, novel biopolymers with increased functionality were developed. Semisynthetic organisms containing unnatural base pairs were created, and proteins containing non-standard amino acids were produced by the organisms. In this talk, by focusing on our research, I introduced unnatural base pair systems and their applications to high-affinity DNA aptamers that specifically bind to target proteins and cells for diagnostics and therapeutics.
Fong Tian Wong
Molecular Engineering Laboratory
Discovery of New Molecules Via CRISPR/Cas9-Mediated Activation of Cryptic Gene Clusters
Natural products (NPs) are complex molecules with a diverse range of functions, where most natural products have evolved medicinal properties. Even though much NPs have been uncovered over the last half a century, discovery has slowed down in the early 2000s. Together with the advancement of genomics and our knowledge of biosynthetic pathways and enzymes, it has been predicted that we have been only probing ~10-20% of all potential NPs. Much of these potential have been locked in biosynthetic genetic clusters which are transcriptionally silent or not detectable under lab conditions. To activate these gene clusters, a well-tested workflow is to sequence their genomes, annotated the gene clusters, perform system-wide or pathway-directed perturbations and then screen for new molecules. To hasten this process, we have established a CRISPR-Cas9 mediated genome editing protocol for promoter knock-in, where we have not only halved the time compared to traditional protocols but also increase knock-in efficiencies. With the latter advantage, we can now even access strains which were genetically inaccessible before.
Synthetic Biology has opened a new perspective on how we view and approach manufacturing. In GSK, we hope to leverage on this frontier to drive sustainable manufacturing and deliver value to our patients. From development in the Lab to Manufacturing, a few examples on how GSK drives synthetic biology efforts within the company.
Wen Shan Yew
National University of Singapore
New Wine from Old Barrels: Repurposing Biology through Synthetic
Synthetic biology is the engineering of biology for purpose. One of the most common techniques in synthetic biology is the repurposing of tools and processes in Nature for a purposeful function. This normally involves the re-wiring of metabolic processes, channelling energy across systems for the purposeful production of therapeutics, nutraceuticals and ingredients, and more recently, in environmental sustainability. In the first part of the presentation, the biosynthesis of polyketides will be discussed. Polyketides are a large class of biomolecules that are naturally produced by bacteria, fungi and plants, and include many clinically important biomolecules with anti-cancer, anti-microbial, anti-oxidant and anti-inflammatory activities. They are biosynthesized from acyl-CoA precursors by polyketide synthases (PKSs), and due to their chemical complexity, are not easily synthesized and structurally manipulated by chemical means. In the second part of the presentation, efforts made in achieving environmental sustainability, allowing our society to progress without effecting environmental pollution and resource depletion, will be illustrated. Electronic waste represents a significant and growing portion of waste in our cities, and thus there is a pressing need to develop sustainable technologies to recycle electronic waste in order to protect our environment and preserve natural resources. Current recycling methods have proved to be environmentally and economically unsustainable; these treatment technologies for electronic waste use strong acids or cyanide that are pollutive in nature. Synthetic biology provides sustainable solutions to address challenges that confront society. Using Nature as inspiration, our synthetic biology-empowered approach will contribute towards Singapore’s efforts to transition to a green economy, furthering her efforts to become an environmentally sustainable city.
Biotransformation Innovation Platform
Total industrial biosynthesis of astaxanthin and other natural products
Efficient optimization of metabolic pathways with large number of genes (>10 genes) is challenging due to numerous biological limitations including, complexity of cellular metabolism and poor expression/activity of enzymes. To address these challenges, we developed a multidimensional heuristic process (MHP). By modularizing all the genes along biosynthetic pathways and controlling different functional dimensions (transcription, translation and enzymatic reactions), MHP provides a focused and systematic approach to balance the different modules (global pathway) and within specific module (local pathway). MHP adopts ‘modular design’ that significantly reduces experimental workload yet without losing the flexibility by simultaneously control different functional dimensions. As a proof-of-concept, we effectively enhanced the production of the 15-step heterologous biosynthetic route of astaxanthin, a high-value carotenoid with multiple clinical benefits. Currently, this is the highest reported productivity (184 mg/L/day) and titer (320 mg/L), paving the way for a sustainable, commercial production of astaxanthin. In addition, we have successfully applied MHP to optimize other natural products. With linalool (a C10 isoprenoid fragrance molecule), the titer was 65 mg/L in flasks, a greater than 684-fold enhancement over that previously reported. With nerolidol (C15 isoprenoid fragrance molecules), the titer in flask was 300 mg/L (comparable to highest reported titer) and with amorphadiene (a C15 isoprenoid, a precursor to the antimalarial drug artemisinin), the titer was 30 g/L (in bioreactors, comparable to highest reported titer). Other natural products that were enhanced by MHP included the production of α-ionone (a C13 apocarotenoid fragrance molecule) with a titer of 0.5 g/L (in bioreactors), β-ionone (a C13 apocarotenoid fragrance molecule) of 0.5 g/L (in bioreactors, >80-fold higher than previously reported) and retinol (a C20, vitamin A) of 0.5 g/L (in bioreactors). Collectively, these examples lend further evidence that MHP is an effective workflow for designing and optimizing complex metabolic pathways and is generally applicable to other biological pathways and products, beyond the scope of our study.
1. Zhang, C.*, Chen, X., Lindley, N. D., Too, H. P*. A Multidimensional Heuristic Process for the Total Biosynthesis of Astaxanthin and other Natural Products. Nat. Commun. (2018) (Accepted).
2. Zhang, C.*, Chen, X., Lindley, N. D. & Too, H.-P*. A ‘plug-n-play’ modular metabolic system for the production of apocarotenoids. Biotechnol. Bioeng. 115, 174–183 (2018).
3. Zhang, C.* & Too, H.-P*. Revalorizing lignocellulose for the production of natural pharmaceuticals and other high value bioproducts. CMC (2017).
4. Zhang, C., Chen, X., Stephanopoulos, G. & Too, H.-P*. Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli. Biotechnol. Bioeng. 113, 1755–1763 (2016).
5. Zhang, C., Zou, R., Chen, X., Stephanopoulos, G. & Too, H.-P*. Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production. Appl. Microbiol. Biotechnol. 99, 3825–3837 (2015).
Meng How Tan
Nanyang Technological University / Genome Institute of Singapore
Development and Evaluation of Novel CRISRP-Cas Genome Engineering Tools
In recent years, the CRISPR-Cas system has emerged as a powerful platform technology for the engineering of complex genomes. To date, multiple natural Cas endonucleases have been successfully used to edit the genomes of plants and animals, including human. Despite its tremendous potential, the CRISPR-Cas system suffers from several shortcomings that hamper its widespread adoption in biomedical and biotechnological applications. For example, the nuclease is known to cleave non-specifically at off-target sites. Additionally, the large size of the Cas protein poses a challenge for in vivo delivery, particularly when it is fused to additional effector domains. In this talk, I will describe our efforts to evaluate the performance of different natural Cas nucleases in mammalian genome editing and also share some of our work on developing novel strategies to overcome the limitations of existing CRISPR-Cas technologies.
Nestlé R&D Center, Singapore
From gene to activity – Exploring the potential of the Nestlé Culture Collection
Providing nutritious, healthy and sustainably produced food is one of the main objectives of food companies such as Nestlé. A culture collection of more than 3000 food grade strains (Nestlé Culture Collection, NCC), has been integrated into an R&D network covering microbiology, fermentation, food processing, nutrition and health, as well as clinical trials. Thanks to this approach and its research and technological capabilities, Nestlé has been able to develop and produce a large range of functional foods containing beneficial microbes.
Today the genomes of all NCC strains have been sequenced, assembled and annotated by using the complementary Illumina or Pacific Biosciences sequencing technologies, followed by assembly and annotation pipelines. Web based bio-informatic software enabling the storage and the analysis of the genomes have been implemented. The resulting genome databank allows direct evaluation and exploration of the NCC microbes for the development of fermented foods with enhanced taste and texture, functional benefits, and/or probiotics. The sequenced NCC can also be used to identify and source specific enzymes. In addition, the gene collection enables an easy screen for the presence/absence of undesired metabolic pathways or antibiotic resistance genes.
In this presentation we will illustrate how the newly developed bacterial genomic platform was used to go from gene to activity, bridging between in silico analysis and preclinical or proof of concept human trials. Examples relate to the use of lactic acid bacteria to deliver functional molecules or enzymes that could alleviate the consequences of specific food adverse reactions; identification and application of lactic acid bacteria as delivery systems for micro nutrients like iron; the use of specific lactic acid bacterial enzymes for the conversion of sugars into fibers; and the identification of metabolic routes for the production of desired flavor molecules.