Technical Program

BD4.0 Program 2018-04-23

 

Speakers

Opening Keynote

Andreas Bommarius
Georgia Institute of Technology


Towards a process model for continuous biotechnological processes

Continuous processes aim to run at a fixed, predetermined operating point. However, in biotechnological processes, there are several phenomena that cause deviations from a stable operating point, such as deactivation of biocatalysts, clogging of membranes, or simply changes in pH value over the course of the reaction or fermentation.
We will present a case that a detailed comprehensive model of the overall process is required for optimum performance or even for stable operation. Such a model is coupled with appropriate Process Analytical Technology (PAT), which establishes the range of probes, whether in-line or at-line, which all continually feed data of the state of the system. Process control allows the use of such data to detect deviations form a stable operating point and take appropriate countermeasures. Stable, model-assisted continuous operation requires models of the kinetics, the state of the cells or biocatalyst, the reactor, and any associated rate processes, such as crystallization. We will discuss cases where either the temperature and/or the pH value vary over time.
The enzyme-catalyzed synthesis of semi-synthetic beta-lactams, such as ampicillin, amoxicillin, or cephalexin, is an established process. However, the process is plagued by high cost of goods (COGS) as well as incomplete selectivity and conversion; the selectivity issue is caused by hydrolyses of both the electrophile (p-hydroxy)phenylglycine ester as well as the final product itself.
We have developed an overall process model that incorporates i) pH effects during the Pen G acylase (PGA)-catalyzed reaction [1], yielding higher accuracy than previous attempts, ii) enzyme deactivation kinetics, based on a new method to distinguish between different deactivation models [2], iii) crystallization kinetics [3], and initial reactor/crystallizer design. We propose reactive crystallization to improve yield, selectivity, and cycle time.[4]

References
[1] MA McDonald, AS Bommarius, RW Rousseau, Chem. Eng. Sci. 2017, 165, 81-88
[2] MA McDonald, L Bromig, MA Grover, RW Rousseau, AS Bommarius, Chem. Eng. Sci., in revision
[3] LG Encarnación-Gómez, AS Bommarius, RW Rousseau, Ind. Eng. Chem. Res. 2016, 55, 2153–2162
[4] LG Encarnación-Gómez, AS Bommarius, RW Rousseau, React. Chem. Eng. 2016, 1, 321-9

Closing Keynote

Zixin Deng
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.

Session 1: Parts Design and Engineering

Zhi Li
National University of Singapore


Engineering new cascade biotransformations for bio-based fine chemical synthesis

Sustainable manufacturing of chemicals from renewable feedstocks is attracting increasing attention because of the oil depletion and global climate change. The advances of synthetic biology have enabled the fermentation of (hemi)cellulose-derived sugars to produce a variety of biobased bulk chemicals. To further extend the scope of synthetic biology for chemical synthesis from renewable feed stock, we have being working on a feasible and potentially general approach for green, efficient, and sustainable production of high-value fine chemicals from the easily available biobased bulk chemical by developing novel types of one-pot cascade biotransformations.
We recently established several new types of regio- and enantio-selective cascade transformations, engineered recombinant Escherichia coli strains expressing the necessary enzymes as simple and active whole-cell biocatalysts for these reactions, and demonstrated the synthetic potential of the developed whole-cell based cascade biotransformations. Some representative examples will be presented: a) asymmetric conversion of L-phenyl alanine to (S)-styrene oxide;[1] b) (S)-1-phenylethane-1,2-diol and (R)-1-phenylethane-1,2-diol;[1] c) (S)-mandelic acid;[1] d) (S)-phenyl glycine and (R)-phenyl glycine;[1-2] and e) 2-phenylethanol.[3-4]

References
[1] Zhou, Y.; Wu, S.; Li, Z. Cascade biocatalysis for sustainable asymmetric synthesis: from biobased L-phenylalanine to high-value chiral chemicals. Angew. Chem. Int. Ed., 2016, 55, 11647 –11650.
[2] Zhou, Y.; Wu, S.; Li, Z. One‐pot enantioselective synthesis of D‐phenylglycines from racemic mandelic acids, styrenes, or biobased L‐phenylalanine via cascade biocatalysis Adv. Synth. Catal. 2017, 359, 4305-4316 (Very important publication).
[3] Wu, S.: Liu, J.: Li, Z. Biocatalytic formal anti-Markovnikov hydroamination and hydration of aryl alkenes. ACS Catalysis, 2017, 7, 5225-5233.
[4] Lukito, B. R.; Wu, S.; Saw J. H. J., Li, Z. Manuscript to be submitted.

Wen Shan Yew
National University of Singapore


New Wine from Old Barrels: Repurposing Biology through Synthetic Biology

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.

Ee Lui Ang
Institute of Chemical and Engineering Sciences


Green Fluorination Technology: Engineering of the Fluorinase and a Coupled Halogenase System

Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and their high specificity for the S-adenosyl-L-methionine (SAM) substrate has restricted their applications as biocatalysts. Herein, I will present our efforts at engineering the enzyme to expand its substrate range and improve its activity on a non-native substrate, 5’-chloro-5’-deoxyadenosine (5’-ClDA), to 5’-fluoro-5’-deoxyadenosine (5’-FDA). The molecular determinants of fluorinase specificity were probed using 5’-chloro-5’-deoxyadenosine (5’-ClDA) analogs as substrates and active site mutants. Modifications at key residues were found to be beneficial towards these modified substrates, including 5’-chloro-5’-deoxy-2-ethynyladenosine, ClDEA (>10-fold activity improvement), and conferred novel activity towards substrates not readily accepted by the wild-type fluorinase. We also discovered two new S-adenosyl-L-methionine (SAM)-dependent chlorinases, ClA1 and ClA2, from soil bacteria by genome mining. These chlorinases are several orders of magnitude more efficient in SAM synthesis from 5’-ClDA than a fluorinase. A coupled chlorinase-fluorinase system was developed for highly improved trans-halogenation of 5’-ClDA to 5’-FDA. The chlorinase also demonstrated the tolerance to the modification at the C-2 position of the adenosine substrate and acted coorperatively with the fluorinase to accelerate the trans-halogenation of ClDEA to 2-ethynyl-FDA (FDEA). The improved enzymes and coupled-enzyme system developed offer the prospect of developing rapid radiolabelling protocols under mild and aqueous conditions.

Session 2: Foundational Tools I

Jin-Soo Kim
Seoul National University


CRISPR Genome Editing

Genome editing with CRISPR systems that allows targeted mutagenesis in cells and organisms is broadly useful in biology, biotechnology, and medicine. Despite broad interest in CRISPR RNA-guided genome editing, Cas9, Cpf1, and Cas9-fused deaminases (a.k.a., Base Editors) are limited by off-target mutations. We developed nuclease-digested whole genome sequencing (Digenome-seq) to profile genome-wide specificities of Cas9 and Cpf1 nucleases and Cas9-fused deaminases in an unbiased manner. Digenome-seq captured in vitro cleavage sites at single nucleotide resolution and identified off-target sites at which indels or base conversions were induced with frequencies below 0.1%. We also showed that these off-target effects could be avoided by using preassembled ribonucleoproteins (RNPs) and modified guide RNAs. Digenome-seq is a robust, sensitive, unbiased, and cost-effective (< USD 1,500) method for profiling genome-wide off-target effects of programmable nucleases and deaminases.

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.

Keiji Nishida
Kobe University


Genome engineering by DNA base editing

In place of nuclease activity of conventional genome editing, DNA base-modifying reactions allow direct introduction of point mutations (base editing). Deaminase-mediated base editing tools (BE and Target-AID) have been developed by tethering DNA cytidine deaminases to nuclease-deficient CRISPR-Cas9 system, enabling pinpoint mutagenesis within the target range of 3-5 bases. The tools now have been applied to wide range of organisms. In mammals and plants, use of nickase Cas9 (D10A), which retains single-strand cleaving activity, greatly increased the efficiency, although it also occasionally induced insertion/deletion (indel). Co-expression of Uracil-DNA glycosylase inhibitor (UGI) further boosted the efficiency and reduced the indel formation. In E.coli, dCas9 is preferred and the use of UGI allows multiplex editing of up to 41 loci of multicopy elements. Several modifications and improvements have been made available for highest efficiency and mitigating unwanted effects, depending on the application.

References
1. Banno S, Nishida K*, Arazoe T, Mitsunobu H, Kondo A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol. Feb 5. doi: 10.1038/s41564-017-0102-6. (2018)
2. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H*, Nishida K*, Ariizumi T*, Kondo A. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol. Mar 27. doi: 10.1038/nbt.3833. (2017)
3. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, Aug 4. pii: aaf8729. (2016)

Session 3: Foundational Tools II

Huimin Zhao
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.

Chueh Loo Poh
National University of Singapore


Optogenetics for cell and cell-free Synthetic Biology

Many biological processes such as metabolism, cellular differentiation, and multicellular development involve precise coordination of gene expression. As a result, having the ability to achieve precise spatiotemporal control of gene expression is of great importance to advance our understanding on how cellular pathways function and could prove useful in biotechnological applications, including the development of cellular factories. To this end, optogenetic systems offer new ways to control gene expressions in precise spatial and temporal manner. However, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these limitations, we have recently engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using a blue light dependent DNA-binding protein EL222. We demonstrated that we can positively and negatively control target genes using a single transcription factor in a single cell depending on light illumination. This has potential in assisting dynamic control of gene expression during the optimization of metabolic pathways, particularly when toxic intermediates are involved. To expand the toolbox, we constructed and characterized a library of synthetic blue-light repressible chimeric promoters of varying strength to precisely tune gene expression in E. coli. Further, because of the portability of our system, we demonstrated that it is possible to achieve light control using the light-repressible promoter in a cell-free system. This opens up opportunity to test genetic circuits in cell-free TXTL system using light before moving to more complex cell systems. Overall, our toolbox of synthetic promoters represents a versatile platform for next-generation light-controllable synthetic biology systems in prokaryotes.

Jeroen Muller
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.

Kentaro Miyazaki
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.

References
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

Session 4: Therapeutics

Matthew Chang
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.

Ichiro Hirao
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.

Steven Tan
GlaxoSmithKline


GSK’s SynBio Efforts towards Sustainable Manufacturing

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.

Session 5: Cell Factories

Jibin Sun
Tianjin Institute of Biotechnology


Understanding, design and rebuild of industrial superbugs for amino acid production

Amino acids occupy millions of tons of worldwide market for food, feed, healthcare, material applications. Fermentative production of amino acids dates back to 1950s. With the recent development of systems and synthetic biology, the long-history industrial strains of amino acids were rediscovered and rebuilt for better production performance typically known as higher titer, higher yield and higher productivity. We worked with industrial partners on bulky or high value added amino acids including glutamate, lysine, threonine, 1,5-pentane diamine, hydroxyproline, 5-aminolevulinic acid and etc. since many years. Here we would like to discuss some key technologies and experiences for fast development/improvement of industrial strains for amino acid production, especially computer aided design, genome editing technology for industrial strains, and biosensor based high throughput screening.

Ji-Sook Hahn
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.

Kang Zhou
National University of Singapore


A Generic Strategy to Maintain Stable, Multi-Member Microbial Consortia

Microbial community is spotlighted recently in metabolic engineering applications, since it has many benefits such as combining strengths of multiple microbial species, accelerating strain development, and reducing metabolic burden on microbial workhorse. Despite the increasing attention from the relevant field, there are still some technical limitations on establishing stable microbial consortia, a feature required for developing industrially relevant processes. For example, current strategies to maintain stable co-culture often require use of certain carbon substrates, which are prohibitively expensive for many processes. Here, we report a generic strategy to maintain stable cell ratio of multi-member co-culture in glucose based medium. Chromogenic and fluorescent proteins have also been used to develop novel ways for visualizing cell ratio in bioreactor.

Simon Zhang
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.

References
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).

Michael Koepke
LanzaTech, Inc


Design and engineering of gas fermenting organisms for commercial scale production of fuels and chemicals from low-cost C1 feedstocks

Gas fermentation using anaerobic, acetogenic bacteria is an emerging technology for sustainable, high volume production of fuels and chemicals from low-cost, non-food feedstocks. Rather than relying on the use of sugars, acetogens have the ability for autotrophic growth on carbon monoxide (CO) and/or carbon dioxide (CO2) plus hydrogen (H2) as their sole source of carbon and energy via the Wood-Ljungdahl pathway, considered to be the most efficient C1 fixation pathway.
LanzaTech has developed a gas fermentation process utilizing a diverse range of feedstocks including waste gases from industrial sources (e.g., steel mills, processing plants or refineries) or syngas generated from any biomass resource (e.g., unsorted and unrecyclable municipal solid waste, agricultural waste, or organic industrial waste). The process has been successfully scaled up from the laboratory bench through in-lab and in-field pilot plants to fully integrated demonstration plants with over 70,000 hours of operation. First full commercial scale production plants (48k MTA) are expected to come online this year.
Less than 10 years ago, acetogens have been considered to be genetically inaccessible. LanzaTech has pioneered the development of a full genetic toolbox for the model acetogen Clostridium autoethanogenum, including large libraries of validated genetic parts, multiplexed genome editing tools and novel high-throughput workflow concepts. These approaches are complemented by the use of computer-aided design tools, predictive integrated models and deep learning approaches, which are enabled by large datasets across scales. Using this platform, production of over 50 molecules directly from gas have been demonstrated and first products have been successful transitioned to scale.

Session 6: Natural Products Discovery and Development

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.

Rebecca Goss
University of St Andrews


Blending Synthetic Chemistry with Synthetic Biology to Access Novel Natural Products

Though natural products represent a treasure trove of medicinally relevant compounds, they are commonly misperceived to be unsuitable for medicinal chemistry. We have an interest in the discovery of novel bioactive natural products and elucidating the biosynthesis of structurally unusual natural products. We are developing new approaches to natural product analogue synthesis by blending together synthetic biology and synthetic chemistry. By complementing the biosynthetic machinery encoding an existing natural product with foreign genes we are able to introduce chemically orthogonal, reactive and selectable functionalisable handles into natural products. We have been developing mild chemical methodologies to enable the chemical derivitisation of these handles
Genochemetics: gene insertion enables the installation of a reactive and chemically orthogonal handle into a natural product, permitting its selective functionalization and diversification
Keywords: GenoChemetics, biosynthesis, natural product, antibiotic, enzymology, halogenase

References
1. A. Deb Roy, S. Grüschow, N. Cairns, R.J.M. Goss, J. Am. Chem. Soc., 2010, 134, 1224-12245.
2. R. T. Bown, P. Poirrot, D. R. M. Smith, C. Cartmell, T. Abou Fayad, R. J. M. Goss, ChemSci, 2017, DOI: 10.1039/C6SC04423A
3. D.R. M. Smith, A. R. Uria, D.Milbredt, K-H van Pée, J. Piel*, R. J. M. Goss* ACS Chem. Biol. 2017
4. E. Marelli*, Y. Renault, S. V. Sharma, S.P. Nolan and R. J. M. Goss*Chem. Eur. J., 2017
5. DS Gkotsi, J Dhaliwal, MMW McLachlan, KR Mulholand, RJM Goss*, Current opinion in chemical biology, 2018 43, 119-126
6. S. V. Sharma, R. J. M. Goss et al., Nature Commun. 2017, 8, 229. DOI: 10.1038/s41467-017-00194-3.