A Synthetic Biology Approach to Bacteria Mediated Tumor Targeting

Development of a drug delivery agent that selectively targets and destroys tumor cells with minimal toxicity to normal tissues is a major challenge in cancer therapy. It has been known for more than 60 years that anaerobic bacteria such as Clostridium can selectively colonize inside the necrotic core of solid tumors. Inoculation of a tumor by wild type Clostridium results in colonization of the necrotic core and consequently significant tumor destruction. This treatment strategy is hampered by the fact that the outer rim of the tumor is typically viable, and so does not present an anaerobic environment. As a result, colonization by Clostridium is unlikely to lead to complete tumor regression, since tumor regrowth occurs from the remaining outer viable rim, as evidenced by clinical trials. This project aims to address the problem of regrowth by developing a novel selectively aerotolerant strain of Clostridium that cannot colonize inside healthy tissue, but that could grow in the viable rim of an infected tumor. We have engineered a gene coding for an aerotolerance enzyme into Clostridium sporogenes. To couple the selective expression of this gene to tumor colonization, it can be placed under the control of a promoter activated by a synthetic quorum sensing circuit. This document describes the foundational work that will allow this system to be implemented. A suitable strain of C. sporogenes was selected, and a cloning technique (via conjugation with E. coli) was implemented. Expression of the aerotolerance enzyme and a synthetic quorum sensing circuit were verified in engineered colonies, and appropriate function was confirmed in both cases. Additionally, a model-based design exercise was carried out in order to better understand the system behavior and to identify key parameters for controlling the bacterial population. This analysis was based on mathematical models of the quorum-sensing circuit and of bacterial growth in the tumor environment. Sensitivity analysis reveals the design parameters that have the most significant impact on the extent and specificity of colonization of the viable rim, and thus provides insights into efficient design of the synthetic mechanism

Green Tea Polyphenols : Potential Antibiotic Synergistic Agents for Combatting Antibiotic Resistant Bacteria

The rise in antibiotic resistant cases has caused a global concern and researchers around the world are trying to find a novel alternative to combat this issue. Green tea with its many health benefits, including antibacterial and antiviral activity, has shown to be one of the most promising candidates to be used as an agent to solve this problem. Green tea commonly found throughout Asia is made from the unfermented leaves of Camellia sinensis and the polyphenols extracted from it can be divided into two categories: (1): Crude, which includes green tea polyphenols (GTP) and lipophilic green tea polyphenols (LTP), and (2): Pure tea polyphenols, which include Epigallocatechin Gallate (EGCG) and lipophilic Epigallocatechin gallate stearate (EGCG-S). All four extracts were used in this study. Twelve antibiotics were selected for this study. Five different bacteria: Gramnegative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), Grampositive Staphylococcus aureus (S. aureus) and Bacillus megaterium (B. megaterium), and Acid Fast Mycobacterium Smegmatis (M. smegmatis) were used. Profiling the effect of four tea polyphenols on 12 antibiotics against five bacteria has been established using a disk diffusion test. Different concentrations of five selected antibiotics and four tea polyphenols on these bacteria were evaluated to select the concentrations achieve LD50. These concentrations were then used for a combination study to determine their inhibitory effect on these bacteria using CFU and microplate assay methods. The Fluoresce microscopic observation and SEM imagery further study the viability and morphological changes. The optimal combination concentrations for E. coli, P. aeruginosa, B. megaterium M. smegmatis is E15 ug / EGCG-S 25 ug/ml, for S. aureus it is Te30 ug / EGCG-S 50 ug/ml. This study suggested that the combination of Erythromycin or Tetracycline with EGCG-S achieves the best antibacterial effect and may be used as a potential therapeutic agent for antibiotic resistant bacteria. Further study using these optimal concentrations to observe their effect on biofilm formation and formed biofilms suggested that higher combination concentrations are needed to efficiently inhibit biofilm. Bioinformatics on four proteins on the bacterial surface suggested that they all have high affinity to EGCG and EGCG-S and support the mode of action that EGCG is inhibiting these proteins

Artificial Golgi reactions for targeted glycosylation

N-linked glycosylation constitutes a Critical Quality Attribute for biotherapeutics. It is known to affect drug efficiency, efficacy and half-life. Glycosylation is a non-templated and complex process owning firstly to the promiscuity of the enzymes involved and secondly to enzyme and nucleotide sugar donor availability. This leads to heterogeneity amongst cell-derived glycoproteins, limiting therapeutic efficacy. Production of biotherapeutics focuses on controlling the glycosylation profile to enhance their activity and produce tailored drugs. Despite the intense efforts to control glycosylation, current methods face important limitations including simplicity, cost and lack of homogeneity. The work presented here addresses the current limitations by developing an Artificial Golgi Reactor (AGR) that allows bespoke N-linked glycosylation of glycoproteins in an artificial environment. Specifically, this novel proof-of-concept system comprises immobilised glycosyltransferase (GnTI, GalT) and glycosidase enzymes (ManII). These enzymes comprise a glycosylation pathway where promiscuity naturally exists. A method to express, in vivo biotinylate and immobilise GnTI and GalT was developed enabling “one-step immobilisation/purification”. ManII was biotinylated using an alternative chemical approach and similarly immobilised. The immobilised enzymes were used in a sequential fashion to reconstruct the N-linked glycosylation pathway on artificial glycans and on a monomeric Fc expressed in glycoengineered Pichia pastoris. The spatiotemporal separation tackled enzyme promiscuity, resulting in increased glycoform homogeneity (>95% conversion). Finally, immobilised GalT was used to enhance the galactosylation profile of three IgGs, yielding 80.2 – 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140-hours. The methods and results outlined in this work demonstrate the application of the AGR as an in vitro glycosylation strategy applied post-expression that is easy to implement, modular and reusable. Furthermore, it has the potential to be expanded and applied for the large-scale manufacture of bespoke biotherapeutics.Open Acces

Direct use of urine as fertilizer : potential risks of loading pharmaceuticals and hormones to field crops

Ecological sanitation or Ecosan is an alternative approach to avoid the disadvantages of conventional water-based sanitation systems. This approach, based on ecosystem functioning and the closure of material flow cycles, includes different sustainable and environmental friendly technologies to provide appropriate sanitation solutions to specific local situations. Perhaps the most relevant aspect of ecological sanitation is that it recognizes human excreta and household water as resources (not as a waste), giving the opportunity for its re-use. This study focused on re-use of human urine as fertilizer and the identification and mitigation of risks of spreading pharmaceutical and hormone residues into the agricultural fields. For that purpose, a case study was performed in Bogotá, Colombia to understand the actual sanitation scenario as well as to identify the limitations and strengths of implemented Ecosan strategies in peri-urban and rural areas of Bogotá. Furthermore, a group of pharmaceuticals frequently used was identified for a small population group. In the search for an analytical tool that was cost effective, and also required low investment in facilities and equipment when compared to other analytical methods, it was decided to test the yeast estrogen screen assay (YES) for analysis of estrogenic compounds in soil and plant material as well as in human urine. The YES assay was a reliable analytical technique for finding estrogenic activity in soil, wheat grains and human urine. However, difficulties were encountered while analyzing estrogenic compounds in some plant tissues. The current research assessed the degradation potential of estrogenic compounds and discovered that estrogens contained in human urine can degrade under both light and dark conditions where exposure to light presented a slightly higher degradation rate. Degradation of 17β-estradiol (βE2) also occurred in the nutrient solution of a hydroponic system. The findings suggest that there is little risk of βE2 accumulation in agricultural fields since it is easily degraded via different pathways (e.g. photodegradation and biodegradation). Consequently, more attention should be paid to persistent compounds such as carbamazepine (CBZ) which was found in the plant leaves and stems. However, concentrations in the edible parts (wheat grains and sunflower seeds) were rather low, and according to the results did not reach concentrations for therapeutic use. Also, Verapamil (VER) which has not been frequently studied as an emergent micropollutant and very little is known about its environmental risk, was found in soils as well as in plants, and - most importantly it occurred in edible parts. Although the concentrations found were very low, there is the need to further investigate the effect and degradation potential of this compound in different environmental systems (e.g. soils and natural water bodies)

ICR ANNUAL REPORT 2020 (Volume 27)[All Pages]

This Annual Report covers from 1 January to 31 December 202

Revelation of Yin-Yang Balance in Microbial Cell Factories by Data Mining, Flux Modeling, and Metabolic Engineering

The long-held assumption of never-ending rapid growth in biotechnology and especially in synthetic biology has been recently questioned, due to lack of substantial return of investment. One of the main reasons for failures in synthetic biology and metabolic engineering is the metabolic burdens that result in resource losses. Metabolic burden is defined as the portion of a host cells resources either energy molecules (e.g., NADH, NADPH and ATP) or carbon building blocks (e.g., amino acids) that is used to maintain the engineered components (e.g., pathways). As a result, the effectiveness of synthetic biology tools heavily dependents on cell capability to carry on the metabolic burden. Although genetic modifications can effectively engineer cells and redirect carbon fluxes toward diverse products, insufficient cell ATP powerhouse is limited to support diverse microbial activities including product synthesis. Here, I employ an ancient Chinese philosophy (Yin-Yang) to describe two contrary forces that are interconnected and interdependent, where Yin represents energy metabolism in the form of ATP, and Yang represents carbon metabolism. To decipher Yin-Yang balance and its implication to microbial cell factories, this dissertation applied metabolic engineering, flux analysis, data mining tools to reveal cell physiological responses under different genetic and environmental conditions. Firstly, a combined approach of FBA and 13C-MFA was employed to investigate several engineered isobutanol-producing strains and examine their carbon and energy metabolism. The result indicated isobutanol overproduction strongly competed for biomass building blocks and thus the addition of nutrients (yeast extract) to support cell growth is essential for high yield of isobutanol. Based on the analysis of isobutanol production, \u27Yin-Yang\u27 theory has been proposed to illustrate the importance of carbon and energy balance in engineered strains. The effects of metabolic burden and respiration efficiency (P/O ratio) on biofuel product were determined by FBA simulation. The discovery of energy cliff explained failures in bioprocess scale-ups. The simulation also predicted that fatty acid production is more sensitive to P/O ratio change than alcohol production. Based on that prediction, fatty acid producing strains have been engineered with the insertion of Vitreoscilla hemoglobin (VHb), to overcome the intracellular energy limitation by improving its oxygen uptake and respiration efficiency. The result confirmed our hypothesis and different level of trade-off between the burden and the benefit from various introduced genetic components. On the other side, a series of computational tools have been developed to accelerate the application of fluxomics research. Microbesflux has been rebuilt, upgraded, and moved to a commercial server. A platform for fluxomics study as well as an open source 13C-MFA tool (WUFlux) has been developed. Further, a computational platform that integrates machine learning, logic programming, and constrained programming together has been developed. This platform gives fast predictions of microbial central metabolism with decent accuracy. Lastly, a framework has been built to integrate Big Data technology and text mining to interpret concepts and technology trends based on the literature survey. Case studies have been performed, and informative results have been obtained through this Big Data framework within five minutes. In summary, 13C-MFA and flux balance analysis are only tools to quantify cell energy and carbon metabolism (i.e., Yin-Yang Balance), leading to the rational design of robust high-producing microbial cell factories. Developing advanced computational tools will facilitate the application of fluxomics research and literature analysis