Programming microbes to treat superbug infection

Superbug infection is one of the greatest public health threat with grave implications across all levels of society. Towards a new solution to combat infection by multi-drug resistant bacteria, this thesis presents an engineering framework and genetic tools applied to repurpose commensal bacteria into “micro-robots” for the treatment of superbug infection. Specifically, a prototype of designer probiotic was developed using the human commensal bacteria Escherichia coli. The engineered commensal was reprogrammed with user-specified functions to sense superbug, produced pathogen-specific killing molecules and released the killing molecules via a lytic mechanism. The engineered commensal was effective in suppressing ~99% of planktonic Pseudomonas and preventing ~ 90% of biofilm formation. To enhance the sensing capabilities of engineered commensal, genetic interfaces comprising orthogonal AND & OR logic devices were developed to mediate the integration and interpretation of binary input signals. Finally, AND, OR and NOT logic gates were networked to generate a myriad of cellular logic operations including half adder and half subtractor. The creation of half adder logic represents a significant advancement of engineering human commensal to be biological equivalent of microprocessor chips in programmable computer with the ability to process input signals into diversified actions. Importantly, this thesis provides exemplary case studies to the attenuation of cellular and genetic context dependent effects through principles elucidated herein, thereby advancing our capability to engineer commensal bacteria.Open Acces

Establishing chromosomal design-build-test-learn through a synthetic chromosome and its combinatorial reconfiguration

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV, synXV. We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics in vivo. Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains. </p

Applying the design-build-test paradigm in microbiome engineering

10.1016/j.copbio.2017.03.021CURRENT OPINION IN BIOTECHNOLOGY4885-9

Engineering a riboswitch-based genetic platform for the self-directed evolution of acid-tolerant phenotypes

Cells are exposed to shifts in environmental pH, which direct their metabolism and behavior. Here the authors design pH-sensing riboswitches to create a gene expression program, digitalize the system to respond to a narrow pH range and apply it to evolve host cells with improved tolerance to a variety of organic acids

Combining Metabolic Engineering and Lipid Droplet Storage Engineering for Improved α‑Bisabolene Production in <i>Yarrowia Lipolytica</i>

Bisabolene is a bioactive sesquiterpene with a wide range of applications in food, cosmetics, medicine, and aviation fuels. Microbial production offers a green, efficient, and sustainable alternative. In this study, we focused on improving the titers of α-bisabolene in Yarrowia lipolytica by applying two strategies, (i) optimizing the metabolic flux of α-bisabolene biosynthetic pathway and (ii) sequestering α-bisabolene in lipid droplet, thus alleviating its inherent toxicity to host cells. We showed that overexpression of DGA1 and OLE1 to increase lipid content and unsaturated fatty acid levels was essential for boosting the α-bisabolene synthesis when supplemented with auxiliary carbon sources. The final engineered strain Po1gαB10 produced 1954.3 mg/L α-bisabolene from the waste cooking oil under shake flask fermentation, which was 96-fold higher than the control strain Po1gαB0. At the time of writing, our study represents the highest reported α-bisabolene titer in the engineered Y. lipolytica cell factory. This work describes novel strategies to improve the bioproduction of α-bisabolene that potentially may be applicable for other high-value terpene products

Enhanced limonene production by metabolically engineered Yarrowia lipolytica from cheap carbon sources

Limonene is a valuable monoterpene widely used in the food and pharmaceutical industries. Previously, we successfully engineered Yarrowia lipolytica to produce limonenes. In this study, we focused on improving the titers of limonenes in Y. lipolytica by optimizing the metabolic flux of the limonene biosynthetic pathway and the medium composition. First, we adopted a combinatorial gene (over)expression strategy to improve the production of limonenes, obtaining the highest titer production strains. Subsequently, the medium composition and fed-batch fermentation were optimized to improve limonene biosynthesis, and it was confirmed that waste cooking oil (WCO) is the superior substrate to produce limonenes in Y. lipolytica. Under optimal fermentation conditions, the titers of D-limonene and L-limonene were improved to 91.24 mg/L and 83.06 mg/L from WCO. These findings provide valuable insights into the engineering of Y. lipolytica for a higher-level production of limonene and its utilization in converting WCO into other industrial products

Immersive Virtual Reality Training of Bioreactor Operations

This paper explores the application of immersive virtual reality (VR) as a training environment for biopharmaceutical engineering processes. An immersive simulation of a real-world industrial bioreactor setup was developed. This allows repeated practice in a safe and contextualized environment and mitigate limited access to equipment for hands-on training opportunities. It is hypothesized that using VR as an authentic context will assist in achieving the learning outcomes of performing operations in industry standard bioreactor setups, and understanding cellular processes. Two preliminary studies were conducted to evaluate the benefits of the VR application. Using pre- and post tests, there were comparable improvements using the VR application when compared to traditional lab lessons. Qualitatively, participants indicated benefits to learning through repeated practice and immersion in a realistic 3D lab environment. The introduction of gamification methods as motivators are areas to be further explored to better integrate students' theoretical and practical understanding compared to traditional approaches