(2ci) Biological Upcycling of Wastes for Sustainable Development

How to achieve sustainable development is the major challenge we are facing now. Biological upcycling – a process that implements a broad range of biotechnologies to reprogram microbes for producing specialty chemicals and compounds from waste feedstocks – has been considered as a promising alternative to support society’s sustainability in the near future. However, the complexities of the biological system have emerged as substantial challenges for constructing and optimizing the microbial chassis with desired functionalities. My research interests revolve around the understanding of intracellular metabolic and regulatory mechanisms and the development and leveraging of cutting-edge synthetic biology techniques to harness the microbial metabolic repertoire for biological upcycling.

Graduate Research

“Direct photosynthetic upcycling of CO₂ to astaxanthin” (Advisor: Dr. Weiwen Zhang, Tianjin University)

I received my PhD training in metabolic engineering to validate the possibility of engineering cyanobacteria for the de novo biosynthesis of value-added chemicals directly from CO₂. I constructed a biosynthetic pathway by introducing β-carotenoid ketolase and hydroxylase in cyanobacterium Synechocystis sp. PCC6803 for producing astaxanthin which is a highly valued carotenoid with potent antioxidant activity. However, due to the endogenous metabolic constraint, the biosynthesis of astaxanthin in engineered cyanobacterial chassis was of low efficiency. To overcome the limitations, I implemented multiple efforts to achieve high-efficient astaxanthin biosynthesis (i.e., bioinformatics, systematic overexpression, multi-omics, and synthetic biology). The multidisciplinary approach enables the final strain to produce up to 29.6 mg/g of astaxanthin (dry cell weight), which is the highest yield reported in the engineered chassis to date (Diao et al., Metabolic Engineering, 2020). Beyond this, the developed genetic elements and the identified metabolic regulation nodes of the Calvin cycle and endogenous MEP pathway in cyanobacteria are additional flavors of this research.

Skills Acquired: Design of heterologous metabolic pathways; identification and resolution of metabolic bottlenecks; multi-omics-based analysis of the regulatory mechanisms.

Post-Doctoral Research

“Upcycling of solid waste feedstocks utilizing synthetic biology approaches” (Advisor: Dr. Tae Seok Moon, Washington University in St. Louis)

I have dedicated my postdoctoral research to developing cutting-edge synthetic biology technologies for the upcycling of solid wastes such as lignin and plastic. To expand the capacity of lignin valorization in Rhodococcus opacus PD630, my research was focused on the identification of the transcription factors involved in regulating aromatic degradation pathways by developing GFP-based sensors and comprehensive deletion analyses. These combined efforts allow us to figure out the regulation patterns of the funneling pathways for various lignin-derived aromatics. Additionally, promoter activity assays revealed that the substrate hierarchy in R. opacus may be ascribed to the transcriptional cross-regulation of the individual aromatic funneling pathways (Diao et al., Communications Biology, 2022). I also developed a simple gene repression tool based on CRISPR interference (CRISPRi), and this tool’s utility was shown by demonstrating the inducible accumulation of muconate from lignin-derived aromatic compound (DeLorenzo and Diao et al., ACS Synthetic Biology, 2021). To address the waste plastic concern, I developed a new biotechnological method for upcycling PET via the identification of the hyperosmotic stress-tolerant microbial chassis Rhodococcus jostii strain RPET (RPET), which can be paired with a highly efficient PET depolymerization method to enable the valorization of waste PET towards the sustainable production of value-added chemicals with the implementation of synthetic biology (Diao et al., Cell Reports 2023). I have also worked on the development of microbial kill switches used as a safe guide to control GEMs when applied in bioremediation, and I am a co-first author on a manuscript in preparation.

Skills Acquired: Construction of biosensors; design and high-throughput screening of RBS/protein-encoding libraries; genetic circuit design and tuning.

Past Proposals:

DARPA B-SURE Program (Co-PI); NSF’s Rules of Life (URoL) Award (Co-PI); USDA Biotechnology Risk Assessment Grants (BRAG) Award (Co-PI)

Future Research

In my research career, I have dedicated to developing and tuning microbial chassis to address societal challenges, and I will use those principles I have developed and the advanced synthetic biology techniques to direct the research of synthetic biology in the following general topics:

Developing autotrophy-heterotrophy consortium for carbon-negative bioproduction

• Assembling an autotrophy-heterotrophy consortium (cyanobacterium-R. opacus) to maximize the carbon yield of mocunate from lignin and ultimately to ensure the overall process is carbon-negative
• Characterization of the breakdown triggers of the consortium to inform future engineering and development of consortium-regulating tools

Developing a microbial supply chain for biodiesel from mixed plastics

• Developing efficient methods to deconstruct mixed plastics under mild conditions.
• Decoupling the lipid biosynthesis from nitrogen starvation to achieve high carbon yields of biodiesel (or other oleochemicals) from waste plastic stream

Teaching Interests:

Teaching experience

Based on my learning and training experiences, I realized that how successful would the students be in the classroom is not only dependent on their commitment to excellence but also on the capacity of their teachers being involved in the teaching and learning processes. Serving as a teaching assistant at Tianjin University, I always helped professor to incorporate the current societal challenges into our course, aiming to make learning becomes an engaging process. In WashU, I also gave invited guest lectures to graduate students talking about how synthetic biology could change our lives. Collectively, these experiences have led me to develop a great sense of duty to educate the next generation of young scientist. My course preference would be Biochemistry, Microbiology, Metabolic Engineering or Synthetic Biology electives.

Mentoring experience

In graduate school, I have had the rewarding experience of mentoring undergraduate students in the iGEM team, aiming to increase the participation of undergraduate students in the science. I also mentored one master student in Tianjin University, and the student wrapped up the training with a first-author publication about the bioproduction of platform chemicals in microbial consortium. In my post-doc training, I also served as the mentor of rotation students at WashU. Now, as a Staff Scientist, I am mentoring two graduate students, one of whom has contributed as the co-first author of another plastic upcycling manuscript. My objective in future mentoring will be both to provide fundamental scientific knowledge and bench work training and to encourage academic independency.

Teaching Philosophy

My teaching philosophy will focus on these four principles: student-centered learning, engaging students, fostering active and collaborative learning, and diversity.

• As teachers we are committed to creating a student-centered learning environment. Examples include, but are not limited to, changing from “Do as I say” to “Based on your needs, let's co-develop and implement a solution to the problem you have”.
• An effective teacher needs to make learning become an engaging process. For students, active engagement in the learning process could allow them to develop a deeper understanding of concepts and ideally of themselves as learners. I will use a class-response system (e.g., Top Hat) for quizzing students and polling them on various issues relevant to their learning experiences.
• Teachers must establish an innocuous environment to foster active and collaborative learning. Active learning usually emphasizes discussion and collaboration that can make the virtual classroom a more inviting place. To realize it, I will engage students by adding “Fun factor” to the process, but without sacrificing the “challenge factor”, and ask students to work on Top Hat questions in groups.
• An effective teacher must embrace diversity. Here, diversity is not confined to the ethnic status, but encompasses the wide range of learning abilities of the individual students. All classes and students are different, which demands that a teacher could be able to read the class at several different levels and teach in a way from which a wide range of students can benefit. In my opinion, this means that we need to timely alter a presentation strategy based on student queries, facial expressions, attention levels, and knowledge base.

Selected Publications:

1. Diao J# (equal contribution), Tian Y#, Hu Y, Moon T* (2023) Development of genetic toolkits in Rhodococcus jostii strain RPET for biocontainment and PET upcycling (In preparation)
2. Manna A#, Diao J# (equal contribution), Moon T* (2023) Developing aromatic dependent kill switches to sustainably remediate phenolic pollutants from waters. (To be submitted)
3. Diao J, Hu Y, Tian Y, Carr R, Moon T* (2023) Upcycling of poly(ethylene terephthalate) to produce high-value bio-bioproducts. Cell Reports 42 (1), 111908
4. Diao J, Carr R, Moon T* (2022) Deciphering the transcriptional regulation of the catabolism of lignin-derived aromatics in Rhodococcus opacus PD630. Communications Biology 5 (1), 1-17
5. Diao J, Song X, Zhang L, Cui J, Chen L, Zhang W* (2020) Tailoring cyanobacteria as a platform for highly efficient synthesis of astaxanthin. Metabolic Engineering 61, 275-287
6. Diao J, Song X, Guo T, Wang F, Chen L, Zhang W* (2020) Cellular engineering strategies toward sustainable omega-3 long chain polyunsaturated fatty acids production: State of the art and perspectives. Biotechnology Advances 40, 107497
7. Diao J, Song X, Cui J, Shi M, Liu L, Wang F, Chen L, Zhang W* (2019) Rewiring metabolic network by chemical modulator based laboratory evolution doubles lipid production in Crypthecodinium cohnii. Metabolic Engineering 51, 88-98
8. Diao J, Song X, Zhang X, Chen L, Zhang W* (2018) Genetic engineering of Crypthecodinium cohnii for increased growth and lipid accumulation. Frontiers in Microbiology 9, 492