Engineering enzymes for upscaling volatile carboxylic acids derived from organic waste

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About This Project

We propose to develop efficient biocatalysts for transforming volatile carboxylic acids (VCA) derived from organic waste into high-value industrial chemicals and biofuels. We will engineer enzymes to meet industrial criteria for productivity and stability. Supported by the Homeworld Garten Grant, we have a 12-month plan to achieve significantly better-performing enzyme variants. Our long-term goal is to build a microbial platform for VCA post-processing.

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What is the context of this research?

The "Carboxylate Platform" is an innovative approach to transform organic wastes (agricultural residues, food scraps, sludge, manure, etc.) into industrially useful volatile carboxylic acids (VCA). The methane-arrested anaerobic digestion (MAAD) catalyzed by microbial consortia progresses without the need for sterilizing biomass or equipment, with a tunable and high yield of VCAs, biogenic CO2, compost, and water as byproducts. However, the carboxylic acid products are currently upgraded to industrial chemicals, products, and fuels using traditional chemistry techniques with a substantial environmental footprint.

What is the significance of this project?

Biochemical post-processing of VCAs offers more sustainable, versatile valorization pathways for the outputs of MAAD. We have recently identified two types of oxidative enzymes that promise highly selective conversions of VCAs to chemicals that find applications as drop-in biofuels, bio-based building blocks, and cosmetic and food ingredients. Yet, the performance of the identified enzymes does not match the demanding industrial criteria regarding productivity, stability, and substrate spectrum. Such catalyst properties can be conveniently tailored by means of protein engineering.

What are the goals of the project?

Our long-term goal is to build a commercial microbial platform for post-processing VCAs. To get there, we first need to identify or create top-performing biocatalysts to equip a suitable microbial chassis.

Supported by the Homeworld Grant, we will carry out two protein engineering campaigns aiming at identifying superior enzymes for the conversion of 1) VCAs to alkenes and 2) VCAs to enantiopure hydroxy carboxylic acids and assessing the commercial potential of the two pathways. We will iterate rational mutant library creation, screening and characterization of the generated diversity to achieve sufficiently improved enzyme robustness (activity, stability), allowing us to create a first-generation whole-cell biocatalyst within a short project frame.


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Enzyme engineering is a resource-intensive and time-consuming activity. Pursuing two campaigns means twice the resources needed.

Large amounts of chemicals/plasticware are inevitably consumed. DNA synthesis/sequencing services are indispensable to conducting engineering campaigns effectively. It is important to allocate a substantial budget here, as this is at the backbone of research activities.

The budget to cover expenses related to meetings and training can also be viewed as a contingency for unexpected expenses.

A budget for protecting intellectual property is needed to cover legal services related to writing and submitting patent applications.

Funding for the project lead ensures that the project is overseen/managed effectively. Please notice we operate out of Switzerland, likely the most expensive country on the planet.

Overhead costs are mandatory for budgeting university research and cover indirect costs (office spaces, utilities, administration).

Project Timeline

Starting December 2023, we propose to conduct a 12-month project with the following work packages:

1. Develop product quantification methods based on gas- and liquid chromatography.

2. Bioinformatic analysis of enzyme targets.

3. Performing 3 iterative rounds of protein engineering in the lab.

4. Detailed performance assessment and benchmarking of identified mutants.

Jan 20, 2024

Quantitative and high-throughput assays for volatile alkenes and short-chain 2-hydroxycarboxylic acids

Feb 20, 2024

Completed computational analyses of enzyme targets. Recommendations for smart mutant library design and library creation.

Sep 30, 2024

Three completed rounds of smart library generation and exhaustive screening of the generated sequence space

Nov 30, 2024

One enzyme variant with significantly improved activity (>10 fold) and increased operational stability for conversion of either 1) VCA to alkene or 2) VCA to enantiopure 2-hydroxy carboxylic acid. Performance benchmarking in vitro and within a living microbial cell environment.

Meet the Team

Tsvetan Kardashliev
Tsvetan Kardashliev

Team Bio

We are Tsvetan and Tobias, a team of biotechnology scientists (Ph.D. level) and aspiring entrepreneurs from the University of Basel, Switzerland. Tsvetan will take the lead in project management and carry out research activities related to alkene biosynthesis. Tobias, who receives modest financial support from Switzerland to work on the enantioselective hydroxylation of volatile carboxylic acids, will support the related lab activities.

Tsvetan Kardashliev

Tsvetan holds a Ph.D. degree in protein engineering from RWTH Aachen University, Germany. He continued his specialization as a postdoctoral fellow in the field of industrial biotechnology at ETH Zurich, Switzerland. He joined the group of Prof. Thomas R. Ward at the University of Basel as a research associate in December 2022. Tsvetan boasts 10+ years of experience in working and engineering (oxidative) enzymes, fermentations, bioprocess development, and scale-up. He is also an experienced scientific project manager and an inventor with patenting and technology transfer know-how.


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