Background 
cis,cis-Muconic acid (ccMA) is a high-value platform chemical that can be readily converted to adipic acid via chemical catalysis for production of nylon-6,6, polyurethane, and other polymers. Currently, adipic acid is primarily produced from petroleum at over 3 million tons annually, and responsible for nearly 10% of global N₂O emissions through its energy-intensive synthesis. Developing a bio-based route to ccMA from renewable lignin-derived feedstocks would enable sustainable adipic acid production, reducing both emissions and energy demands while valorizing forestry byproducts into high-value materials. Pseudomonas putida KT2440 has emerged as a promising chassis organism due to its robust aromatic catabolism, high stress tolerance, and genetic tractability. Recent studies demonstrate successful engineering of P. putida for ccMA production from glucose and xylose, achieving titers up to 47 g/L. However, the efficient bioconversion of lignin-derived monomeric aromatics (LDMA), present as complex mixtures in varying ratios, requires systematic optimization of multiple ccMA-producing pathways while balancing energy demands and toxicity, to maximize yield and productivity.

Description 
This thesis project aims to develop and optimize engineered P. putida strains for efficient bioconversion of LDMA to ccMA at laboratory and pilot scale. The research will focus on constructing strains through targeted genetic modifications, incorporating heterologous genes from diverse bacterial species to expand substrate range and enhance pathway efficiency. These newly constructed strains will then be systematically screened for efficient conversion of target LDMA compounds.

The project scope will be adapted based on thesis credit level:

• 30-credit Master's Thesis: Focus on molecular cloning, variant library construction, and shake flask screening

• 60-credit Master's Thesis: Extended to include bioreactor cultivation, robotic automation, process optimization, and scale-up studies

Key Responsibilities

• Design and construct expression vectors for heterologous enzyme variants.

• Perform molecular cloning using CRISPR-Cas9, markerless sacB/KanR counter-selection or other suitable molecular cloning methods.

• Conduct shake flask cultivations to evaluate strain performance with LDMA compounds with individual substrates and mixtures.

• Perform strain characterization including growth kinetics and yield calculations.

• Adaptation of construction and or screening methods to our high-throughput screening facility.

• Quantify ccMA production and substrate consumption using HPLC or GC-MS.

• Scale up promising strains to 3L and 7L bioreactor cultivations

Expected Outcomes

• Construction of improved P. putida KT2440 strains with enhanced ccMA production from LDMA.

• Identification of optimal enzyme variants for key metabolic conversions.

• Characterization of strain performance with individual and mixed LDMA substrates.

• Development of cultivation protocols suitable for pilot-scale implementation.

Qualifications

• Currently enrolled in a master’s program in Biotechnology, Molecular Biology, or related field, requiring a thesis project of at least 30 ECTS credits to graduate.

• Previous practical experience in molecular cloning techniques (PCR, digestions and ligations, cloning and transformation) will be preferred.

• Basic knowledge of bacterial fermentation will be preferred.

• Familiarity with analytical chemistry methods (HPLC, GC) is advantageous.

• Strong attention to detail and good laboratory practice is must.

• Ability to work independently and as part of a multidisciplinary team

• Must be legally eligible to work/study in Sweden (i.e., no VISA sponsorship required).

Additional Information
This project is part of a larger initiative to valorize lignin-derived streams from biorefinery processes. The successful candidate will work as part of a team and contribute to the development of sustainable bio-based chemicals production.

Terms
Compensation: 1333 SEK/HP (before tax, when the Master Thesis is completed and approved by the University). For 30/60 credits: 39990SEK/79980 SEK

Location: RISE Processum, Örnsköldsvik

Last day of application: 30-November-2025

Expected start: January 2026

Contact: andreas.s.johansson@ri.se or vaskar.mukherjee@ri.se for additional details

We are looking forward to your application!