Production of Aromatics in Microbes

Aromatics are amongst the most important bulk feedstocks for the chemical industry, however, no viable bioprocess exists today and production is still dependent on petro-chemistry. We have established toxicity levels and baseline production of two key molecules, p-hydroxybenzoic acid (PHBA) and p-amino benzoic acid (PABA), in Saccharomyces cerevisiae [1]. In order to further improve this baseline production we employed in-silico analysis for the prediction of suitable targets for strain improvement for products of the shikimate pathway [2]. A key reaction in metabolism was the knockout of pyruvate kinase, which is known to be non-viable on glucose. In addition, the knockout of competing pathways draining carbon from the shikimate pathway led to very poor growth and auxotrophic strains. To address these problems we have engineered dynamic regulation using a synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit activates gene expression at a high population density, and is linked with an RNA interference (RNAi) module to enable targeted gene silencing. The circuit was used to control flux through the shikimate pathway for the production of PHBA, showing that wild-type equivalent growth was observed prior to activating the circuit while reaching PHBA titers of over 1.1 mM in production phase [3]. Building on this, the strain was further engineered, a feeding strategy was developed and a fed-batch process finally achieved PHBA concentrations over 20mM. Two current limitations of the yeast system is the Crabtree effect causing production of ethanol in batch processes and the toxicity of PHBA. We have recently implemented PHBA production in Pseudomonas putida, known for its resistance to aromatics and achieved over 12 mM PHBA and very low by-product formation in first generation strains.

1. Krömer, J.O.; Nunez-Bernal, D.; Averesch, N.J.; Hampe, J.; Varela, J.; Varela, C. (2013) Production of aromatics in Saccharomyces cerevisiae--a feasibility study. J Biotech, 163, 184-193.
2. Averesch, N.J. and Krömer, J.O. (2014) Tailoring strain construction strategies for muconic acid production in S. cerevisiae and E. coli. Metab Eng Comm, 1, 19-28.
3. Williams, T.C.; Averesch, N.J.; Winter, G.; Plan, M.R.; Vickers, C.E.; Nielsen, L.K.; Krömer, J.O. (2015) Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae. Metab Eng, 29, 124-134.