Bioconversion of p -coumaric acid to cis,cis -muconic acid using an engineered A. baylyi ADP1 - E. coli co-culture

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

ABSTRACT Lignin-derived aromatics are abundant in depolymerized lignin but remain remain untilized as carbon sources for commercial production of bulk chemicals. Among these aromatics, p -coumaric acid can be funnelled through the β-ketoadipate pathway toward cis,cis -muconic acid (ccMA), a precursor of bio-based adipic and terephthalic acids. However, efficient ccMA production by Acinetobacter baylyi ADP1 is constrained by toxicity of catechol (the immediate precursor of ccMA), inefficient channelling of protocatechuate (PCA) metabolism towards ccMA production, and absence of PCA decarboxylase for converting PCA to catechol. Therefore, in this study, we engineered a modular co-culture system, combining engineered strains of A. baylyi and E. coli , for ccMA production from synthetic p -coumaric acid. Deletion of catB and catC genes and overexpression of catA in A. baylyi GJS_ catA strain enabled near-stoichiometric conversion of catechol to ccMA (∼90% carbon yield) with titres up to 56.4 mM (∼ 8 g/L) under controlled fed-batch feeding. The strain was further engineered ( A. baylyi GJS2_ catA ) to convert p -coumaric acid to PCA. Due to the inactivity of heterologous PCA decarboxylase ( aroY gene) in A. baylyi , this gene was incorporated in E. coli where it exhibited activity through PCA to catechol conversion. Upon its production by E.coli_aroY in the co-culture, catechol is instantaneously converted to ccMA by A. baylyi GJS2_ catA strain. In a two-step process, 22 mM p -coumaric acid was initially converted to 20.6 mM PCA ( A. baylyi GJS2_ catA ), which was further converted to catechol ( E.coli_aroY ) and finally to 18.55 mM ccMA (2.63 g L⁻¹) by A. baylyi GJS2_ catA . This process was validated by the valorization of lignin-derived p -coumaric acid to ccMA. While the modular strategy developed in this study substantially improves ccMA titres, it also highlights the bottlenecks in A. baylyi metabolic pathway engineering for lignin valorization. Abstract Figure

Authors 4

1. Suchismita Maiti Aachen Institute of Applied Microbiology

   RWTH Aachen University · Indian Institute of Technology Madras

   Affiliation as printed

   Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India

   Institute of Applied Microbiology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

2. T. Priyadharshini

   Indian Institute of Technology Madras

   Affiliation as printed

   Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India

3. Lars M. Blank Aachen Institute of Applied Microbiology

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

4. Guhan Jayaraman corresponding

   Indian Institute of Technology Madras

   Affiliation as printed

   Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India