Rational orthologous pathway and biochemical process engineering for adipic acid production using Pseudomonas taiwanensis VLB120

• Published in: Metabolic engineering 2022-03, Vol.70, p.206-217
• Main Authors: Bretschneider, Lisa, Heuschkel, Ingeborg, Bühler, Katja, Karande, Rohan, Bühler, Bruno
• Format: Article
• Language: English
• Subjects: Adipates - metabolism; Biocatalysis; Biotransformation; Enzyme cascade; Metabolic Engineering; Nylon 6,6; Orthologous pathway engineering; Pseudomonas - genetics; Pseudomonas - metabolism; Reaction engineering; Whole-cell biocatalyst
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2022.01.014

Microbial bioprocessing based on orthologous pathways constitutes a promising approach to replace traditional greenhouse gas- and energy-intensive production processes, e.g., for adipic acid (AA). We report the construction of a Pseudomonas taiwanensis strain able to efficiently convert cyclohexane to AA. For this purpose, a recently developed 6-hydroxyhexanoic acid (6HA) synthesis pathway was amended with alcohol and aldehyde dehydrogenases, for which different expression systems were tested. Thereby, genes originating from Acidovorax sp. CHX100 and the XylS/Pm regulatory system proved most efficient for the conversion of 6HA to AA as well as the overall cascade enabling an AA formation activity of up to 48.6 ± 0.2 U gCDW−1. The optimization of biotransformation conditions enabled 96% conversion of 10 mM cyclohexane with 100% AA yield. During recombinant gene expression, the avoidance of glucose limitation was found to be crucial to enable stable AA formation. The biotransformation was then scaled from shaking flask to a 1 L bioreactor scale, at which a maximal activity of 22.6 ± 0.2 U gCDW−1 and an AA titer of 10.2 g L−1 were achieved. The principal feasibility of product isolation was shown by the purification of 3.4 g AA to a purity of 96.1%. This study presents the efficient bioconversion of cyclohexane to AA by means of a single strain and thereby sets the basis for an environmentally benign production of AA and related polymers such as nylon 6,6. •Efficient adipic acid production from cyclohexane via orthogonal pathway engineering.•Expression system engineering enables 96% conversion and 100% AA yield.•Stable adipic acid formation requires intact energy and redox metabolism.•Gas-phase feed of cyclohexane enables stable operation at 1 L bioreactor scale.•Proof of concept from pathway design to isolated product.