Category: Research paper

Lisa Bretschneider, Ingeborg Heuschkel, Katja Bühler, Rohan Karande, Bruno Bühler 

Abstract

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 g_CDW^-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 g_CDW^-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.

PMID: 35085781
DOI: 10.1016/j.ymben.2022.01.014

Lisa Bretschneider, Ingeborg Heuschkel, Afaq Ahmed, Katja Bühler, Rohan Karande, Bruno Bühler

Abstract

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer–Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, that is, isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for K_S (9.3-fold higher) and k_cat (4.8-fold lower) compared with corresponding K_M and k_cat values of isolated CHMO, but a favorable K_I for cyclohexanone (5.3-fold higher). The unfavorable K_S and k_cat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8-fold higher K_S, but 1.3- and 4.2-fold higher k_cat and K_I values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

doi.org/10.1002/bit.27791