Conversion of Cyclohexane to 6-Hydroxyhexanoic Acid Using Recombinant Pseudomonas taiwanensis in a Stirred-Tank Bioreactor

Abstract

6-hydroxyhexanoic acid (6HA) represents a polymer building block for the biodegradable polymer polycaprolactone. Alternatively to energy- and emission-intensive multistep chemical synthesis, it can be synthesized directly from cyclohexane in one step by recombinant Pseudomonas taiwanensis harboring a 4-step enzymatic cascade without the accumulation of any intermediate. In the present work, we performed a physiological characterization of this strain in different growth media and evaluated the resulting whole-cell activities. RB and M9* media led to reduced gluconate accumulation from glucose compared to M9 medium and allowed specific activities up to 37.5 ± 0.4 U gCDW−1 for 6HA synthesis. However, 50% of the specific activity was lost within 1 h in metabolically active resting cells, specifying growing cells, or induced resting cells as favored options for long-term biotransformation. Furthermore, the whole-cell biocatalyst was evaluated in a stirred-tank bioreactor setup with a continuous cyclohexane supply via the gas phase. At cyclohexane feed rates of 0.276 and 1.626 mmol min−1 L−1, whole-cell biotransformation occurred at first-order and zero-order rates, respectively. A final 6HA concentration of 25 mM (3.3 g L−1) and a specific product yield of 0.4 g gCDW−1 were achieved with the higher feed rate. Product inhibition and substrate toxification were identified as critical factors limiting biocatalytic performance. Future research efforts on these factors and the precise adjustment of the cyclohexane feed combined with an in situ product removal strategy are discussed as promising strategies to enhance biocatalyst durability and product titer and thus to enable the development of a sustainable multistep whole-cell process.

A 3D‐Printed Customizable Platform for Multiplex Dynamic Biofilm Studies

“…Such biofilms can sometimes be beneficial for the environment and some industries. In fact, biofilms have been utilized for microbial bioprocessing, [36,37] microbial fuel cells, [38,39] bioremediation, [40,41] biosensor design, [42,43] and biohydrogen production. [44,45] However, a tendency of bacterial accumulation on surfaces is in charge of a diverse spectrum of local/systemic diseases and industrial biofouling.…”

Recent advances in the sustainable production of α,ω-C6 bifunctional compounds enabled by chemo-/biocatalysts

“…Subsequently, physiological characterization of this strain (engineered P. taiwanensis (pSEVA_6HA_2)) in different growth media was compared and a stirred-tank bioreactor with a continuous supply of gaseous CH was applied for the evaluation of activity. 65 Both RB and M9* media (RB medium contains more ammonium than M9 medium and M9* medium contains 3-fold higher phosphate salt concentrations than M9 medium) were found to be more suitable than M9 media, with less gluconate formation from glucose, which enabled faster growth of strain and higher specific activity for HHA synthesis (up to 37.5 ± 0.4 U g −1 CDW). When the gaseous CH was feed at a rate of 1.626 mmol min −1 L −1 , a final HHA concentration of 25 mM and a specific product yield of 0.4 g g −1 CDW were achieved at 6 h. 65 In addition, product inhibition and substrate toxification (growth rate reduced by half at ∼450 μM CH) were identified.…”

“…The fine-tuning enzyme cascade system within Acidovorax sp. CHX100 was widely utilized for the production of CHOL, 25 ACA, 62 lactone 63 and HHA, 32,64,65 via in situ or heterologous expression. 32,64…”

Contribution of Fermentation Technology to Building Blocks for Renewable Plastics

“…[185]. Fermentation of 6HA by recombinant P. taiwanensis in stirred-tank bioreactor with a continuous cyclohexane supply was reported to reach a final concentration of 25 mM (3.3 g/L) [186].…”