Introduction

The utilization of carbohydrate substrates for chemical production is often criticized, as it competes with food. Thus, aerobic gas fermentation to produce chemicals from C1 units is an interesting approach. One promising candidate for such processes is the carboxydotrophic bacterium Hydrogenophaga pseudoflava DSM 1084. This Gram-negative β‑proteobacterium shows comparable high growth rates on CO, H₂ and CO₂ and mixtures thereof under aerobic conditions. Moreover, a basic genetic engineering toolbox is available which renders this bacterium an interesting host for biotechnological application. [1, 2]

Objectives

To analyze the electron transfer and its impact on energy and redox metabolism, different key enzymes of the autotrophic pathways were deleted. These mutants were cultivated with CO₂ and H₂ or CO gas mixtures. Beside the effect on growth, also the overall hydrogenase and CO dehydrogenase activity was determined.

Materials & methods

Cells were harvest in the exponential phase, stored at -80 °C and later lysed via sonication. According to Meyer and Schlegel (1980) the oxidation of CO or H₂ was measured photometrical using methylene blue as electron acceptor.

Results

The hydrogenase activity exhibited substrate-dependent behavior, with notably high activity recorded on 1 % fructose (1598 ± 201 nmol mg^-1_Protein min^-1). Under autotrophic conditions (86 % H₂, 10 % CO₂ and 4 % O₂) the activity doubled compared to heterotrophic conditions (3021 ± 452 nmol mg^‑1_Protein min^-1). The deletion of the membrane-bound hydrogenase led to no growth under these conditions, emphasizing its crucial role. On the other hand, CO dehydrogenase 1 was identified as essential for growth on 80 % CO gas mixture.

Conclusion

Relevant enzymes for the autotrophic metabolism of H. pseudoflava have been identified.

[1] Grenz et al., Metab. Eng., Vol. 55 (2019)

[2] Siebert et al., Adv. Biochem. Eng. Biotechnol., Vol. 180 (2022)

[3] Meyer and Schlegel, J. Bacteriol., Vol. 140 (1980)