Hemoglobin expression reduces aerobic bioconversion of guaiacol in Pseudomonas putida despite non-limiting oxygen

Efficient lignin valorization relies on oxidative enzymatic reactions, making oxygen availability a critical determinant of process performance. Pseudomonas putida, an obligate aerobe and promising host for valorization of aromatic compounds, experiences reduced growth and conversion rates under oxygen limitation. Here, two plant hemoglobin genes originating from Arabidopsis thaliana (AHb2) and Beta vulgaris (BvHb2) were expressed, and the resulting strains were evaluated for growth on glucose and vanillin, and for whole-cell bioconversion of guaiacol to muconic acid. Expression of hemoglobin showed a tendency toward improved growth on glucose, but not on vanillin, in shake flask cultivations. In contrast, expression of AHb2 in a strain co-expressing gcoAB for conversion of guaiacol to cis, cis-muconic acid resulted in reduced growth and respiration under well-controlled bioreactor conditions, despite non-limiting oxygen. Furthermore, lower conversion rates of guaiacol to cis, cis-muconic acid were observed without affecting product yield, indicating that hemoglobin expression influences how oxygen is utilized within the cell rather than improving its availability for bioconversion.