Design of Pseudomonas putida fermentations for robust biomanufacturing

The ability of microorganisms to convert renewable resources to biobased chemicals as an alternative to traditional fossilbased chemical production has been the primary driver of the increased focus on industrial biotechnology. Generally, a small set of microorganisms such as Escherichia coli and Saccharomyces cerevisiae have been the center of attention in the early phases of industrial biotechnology. Nonetheless, they may not be the most suitable production hosts for any given product or production environment. Therefore, research has lately focused on developing alternative species such as Pseudomonas putida to broaden the range of microorganisms matured for industrial application. The bacterium is characterized by robustness and a versatile metabolism that allows it to adapt and cope with high demands of reducing power, well suited for producing highly reduced or toxic chemicals. This thesis sought to investigate the effect of industrial production conditions such as oxygen supply and medium components on the genome reduced P. putida SEM10 strain and benchmark it against the wild type strain KT2440.

The P. putida wild type strain KT2440 and the genome reduced strain SEM10 were subjected to different oxygen partial pressures (pO2) in the aeration gas to study the effect of low oxygen availability on their growth characteristics. Both strains showed an 810 % increase in Y_X/S during exponential growth at low pO₂ (0.0525 atm) compared to growth at high pO₂ (0.21 atm), despite showing a slightly lower growth rate. However, yields diminished as dissolved oxygen became limiting during growth at low pO₂, reaching overall biomass yields similar to growth at high pO₂. At the end of the cultivation at low pO₂, KT2440 achieved an overall Y_X/S of 0.352±0.027 g∙g^-1, similar to the Y_X/S of 0.383±0.016 g∙g^-1 at high pO₂. Likewise, SEM10 achieved a similar overall Y_X/S at both high and low pO₂ of 0.432±0.015 g∙g^-1 and 0.434±0.008 g∙g^-1, respectively. This showed that the genome reduced strain, SEM10, retained its advantageous growth characteristics regardless of the applied pO₂.

A medium for high cell concentration fedbatch cultivation of P. putida was developed and the inhibitory effects of the medium components were investigated for KT2440 and SEM10. Ammonium salts, phosphate buffer, glucose, and slightly acidic pH showed inhibitory effects. Growth inhibition was pronounced for both ammonium sulfate and ammonium chloride concentrations exceeding 0.121 M nitrogen, and growth was absent above 0.969 M and 0.484 M, respectively. Phosphate buffer concentrations above 0.108 M showed inhibition. Uninhibited growth was observed for glucose concentrations up to 25 g∙L^-1, and growth was absent at glucose concentrations exceeding 125 g∙L^-1. Lastly, the optimum medium pH was between pH 7.0 and 8.0. Both strains showed similar inhibitory trends across the tested conditions.

The wild type KT2440 and genome reduced SEM10 strains were applied in the designed fedbatch medium to evaluate the genome reduced strain under industrially relevant cultivation conditions. During DO limitation and glucose in excess, SEM10 more efficiently compared to KT2440. However, the growth of SEM10 diminished later in the feeding phase, and the strains obtained comparable final biomass concentrations of 26.15±1.04 g∙L1 and 28.15±0.39 g∙L^-1 for SEM10 and KT2440, respectively. The growth profiles indicated that the genome reduced strain experienced a demanding dual imitation of glucose and DO in the late feeding phase, which diminished the improved growth characteristics of SEM10. Moreover, SEM10 showed a 53% and 29% increased viability, compared to KT2440, in the batch and stationary phases, respectively.

In summary, the genome reduced P. putida strain SEM10 showed promise when benchmarked against the wild type KT2440 under industrially relevant conditions such as oxygen limitation and fedbatch cultivation