Bacillus pumilus als Plattform für die Produktion technischer Enzyme

• Bacillus pumilus as a platform for the production of technical enzymes

Küppers, Tobias; Schwaneberg, Ulrich (Thesis advisor); Wiechert, Wolfgang (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

The scope of this thesis is the development of an alternative production platform for the production of alkaline hydrolases, which are used in modern washing and cleaning agents. For this purpose, the strain Bacillus pumilus Jo2 was provided and should be developed to produce competitive enzyme yields in reference to a downscaled production process of Henkel’s established industrial workhorse Bacillus licheniformis E.Based on alkaline protease BL18, which was chosen as a well-characterized reporter molecule, B. pumilus Jo2 was initially developed to produce protease in reference to B. lichniformis E. Thereby, the iterative process development and adjustments, as well as genetic modifications, enabled to reach a final product titer of 114.5% in reference to B. licheniformis E. at the same scale. Further homologues promoter sequences were identified, and their use was claimed for various enzyme expressions (WO2013113689). In a final exploratory setup, the production capacity of the generate Jo2 expression platform was re-evaluated for its performance in producing non-proteolytic hydrolases. Therefore, Bacillus spec A7-7s α-amylase was chosen as a suitable reporter molecule. Besides minor pH and glucose adjustments, the developed B. pumilus Jo2 extemporaneously reached a maximum amylase titer of about 65.7% in comparison to B. licheniformis E protease titer at the same scale. This amylase titer, which is by far more than 4 g/L, indicates the outstanding potential of the developed B. pumilus platform, in due regard to its further optimization potential for amylase production. In addition to its development, the B. pumilus Jo2 platform was characterized in regard to its substrate utilization, amino acid synthesis and to identify optimization targets for further optimizations employing 13C-flux analysis and proteomics. Thus, among other results, it was shown, that in the subtilisin BL18 production process approximately 41.1 ± 4.2% of subtilisin BLl8’s carbon is derived from glucose. Based on results of amino acid pools, aspects for further media and substrate optimization are presented, as for instance precursor availability of alanine, asparagine/aspartic acid, serine and valine. As finding of the proteomic approach various optimization targets were proposed for subsequent strain improvements such as to avoid the formation of flagella protein Hag and extracellular enzymes (BglS, PelB).