Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer

Abstract

Background: Since volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis.

Results: To develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel's industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an α-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer.

Conclusions: In this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the basis for a next generation production host, since the strain has still a large potential for further genetic engineering. The final amylase titer of 65% in reference to B. licheniformis protease titer suggests that the developed B. pumilus expression platform is also suitable for an efficient production of non-proteolytic enzymes reaching a final titer of several grams per liter without complex process modifications.

Figure 1

Fed batch cultivation of B. pumilus Jo2.1/pHP49 at lab scale. Adapted cultivation process based on a down-scaled production process of B. licheniformis. Colony forming Units (CFU) (blue triangle) [L^-1], glucose concentration (orange diamond) [g/l], acetate concentration (green square) [g/l] and protease titer (gray square) were measured at line. The pH-value (light blue circle) was monitored online. Yielded enzyme concentrations scaled in correlation to the subtilisin BL18 protease titer of the current production host B. licheniformis in a lab scale cultivation serving as industrially relevant reference in this contribution.

Figure 2

Protease titer of B. pumilus Jo2.1/pHP49 cultivations under adopted (gray square) and optimized (black circle) process conditions at lab scale. Yielded enzyme concentrations scaled in correlation to the subtilisin BL18 protease titer of the current production host B. licheniformis/pHP49 in a lab scale cultivation serving as industrial relevant reference in this contribution. Error bars represents the standard deviation of three biological replicates.

Figure 3

Promoter optimization in the pHP49 backbone for improving the protease titer in B. pumilus Jo2.1 cultivations. Used promoters: P_ref, P_{aprE1 I}, P_{aprE1 II}, P_{aprE1 III}, P_{aprE1 IV}, P_mpr and P_aprE2. Cultivations were carried out in the optimized fed batch fermentation process. Yielded enzyme concentrations scaled in correlation to the subtilisin BL18 protease titer of the current production host B. licheniformis/pHP49 in a lab scale cultivation serving as industrial relevant reference in this contribution. Error bars represents the standard deviation of at least three biological replicates. P_aprE2 represents the mean value of only two independent cultivations (with this promoter the two fermenter yields were essentially identical).

Figure 4

Maximum amylase titer of B. pumilus Jo2.1 cultivations deploying various promoters in the pHP49 backbone. Jo2.1/pHP5-31 (P_ref) was initially cultivated under optimized protease process conditions (bar with grey background). Strains containing the plasmid encoded promoter P_ref, P_mpr,P_{aprE1 III} and P_aprE2 were cultivated under aligned process conditions in respect to modified pH setpoint (bars with white background). Based on a harshly decreased process robustness expressing the amylase in the protease process, bars represent the maximum achieved amylase titer of four independent cultivations per strain. Yielded amylase concentrations are scaled to B. licheniformis protease BL18 titer at the same scale serving as industrial relevant reference.

Figure 5

Maximum enzyme titer of B. pumilus Jo2.1 cultivations in reference to the current production host B. licheniformis at lab scale: Overexpression of subtilisin BL18 (B. licheniformis) vs. subtilisin BL18 (B. pumilus Jo2.1) and A7-7 α-amylase (B. pumilus Jo2.1), respectively. Process conditions and specific plasmid configurations are given below the figure. Based on a harshly decreased process robustness expressing the amylase, bars represent the maximum achieved amylase titer of four independent cultivations per strain. Yielded amylase concentrations are scaled to B. licheniformis protease titer at the same scale serving as industrial relevant reference for enzyme production.