High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

Abstract

Background: During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of beta-lactam antibiotics were systematically improved.

Results: For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached.

Conclusion: The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.

Figure 1

Influence of calcium ions on PGA production and export in B. megaterium. MS941 carrying pRBBm23 (encoding SP_pga-PGA) was cultivated in LB medium with indicated concentrations of CaCl₂. Proteins from 1.5 mL cell-free growth medium were precipitated by ammonium sulfate, analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue G250. Lane M shows Precision Plus Protein Standard (Bio-Rad, Muenchen, Germany).

Figure 2

B. megaterium YYBm1 is deficient in xylose utilization. Shaking flask cultivation of B. megaterium strain MS941 (ΔnprM) (■), YYBm1 (ΔnprM, ΔxylA) (□), WH320 (▲), and WH323 (ΔxylA) (△) in minimal medium with glucose as initial carbon source. At the beginning of the stationary phase 5 g L^-1 xylose was added as second carbon source into the growth medium (indicated by arrow).

Figure 4

Comparison of different leader peptides for the production and export of B. megaterium PGA. PGA was produced in shaking flask cultivation of B. megaterium MS941 and YYBm1 carrying either pRBBm23 (encoding SP_pga-PGA) or pRBBm49 (encoding SP_lipA-PGA) in LB medium containing tryptone from different companies. At OD_578nm of 0.4 pga expression was induced by the addition of 5 g L^-1 xylose to the growth medium. Samples were taken at various time points after induction. Proteins from 10 μL unconcentrated growth medium were separated by SDS-PAGE and stained with Coomassie Brilliant Blue G250. Biomass concentration and PGA volumetric activity 24 h after induction of recombinant gene expression are shown.

Figure 3

Comparison of growth media for PGA production and export using B. megaterium. MS941 carrying pRBBm23 (encoding SP_pga-PGA) grew in LB (square), A5 (circle), and MOPSO (triangle) medium. The pga expression was induced at OD_578nm of 0.4 by adding 5 g L^-1 xylose. (A) Solid symbols represent the measured growth curve. (B) open symbols represent specific PGA activity.

Figure 5

Cultivation and PGA production in microtiter plates. YYBm1 carrying pRBBm49 (encoding SP_lipA-PGA) was cultivated in LB medium using microtiter plates and shaking flasks. OD_578nm from microtiter plate cultivation was measured with a spectrophotometer and Multiskan Ascent photometer. PGA activity measurements were performed as described in material and methods.

Figure 6

The influence of the concentration of amino acids supplementation on cell dry weight and PGA activity. Shaking flask cultivation of YYBm1 carrying pRBBm49 (encoding SP_lipA-PGA) was employed.

Figure 7

Upscaling of PGA production and export using B. megaterium and a 2 L bioreactor. The pH controlled batch cultivation of B. megaterium YYBm1 carrying pRBBm49 (encoding SP_lipA-PGA) was performed in complex medium (square) and optimized minimal medium (circle). B. megaterium MS941 carrying pRBBm23 (encoding SP_pga-PGA) was grown in semi-defined A5 medium (triangle). For induction of recombinant gene expression 5 g L^-1 xylose were added at the beginning of the cultivation. Samples were taken at indicated time points to determine cell dry weight (open) and PGA volumetric activity (solid).