Abstract
BackgroundDuring 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 β-lactam antibiotics were systematically improved.ResultsFor 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.ConclusionThe 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.
Highlights
During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium
The leader peptide of the extracellular lipase LipA from B. megaterium was tested for the improvement of penicillin G amidase (PGA) export
Optimal PGA production was obtained in minimal medium with 1 × addition of the amino acid solution, which was verified by SDS-PAGE analysis of extracellular proteins. These results indicated that amino acids were essential for PGA production, but the higher concentration of amino acid, here 2 ×, limited PGA production
Summary
During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved. The Gram positive bacterium Bacillus megaterium has several advantages over other microbial host-systems for the production and secretion of recombinant proteins [1]. Production of heterologous exoproteins by B. megaterium was further improved by use the exoprotease NprM-deficient B. megaterium strain MS941 [5,6] and by the coexpression of the signal peptidase gene sipM [7]. Some bottlenecks were still observed for the production and secretion of some of the studied heterologous proteins.
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