Yang Yang, Biedendieck Rebekka, Wang Wei, Gamer Martin, Malten Marco, Jahn Dieter, Deckwer Wolf-Dieter
Biochemical Engineering, TU-BCE, HZI-Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
Microb Cell Fact. 2006 Nov 28;5:36. doi: 10.1186/1475-2859-5-36.
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.
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.
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.
在过去几年中，巨大芽孢杆菌被持续开发作为将蛋白质分泌到生长培养基中的生产宿主。在此，用于β-内酰胺抗生素逆向合成的巨大芽孢杆菌ATCC14945青霉素G酰化酶（PGA）的重组生产和分泌得到了系统改进。
为此，将PGA前导肽替换为巨大芽孢杆菌LipA对应的肽段。构建并使用了一株细胞外蛋白酶NprM缺失且木糖利用缺陷的生产菌株，以防止基因诱导剂缺乏。在小规模培养中开发了一种含有钙离子和特定氨基酸补充剂的缓冲矿物培养基，用于优化PGA生产，并扩大到2升生物反应器。每升生长培养基的PGA产量达到了40毫克。
基因和培养基优化相结合使巨大芽孢杆菌中PGA的生产和分泌总体提高了7倍。从基本培养基中排除某些氨基酸首次导致比复杂培养基培养更高的体积PGA活性。
