Establishment of a novel anabolism-based addiction system with an artificially introduced mevalonate pathway: complete stabilization of plasmids as universal application in white biotechnology.

摘要

Plasmid stability in recombinant microorganisms is a very important requirement for highly efficient plasmid-based production processes in biotechnology. To stably maintain plasmids, we developed in this study an efficient and stringent novel anabolism-based addiction system, which can be widely used. This novel addiction system is based on two components: (i) an Escherichia coli HMS174(DE3) knockout mutant of the ispH gene coding for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2) of the deoxyxylulose 5-phosphate (DXP) pathway, impairing the synthesis of isopentenyl pyrophosphate (IPP) and (ii) a completely synthetic and episomal mevalonate (MVA) pathway as an alternative supplier of essential IPP. The latter is encoded by a plasmid that contains the genes for HMG-CoA reductases from Lactococcus lactis and Staphylococcus aureus plus HMG-CoA-synthase, MVA kinase, MVP kinase and MVPP decarboxylase from S. aureus. This plasmid should then also harbor the genes for the protein or for the pathway that will be produced or that will be utilized for production of a chemical. To demonstrate the functionality of this addiction system, a mutated cyanophycin synthetase gene (cphA(6308)C595S) was used. To determine plasmid stabilities, flasks experiments in media supplied or not supplied with antibiotics were carried out with the knockout mutant and two control strains, one harboring plasmid pCOLADuet-1::MVA1-5::cphA(6308) and the other harboring a conventional expression plasmid pET-23a::cphA(6308). As revealed by measuring the colony-forming units of aliquots spread on solid media with or without antibiotics, the knockout mutant revealed a plasmid stability of 100% whereas the control strains exhibited plasmid stabilities of only 64% and 2%, respectively. Radiometric enzyme activity measurements for CphA revealed only 95% and 12.5% of the activity in the control strains harboring pCOLADuet-1::MVA1-5::cphA(6308) and pET-23a::cphA(6308), respectively, in comparison to the activity measured in the knockout mutant. The knockout mutant synthesized 9.5% (w/w of cell dry weight (CDW)) of cyanophycin, and the control strain harboring pCOLADuet-1::MVA1-5::cphA(6308) synthesized 13.6% (w/w of CDW) after growth without antibiotics.