Considerations and application status of selection marker for generation of recombinant biologics producing mammalian cell lines / 药学学报

The major challenge in the development of recombinant biologics lies in generating and isolating rare high-producing stable single clone in a short period of time. The selection marker is an essential component of the plasmid vector, it plays an important part in the generation and screening of producing cell lines. Engineering the selection marker to enhance the stringency of selection for high producing cells is one of the most effective approaches to improve the cell line development process. Here, using Chinese hamaster overy (CHO) cells as an example, we introduce the application of selection marker for generation of recombinant biologics producing mammalian cell lines, methods of engineering the selection markers to enhance the selection stringency, and propose considerations on cell substrate stability and selection marker safety, in order to provide references for high-efficiency development of recombinant biologics.

Establishment of a plant phosphorus utilization and weed control system based on phosphite and its dehydrogenase / 生物工程学报

Nowadays, available phosphorus (P) deficiency in soil and weed resistance to herbicides have emerged as two severe limiting factors for sustainable agriculture. Therefore, it is of urgent needs to improve plant absorption/utilization ability of the soil P, seek phosphate (Pi)-alternative P fertilizers, and develop new forms of weed control systems. Phosphite (Phi), as a P resource of relatively high amount only less than Pi in Earth, can be converted to utilizable Pi uniquely in some bacterial species by oxidization via its specific dehydrogenase (PTDH), but inhibits plant growth and development. This implies that Phi might rather become a suitable P fertilizer for plants if introducing a PTDH detoxifier from bacteria. Herein, we created the transgenic tobaccos harboring a Pseudomonas PTDH gene (PsPtx) amplified from the soil metagenome previously. RT-PCR showed that the exotic PsPtx gene could express similarly in root, stem and leaf tissues of all transgenic lines. PsPtx transgenic tobaccos could utilize Phi by oxidization as the sole Pi supply, and also outperformed wild-type tobacco with a remarkably dominant growth under Phi stress conditions. Moreover, the PsPtx gene was preliminarily evaluated with a notable quality as a potential candidate of the selection marker in plant genetic transformation. Conclusively, PsPtx and its encoded phosphite dehydrogenase might be applicable for developing a dual system of plant phosphorus utilization and weed control using Phi as P fertilizer and herbicide, and provide an effectual solution to some obstacles in the current crop transgenic studies.

Construction and high-density fermentation of alkaline pectate lyase high-yield yeast / 生物工程学报

Pectate lyase is widely applied in ramie degumming and fabric bioscouring in the textile industry. Compared to conventional processes that involve high alkaline and high temperature treatment, enzyme based treatments have significant advantages in fibers protectiveness, improved efficiency of refining, reduced energy consumption and pollution. Hence, it would be highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost. In the previous study, pectate lyase gene pel from Bacillus subtilis168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905A. To improve the expression level, the vector pHBM905BDM with optimized promoter and signal peptide was used to express the optimized gene pels in GS115. The transformant had increased activity from 68 U/mL to 100 U/mL with the improvement in the transcription level by 27% measured by qPCR. The transformants were further screened on pectin plates, where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/mL. Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells. Subsequently, high-copy transformant was screened by using the medium containing antibiotics G418, strain GS115-pHBM905BDMpPIC9K- pels1 was identified with increased activity of 770 U/mL and the copy number of pels was 7 confirmed by qPCR. Finally, the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1reached to 2 271 U/mL in a 5-L fermentor. The activity of pectate lyase in our study reached the highest level of expression in P. pastoris, showing good application potential in the textile industry.