Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak.

The mechanisms of adaptation to inactivation of essential genes remain unknown. Here we inactivate E. coli dihydrofolate reductase (DHFR) by introducing D27G,N,F chromosomal mutations in a key catalytic residue with subsequent adaptation by an automated serial transfer protocol. The partial reversal G27- > C occurred in three evolutionary trajectories. Conversely, in one trajectory for D27G and in all trajectories for D27F,N strains adapted to grow at very low metabolic supplement (folAmix) concentrations but did not escape entirely from supplement auxotrophy. Major global shifts in metabolome and proteome occurred upon DHFR inactivation, which were partially reversed in adapted strains. Loss-of-function mutations in two genes, thyA and deoB, ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP. Multiple evolutionary pathways of adaptation converged to a suboptimal solution due to the high accessibility to loss-of-function mutations that block the path to the highest, yet least accessible, fitness peak.

Tetrahydrofolate increases suspension growth of dihydrofolate reductase-deficient chinese hamster ovary DG44 cells in chemically defined media.

Adaptation of dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time-consuming and labor-intensive process because nonadapted DHFR-deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension-adapted DHFR-deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2-359 µM enhanced cell growth in in-house CDM. Addition of 3.6 µM THF to in-house CDM resulted in a more than 2.5-fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 µM THF yielded up to 2.9-fold enhancement of maximum viable cell density. An anchorage- and serum-dependent DHFR-deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in-house CDM supplemented with 3.6 µM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 µM THF can help achieve a high density of suspension-adapted DHFR-deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR-deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539-1546, 2016.

Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR-/- cells in fed-batch processes - Implications for designing novel CHO-based expression platforms.

DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative.

[Expression of truncated fragment of human OPG in CHO-DHFR- cells and its bioactivity characterization].

AIM: To obtain high level expression of recombinant human truncated osteoprotegerin (TOPG) with higher bioactivity in CHO-DHFR(-) cells. METHODS: The recombinant vector pcDNA3.1/DHFR-TOPG was constructed and transfected into CHO-DHFR(-) cells by the directions of LipofectAMINE 2000 for stable expression. The stable expression cell strains were screened by selective medium IMDM with 50 mL/L FCS, then serially passed in methotraxate (MTX) for gene amplification. The expression were analyzed by ELISA and RT-PCR. At last, the bioactivity analysis was performed in vitro. RESULTS: The expression level of recombinant truncated human OPG was up to 6 mg/L x 72 h, and it had significant suppression effect on the formation of OLC (P<0.05). CONCLUSION: Recombinant truncated human OPG has high expression and bioactivity. The results make it possible for further studying and clinical implying of OPG.

Development of a serum-free medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44) using a statistical design: beneficial effect of weaning of cells.

To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10 serum medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 x 10(5) cells/ml, a maximum viable cell concentration of 6.4 x 10(3)) cells/ ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1 x 10(5) cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium. In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.

Growth properties of a folA null mutant of Escherichia coli K12.

In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate. The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant. Unlike earlier deletions, this mutation did not disrupt flanking genes. When the mutation was transferred into a wild-type strain and a thymidine-(thy) requiring strain, the resulting strains were viable but slow growing on rich medium. Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid. The derivative of the wild-type strain did not grow on any defined minimal media tested. In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements. These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions.