An LDHa single allele CHO cell mutant exhibits altered metabolic state and enhanced culture performance

Abstract

BACKGROUND Reducing lactate production in animal cell cultures has been reported to improve cell culture performance and productivity of recombinant protein. A novel genome editing tool, CRISPR/Cas, has been used widely to induce double‐strand breaks in the genome and introduce targeted mutations efficiently. RESULTS In the present work, we used a publicly available human‐codon optimized CRISPR/Cas system to introduce mutations in one of the LDHa gene copies to obtain an LDHa single‐allele knockout Chinese hamster ovary (CHO) cell clone to analyze its effect over cell metabolism. Fed‐batch cultures were conducted in order to evaluate the culture performance of mutant cells. Results show that cell growth was reduced and metabolism was modified by the LDHa single‐allele knockout, whereas specific protein rate and volumetric production were greatly enhanced. Additionally, the first in‐depth analysis of the metabolic effects of LDHa single allele knockout is presented. CONCLUSION By using CRISPR/Cas we were able to knock down LDHa, showing improved culture productivity. Metabolic flux analysis revealed higher fluxes in the TCA cycle in the mutant cell than in the parental cell, indicating that LDHa mutant cells have an enhanced energy metabolism, which can be attributed mainly to a higher catabolism of amino acids. © 2018 Society of Chemical Industry

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis, gating pyruvate generation, and increasing pyruvate flux into the TCA cycle

“…PKM1 or PKM2 are not the only genes in glycolysis pathway that their deletion results in increased flux of glycolysis intermediates into the TCA cycle 23,28 . A similar effect was also observed when one allele of LDHa was deleted, triggering increased pyruvate flux into the TCA cycle, suppressing cell growth, while increasing the recombinant protein expression 37 . Higher glycolysis intermediate flux into the TCA cycle is often linked to slower cell growth, similar to when CHO cells reach stationary phase during a production process 1–5 .…”

“…23,28 A similar effect was also observed when one allele of LDHa was deleted, triggering increased pyruvate flux into the TCA cycle, suppressing cell growth, while increasing the recombinant protein expression. 37 Higher glycolysis intermediate flux into the TCA cycle is often linked to slower cell growth, similar to when CHO cells reach stationary phase during a production process. [1][2][3][4][5] It remains to be determined whether or not the metabolic switches that shift pyruvate flux to the TCA cycle at the stationary growth phase during production are the same ones that are triggered when PKM or PKM1 expression is attenuated.…”

CRISPR‐interceded CHO cell line development approaches

“…Lactate and ammonia are known to be major byproducts that limit cell growth and productivity and can affect glycosylation patterns at certain quantities in supernatant (Mulukutla et al, 2019). Alternative culture methodologies, media design, and metabolic engineering have all been employed in the past to lower lactate production (Tang et al, 2021; Wilkens et al, 2019).…”