Figure 4. Optimization of prototrophic functionality reduces pathway bottlenecking and stabilizes valine availability at late time points in valine-free conditions.

(A) Infection of pCtrl and pMTIV cells using a lentivirus carrying extra copies of ilvD led to the generation of the pCtrl-ilvD + and pMTIV-ilvD + cell lines. (B) ilvD qPCR on gDNA and cDNA from each cell line (Figure 4—source data 1). Fold change levels were relativized to pMTIV. cDNA was reverse transcribed using oligo(dT) primers from RNA templates collected from each cell line. Error bars show SD of three technical replicates. (C)¹³C-labeling of valine in pMTIV and pMTIV-ilvD + samples collected on day-2 and day-24 of culture in unconditioned, reduced-isoleucine (0.06 mM), valine-free RPMI medium containing ¹³C₆-glucose with 2 mM ¹²C-sodium pyruvate spiked-in on plates coated with 0.1% gelatin (Figure 4—source data 1). Error bars represent data from two replicates. (D) Relative abundance of pathway intermediate 2,3-dihydroxy-isovalerate in pMTIV and pMTIV-ilvD + cells cultured as in (C) (Figure 4—source data 1). Error bars represent data from two replicates. (E) Molar concentration of valine in pMTIV and pMTIV-ilvD + cells cultured as in (C) (Figure 4—source data 1). ¹²C-labeled refers to valine carrying ¹²C exclusively while ¹³C-labeled refers to valine carrying at least one ¹³C. Error bars represent data from two replicates.

Figure 4—figure supplement 1. Long-term culturing of cells in unconditioned isotopically heavy valine-free RPMI medium.

(A) Schematic outline of the ¹³C-labeling strategy for detection of biosynthesized valine. Culturing regimen in isotopically heavy RPMI using ¹³C₆-glucose and ¹²C_3-sodium pyruvate should in principle result in the generation of ¹²C₅-valine (from 2 ¹²C₃ sodium pyruvate), ¹²C₃¹³C₂ valine and ¹²C₂¹³C₃ valine (from 1 ¹²C₃ and 1 ¹³C₃-sodium pyruvate), and ¹³C₅ (from 2 ¹³C₃-sodium pyruvate). (B) pMTIV cells cultured in valine-free RPMI (0.38 mM Ile, 0.38 mM Leu), reduced-isoleucine valine-free RPMI (0.06 mM Ile, 0.38 mM Leu), reduced-leucine valine-free RPMI (0.38 mM Ile, 0.2 mM Leu), and in reduced-isoleucine reduced-leucine valine-free RPMI (0.06 mM Ile, 0.2 mM Leu) (Figure 4—figure supplement 1—source data 1). Triplicate values are plotted. (C) Population doubling level of pMTIV and pMTIV-ilvD + cells cultured in isotopically heavy valine-free RPMI medium as described in (A). Error bars represent data from three replicates. (Figure 4—figure supplement 1—source data 1). (D) % of valine carrying at least one ¹³C in pMTIV and pMTIV-ilvD + cells cultured on unconditioned, reduced-isoleucine, valine-free RPMI medium containing ¹³C₆-glucose and 2 mM ¹²C-sodium pyruvate on plates coated with 0.1% gelatin. Error bars represent data from two replicates. (Figure 4—figure supplement 1—source data 1).