Derive the respiratory-chain kcats from the data, and compare with the Xia et al. (2022) series

[edkerk]

Summary

Rebuilds this study's results with respiratory-chain kcats derived from the data and with enzyme complexes treated as one unit. Then runs the same analysis, for comparison, on an independent dataset: three conditions of the glucose-limited chemostat series of Xia et al. (2022).

From proteomics to enzyme caps (overflow.complexes, overflow.flexibilize):

1. Complex levelling. Each complex gets one abundance: the median per copy over its measured subunits. Absolute proteomics recovers subunits present 1:1 up to tenfold apart, while their replicates agree within a few percent. Proteins that serve several complexes or also work alone keep their own measurement; examples are cytochrome c, thioredoxin and LPD1.
2. Minimum usage. A complex the model cannot run at the dilution rate for less than its measured abundance is raised to what it needs.
3. Reaching the dilution rate. One LP finds the least relaxation that reaches the dilution rate. It minimises the summed fold increases of the measured caps, one per complex, and lets the protein pool grow at 1000 times that price. The result does not depend on the order in which limiting enzymes are found.
4. Measured rates. The same LP, run under the measured exchange rates, makes the least further relaxation that reproduces them.

Respiratory-chain kcats (overflow.respiratory_kcats, a new step): for complex III, complex IV and ATP synthase, the apparent turnover in each condition is the sampled flux (conventional model at the measured rates, no kcats) over the levelled complex abundance. The highest across a dataset's conditions becomes the kcat, which overflow.build --respiratory-kcats applies to every isozyme variant. No other kcat changes.

Derived kcat (1/s)	This study	Xia et al. 2022	ecModel
Complex III	1452 (CN75)	330 (μ0.33)	290
Complex IV	3242 (CN75)	842 (μ0.33)	1376
ATP synthase	1608 (CN75)	379 (μ0.33)	85

The condition that sets a kcat runs that complex at full capacity, so each derived kcat is a lower bound. Each dataset's kcats come from its own abundances, so the difference in absolute calibration between the datasets cancels for the respiratory chain.

Files

• results/: this study, rebuilt (sampling unchanged).
• literature/xia2022/:
  • the series in the pipeline's formats (data/, scripts/prepare_data.py);
  • a wrapper that runs any step on it (scripts/run_step.py);
  • its results, with its own derived kcats;
  • comparison/, with six figures and six tables covering both datasets.

Results

The respiratory limit of a condition is the highest glucose uptake its measured proteome can respire completely at the condition's growth rate. Above it, the model has to secrete overflow carbon.

Condition	D [1/h]	Measured glucose	Measured ethanol	Respiratory limit	Respiratory-chain usage	Overflow predicted	Observed
CN4	0.10	1.12	–	1.95	20%	no	no
CN22	0.10	1.48	–	1.56	31%	no	no
CN38	0.10	2.01	0.79	1.78	45%	yes	yes
CN75	0.10	3.23	2.28	none	86%	yes	yes
hGR	0.29	5.42	3.68	none	68%	yes	yes
μ0.10	0.10	1.14	–	1.87	43%	no	no
μ0.28	0.28	3.14	–	4.09	76%	no	no
μ0.33	0.33	4.88	2.19	none	87%	yes	yes

Notes on the table:

• Glucose uptake is in mmol/gDW/h.

• "None" means the model overflows at every feasible uptake.

• Respiratory-chain usage is the enzyme mass used over the measured capacity of its enzymes.

• CN: this study; μ: Xia et al. (2022).

• In this study, the onset of overflow lies at a glucose uptake of 2.0 ± 0.3.

• When overflow starts. The respiratory limit predicts whether the cells make ethanol in all eight conditions. At D = 0.1, the limits of CN22 (1.56) and CN38 (1.78) lie close to the measured onset, CN22's just above its uptake (1.48) and CN38's below its uptake (2.01).

• Unused capacity at low flux. At D ≈ 0.1, both datasets leave much of their measured respiratory chain and ribosome unused. Usage rises with glucose uptake in both datasets. The ribosome and amino-acid metabolism follow the same trend.

• Fast growth. The apparent turnover of the respiratory chain needs no kcat.

  • In the Xia series it reaches its maximum where overflow starts: 0.79–0.85 of the maximum at μ0.28, without ethanol, and 1.0 at μ0.33, with ethanol.
  • In this study the maximum is at CN75 (D = 0.1). At hGR the respiratory chain runs at 0.65–0.90 of it.
  • At hGR and μ0.33 the model overflows at every feasible uptake. The caps that force it are outside the respiratory chain:
    • hGR: RPE1, LPD1, PPA2, ACS1;
    • μ0.33: YHM2, RKI1, CTP1, PPA2.
  • Raising the respiratory chain's capacity tenfold leaves the forced overflow unchanged in both conditions.

• ATP supply. The respiratory share of ATP is 86–88% without ethanol and at CN38, and falls as ethanol rises: 81% at CN75, 74% at hGR and 82% at μ0.33.

The figures and tables are in literature/xia2022/comparison/.