Synthetic Pathway Designer: Engineering a Novel Metabolic Route

Rate-Limiting Enzyme Variant

Inducible Promoter

Inducer (aTc) concentration 40%

Substrate feed rate 1.00×

Knock out competing host branch

Live Readouts

Product flux

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Byproduct fraction

Steady-state yield (P / S fed)

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Product

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Byproduct

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How it works

Three heterologous enzymes are wired into an engineered cell to build a synthetic route S → I1 → I2 → Product that does not exist naturally in the host. Each step follows Michaelis-Menten kinetics:

v = (Vmax · [expr]) · [substrate] / (Km + [substrate])

Enzyme expression is under an inducible promoter — a Hill-type dose-response to inducer concentration models real systems like the TetR/aTc or LacI/IPTG switches used to build genetic circuits:

expr(u) = leak + (1-leak) · u^n / (K^n + u^n)

Swapping the rate-limiting enzyme (step 2, I1 → I2) between directed-evolution variants changes its kinetic constants:

Wild-type — baseline Vmax and Km.
High-k_cat — 3× higher turnover rate, same substrate affinity.
High-affinity — much lower Km (binds I1 tightly even at low concentration), same Vmax.

The host cell also runs a native, constitutively-active branch that drains intermediate I2 into an unwanted byproduct — exactly the kind of metabolic cross-talk that eats into yield in real strain engineering. Knocking it out removes that competing flux entirely, at the cost of losing whatever native function it served. Substrate is replenished at a feed rate and consumed by step 1; concentrations are integrated forward every frame and the particle streams flowing through the 3D cell are drawn with a speed and density proportional to each reaction's instantaneous flux, so a bottleneck step visibly backs up molecules behind it.

Substrate S Intermediate I1 Intermediate I2 Product Byproduct (drain)

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