Computational Validation Complete
From a genome-scale metabolic model to 16 chip-eligible strains, a structurally validated optogenetic valve, and a cross-species proof point — every computational phase is done and now backed by a filed provisional patent.
10,440
KO combinations screened
16
Chip-eligible strains
2
Species validated
91/88
pLDDT — LOV2 / HEX1
iJDZ836 Genome-Scale Metabolic Model
The iJDZ836 model (836 genes, ~1,200 reactions) provides a comprehensive picture of N. crassa metabolism. Full flux-balance analysis predicted growth across 78 candidate carbon sources and quantified the metabolic impact of every knockout combination before any wet-lab work.
| Model Property | Value |
|---|---|
| Total genes modeled | 836 |
| Metabolic reactions | ~1,200 |
| Carbon sources tested | 78 |
| Carbon sources supporting growth | 24 |
| Permissive chow source | Xylose |
| Pairwise KO combinations | 10,440 |
Key finding: Of 78 carbon sources, only 24 support growth. Xylose is selected as the permissive chow feed — knockouts then block the common reagent sugars while preserving xylose utilization, the basis of metabolic orthogonality.
Carbon Source Preference (relative)
Relative predicted growth preference, normalized to xylose.
Screening Funnel
16 Chip-Eligible Strains
From 10,440 pairwise knockout combinations across a 145-gene pool, each pair was ranked by the Composite Orthogonality Score (COS). Sixteen pairs clear the chip-integration threshold of RBS ≥ 0.868 — these are the chassis candidates that can colonize a chip while blocking reagent metabolism.
Composite Orthogonality Score
COS = √( CPS × RBS )
CPS (Chow Preservation Score) measures retained xylose growth. RBS (Reagent Block Score) = 1 − (reagent growth ÷ WT xylose growth). The geometric mean rewards pairs that block reagents and stay alive on chow.
Exemplary Chip-Eligible Pair
A central-glycolysis enzyme knockout paired with a respiratory-chain subunit knockout — strong reagent blocking with enough residual xylose growth to colonize.
0.185
CPS
0.868
RBS
~18.5%
Residual xylose vs WT
COS Tradeoff — CPS vs RBS
X: Chow Preservation (CPS) · Y: Reagent Blocking (RBS) · ● 16 chip-eligible (RBS ≥ 0.868)
Honest constraint: no pair reaches the upper-right corner — none achieves both CPS > 0.5 and RBS > 0.8 at once. This is an inherent biological tradeoff: deeper reagent blocking costs chow fitness. The 16 chip-eligible strains are the best available balance.
The Six-Gene Chassis
A chip-eligible pair is only the metabolic core. The full chassis layers in four more knockouts for kinetics, morphology, and optogenetic isolation — six engineered edits that together make a clean, light-controllable fluidic network.
Central glycolysis enzyme
Knocked out to break glucose-route flux — forces dependence on the xylose chow feed.
Respiratory-chain subunit
Paired knockout that deepens reagent blocking while preserving residual xylose growth.
Carbon-catabolite-repression regulator
Removed to lift repression on xylose utilization — a kinetic growth benefit on the chow source.
Aerial-hyphae suppressor (i)
Keeps growth confined to the channel plane so the network stays a clean fluidic conduit.
Aerial-hyphae suppressor (ii)
Second morphology control that reinforces channel integrity during colonization.
Blue-light photoreceptor
Removed so the native light response cannot interfere transcriptionally with the 450 nm optogenetic valve.
Validated in a Second Species
Metabolic orthogonality is a platform strategy, not a one-organism trick. The same approach was re-run in silico on Aspergillus nidulans using the published iMA871 model (871 genes) — a fungus separated from N. crassa by roughly 170 million years of evolution.
Orthologous knockouts (a sugar transporter + a central-carbon aldolase, identified by reciprocal BLASTP) reproduce the xylose-selective phenotype — and actually block reagents more strongly than the N. crassa equivalent.
| A. nidulans double KO | Result |
|---|---|
| Growth on xylose | 0.198 h⁻¹ (27% WT) |
| Growth on glucose | 0.031 h⁻¹ (4.2% WT) |
| Reagent blocking | 95.8% |
| CPS | 0.270 |
| RBS | 0.958 |
| COS | 0.508 |
Why it matters: A. nidulans clears the chip-integration threshold (RBS 0.958) where N. crassa falls short — direct evidence the strategy generalizes and the addressable organism set is broad.
Composite Orthogonality Score
Reagent Block Score: 0.958
Reagent Block Score: 0.75
Evolutionary Distance
N. crassa
A. nidulans
Two genera, ~170M years apart — the same orthogonality phenotype is reachable in both.
ColabFold Structural Validation
Both valve components were predicted with ColabFold (AlphaFold2-based). High pLDDT scores (>80) on the functional domains validate the architecture before any wet-lab synthesis.
91
LOV2 domain
Very high
88
HEX1 domain
Very high
76
Jα helix
Flexible hinge
| Construct | Size | pLDDT | Key structural feature |
|---|---|---|---|
| CompA_v3 (LOV2-HEX1 plug) | 334 aa · 36.9 kDa | 91 | LOV2 β-sheet + Jα helix intact |
| CompB (SPA1-ePDZ anchor) | 358 aa · 39.4 kDa | 88 | PDZ binding groove correctly folded |
Next step: SEC-MALS (M4) confirms the oligomeric state of CompA_v3 with target R_h ≥ 28 nm, followed by BLI/SPR binding confirmation (M6) targeting K_d ≤ 1 µM in the lit state.
pLDDT Confidence Scale
The functional domains score 91 and 88 — both "very high". The Jα helix scores 76, exactly as expected for a flexible photoswitching hinge.
CompA_v3 Domain Architecture (N→C)
from Avena sativa phototropin 1
binds ePDZ in the lit state
~16 aa Gly-Ser
Woronin-body pore-occluding domain
N-terminal LOV2 placement leaves the HEX1 C-terminal crystal contacts free to self-assemble — verified by structural superposition (6.5 Å clearance).
With $1.5M Seed
Six clear milestones from chassis construction to first customer. Each has a concrete, measurable success metric.
Construct six-gene chassis
PCR-verified knockouts
SEC-MALS on CompA_v3
R_h ≥ 28 nm
BLI/SPR Jα-ePDZ binding confirmed
K_d ≤ 1 µM lit, ≥10× dark/lit ratio
Valve functional (microscopy)
≥50% pore closure at 450 nm
Prototype chip colonization
Fluorescent flow through network
First customer — academic beta site
LOI from 3 labs
Provisional Patent Filed
A provisional patent covering the metabolically orthogonal chassis, the optogenetic septal-pore valve, and the H-tree scaffold has been filed. A full methods manuscript describing the screening pipeline and valve design is in preparation.
In Preparation
"Self-assembling microfluidic chips via metabolically orthogonal Neurospora crassa with optogenetic flow control"
McKenna, M. — Target: Nature Biotechnology / Lab on a Chip