elow is a step-by-step workflow of strain engineering, integrating both traditional
and modern approaches (e.g., rational design, adaptive evolution, and multi-omics).
This pipeline is widely used in academia and industry to develop high-performance
microbial strains.
Typical Strain Engineering Workflow
1. Define Objective
Goal: Identify the desired trait (e.g., higher product yield, stress tolerance,
substrate utilization).
Example: Engineer E. coli to produce succinic acid at industrial scale.
2. Literature & Pathway Analysis
Review existing knowledge:
Metabolic pathways (e.g., TCA cycle for succinate production).
Known genetic modifications (e.g., knockout of competing pathways like ldhA for
lactate).
Tools: KEGG, MetaCyc, PubMed.
3. In Silico Design (Computational Modeling)
Genome-scale metabolic models (GEMs):
Simulate gene knockouts/overexpressions (e.g., using COBRA Toolbox).
Predict flux distributions for optimal succinate yield.
Machine Learning (optional):
Predict promising gene targets from omics datasets.
4. Genetic Modification
Choose one or more strategies:
Rational Engineering:
CRISPR-Cas9: Knock out pflB (reduces formate byproduct).
Promoter Engineering: Overexpress pyc (pyruvate carboxylase).
Random Mutagenesis (if targets are unknown):
UV/chemical mutagenesis + screening.
Synthetic Biology:
Introduce heterologous genes (e.g., Corynebacterium glutamicum’s succinate
exporter).
5. Strain Construction & Transformation
Clone plasmids or integrate genes into the genome.
Verify edits: PCR, sequencing.
6. Lab-Scale Cultivation & Screening
Batch fermentation in bioreactors.
High-throughput screening (HTS):
Microplates + robotics for rapid phenotype testing.
Biosensors (e.g., GFP reporters for product titer).
7. Adaptive Laboratory Evolution (ALE) (Optional)
If strain lacks robustness:
Serial passaging under stress (e.g., low pH for succinic acid tolerance).
Select improved variants via chemostat or turbidostat.
8. Multi-Omics Analysis (If Needed)
Multi-omics integration to troubleshoot bottlenecks:
Genomics: Check for unintended mutations.
Transcriptomics: Identify dysregulated genes.
Metabolomics: Detect intermediate accumulation.
Example: If succinate yield stalls, metabolomics may reveal oxaloacetate depletion,
prompting gltA overexpression.
9. Iterative Optimization
Cycle steps 4–8 until performance meets targets.
10. Scale-Up & Industrial Validation
Fed-batch fermentation at pilot scale (100L+).
Process optimization:
Aeration, feeding strategy, cost analysis.
Final strain: Patent and deploy!
Example Case: Succinic Acid-Producing E. coli
Objective: High succinate yield from glucose.
Design:
In silico model predicts knockout of ldhA, adhE, ackA (reduces
lactate/ethanol/acetate).
Genetic Edits:
CRISPR knockout of competing pathways.
Overexpress pyc + mdh (malate dehydrogenase).
ALE: Evolve strain at low pH.
Multi-Omics:
RNA-seq reveals sucCD (succinyl-CoA synthetase) is limiting → overexpress it.
Result: 80 g/L succinate, ~90% theoretical yield.
Tools & Technologies
Step Tools
Design COBRA, OptKnock, DESHARKY
Genetic Editing CRISPR-Cas9, Golden Gate Assembly
Screening FACS, Raman spectroscopy
Omics RNA-seq (Illumina), LC-MS (metabolomics)
Fermentation BioLector, DASGIP bioreactors
Challenges & Solutions
Metabolic Burden: Use dynamic regulation (e.g., quorum-sensing promoters).
Unintended Mutations: Genome resequencing post-ALE.
Scale-Up Failures: Test early in bioreactors, not just flasks.
