American Society of Hirudotherapy

Combinatorial Evolution of Enzymes and Synthetic Pathways Using One-Step PCR

Synthetic biology paper published in ACS Synthetic Biology (2016)

Last Updated: June 18, 2026Reviewed by: ASH Editorial Board
Research article — evidence reviewArticle reference
Evidence: Preclinical (animal)Drug DevelopmentGenomics & ProteomicsJin P et al. · ACS Synthetic Biology, 2016

Abstract

DNA engineering is the fundamental motive driving the rapid development of modern biotechnology. Here, we present a versatile evolution method termed "rapidly efficient combinatorial oligonucleotides for directed evolution" (RECODE) for rapidly introducing multiple combinatorial mutations to the target DNA by combined action of a thermostable high-fidelity DNA polymerase and a thermostable DNA Ligase in one reaction system. By applying this method, we rapidly constructed a variant library of the rpoS promoters (with activity of 8-460%), generated a novel heparinase from the highly specific leech hyaluronidase (with more than 30 mutant residues) and optimized the heme biosynthetic pathway by combinatorial evolution of regulatory elements and pathway enzymes (2500 ± 120 mg L(-1) with 20-fold increase). The simple RECODE method enabled researchers the unparalleled ability to efficiently create diverse mutant libraries for rapid evolution and optimization of enzymes and synthetic pathways.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Publication typeJournal ArticleResearch Support, Non-U.S. Gov't
Indexed MeSH termsAmino Acid SequenceAnimalsBase SequenceCloning, MolecularDNA LigasesDNA-Directed DNA PolymeraseDirected Molecular EvolutionGenetic EngineeringHeparin LyaseHyaluronoglucosaminidaseLeechesMolecular Sequence Data

Summary

RECODE one-step PCR enzyme evolution method generated a novel heparinase from highly specific leech hyaluronidase via more than 30 mutant residues — repurposing a leech salivary enzyme into a different antithrombotic biopharmaceutical raw material.

Why This Matters for Hirudotherapy

This methodological study presents RECODE, a method for introducing combinatorial mutations into target DNA using a thermostable high-fidelity DNA polymerase and a thermostable DNA ligase in one reaction system. Among its demonstrations, the authors generated a novel heparinase from leech hyaluronidase through directed evolution involving more than 30 mutant residues. For ASH, this illustrates the use of leech hyaluronidase as a starting scaffold for enzyme engineering. CAVEAT: This is a genetic engineering methodology paper with no clinical or therapeutic leech therapy content; leech hyaluronidase serves only as a protein template, and findings are biochemical with no direct hirudotherapy relevance.

Citation

Combinatorial Evolution of Enzymes and Synthetic Pathways Using One-Step PCR.

Jin P et al. · ACS Synthetic Biology, 2016

DOI

Added to ASH library: May 27, 2026 · Site last updated: June 18, 2026

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