Optimization of IspS_ib stability through directed evolution to improve isoprene production

Area of Science:

• Biotechnology
• Enzyme Engineering
• Metabolic Engineering

Background:

• Enzyme stability is crucial for microbial production of chemicals and enzymes.
• The instability of isoprene synthase from Ipomoea batatas (IspS_ib) limits isoprene production in engineered Escherichia coli.

Purpose of the Study:

• To improve the thermostability of IspS_ib using directed evolution.
• To enhance isoprene production in engineered E. coli.

Main Methods:

• Construction of a lac'-IspS_ib-'lac tripartite protein folding system for high-throughput screening.
• Directed evolution involving random and site-saturation mutagenesis of IspS_ib.
• In vitro thermostability tests and melting temperature analysis.

Main Results:

• Three IspS_ib variants (IspS_ib^{N397V A476V}, IspS_ib^{N397V A476T}, IspS_ib^{N397V A476C}) with increased thermostability were identified.
• Melting temperatures of variants were 45.1–47.2°C, compared to 41.5°C for wild-type.
• Isoprene production increased by 1.94-fold to 1,335 mg/L using IspS_ib^{N397V A476T}.

Conclusions:

• Directed evolution successfully enhanced IspS_ib thermostability and isoprene production.
• The developed tripartite system enables enzyme stability screening without detailed structural information.
• This approach is applicable to other terpene synthases for isoprenoid production.