Directed -in vitro- evolution of Precambrian and extant Rubiscos

Area of Science:

• Biochemistry
• Molecular Biology
• Evolutionary Biology

Background:

• Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a crucial enzyme in the carbon cycle, but its slow catalytic rate limits agricultural productivity.
• Current Rubisco engineering relies on in vivo systems, restricting the exploration of traits not essential for cell survival.

Purpose of the Study:

• To develop and apply a directed in vitro evolution platform for engineering Rubisco.
• To improve Rubisco's thermostability and catalytic efficiency by exploring ancient and extant forms.

Main Methods:

• Utilized a directed in vitro evolution platform to isolate Rubisco from its biological context.
• Applied adaptive evolution, focused evolution, and genetic drift strategies to Precambrian and extant form II Rubiscos.
• Employed a highly mutagenic environment using the proteobacterial ancestor.

Main Results:

• Identified a panel of thermostable Rubisco mutants.
• Discovered variants that deviate from typical trade-offs between CO2-fixing speed and specificity.
• Demonstrated that ancient proteobacterial ancestors exhibit unique tolerance to high mutation rates.

Conclusions:

• The in vitro evolution platform offers a new avenue for Rubisco engineering, overcoming limitations of in vivo systems.
• This approach facilitates the identification of Rubisco variants with enhanced catalytic evolution potential.
• Findings pave the way for designing more efficient Rubisco enzymes for agricultural applications.