Pyruvate Carboxylase Variants Enabling Improved Lysine Production from Glucose Identified by Biosensor-Based High-Throughput Fluorescence-Activated Cell Sorting Screening

Abstract

Pyruvate carboxylase is an anaplerotic carbon dioxide-fixing enzyme replenishing the tricarboxylic acid cycle with oxaloacetate during growth on sugars. In this study, we applied a lysine biosensor to identify pyruvate carboxylase variants in Corynebacterium glutamicum that enable improved l-lysine production from glucose. A suitable reporter strain was transformed with a pyc gene library created by error-prone PCR and screened by fluorescence-activated cell sorting (FACS) for cells with increased fluorescence triggered by an elevated cytoplasmic lysine concentration. Two pyruvate carboxylase variants, PCx^T343A,I1012S and PCx^T132A were identified allowing 9% and 19% increased lysine titers upon plasmid-based expression. Chromosomal expression of PCx^T132A and PCx^T343A variants led to 6% and 14% higher l-lysine levels. The new PCx variants can be useful also for other microbial strains producing TCA cycle-derived metabolites. Our approach indicates that a biosensor such as pSenLys enables directed evolution of many enzymes involved in converting a carbon source into the target metabolite.