Molecular Characterization and Directed Evolution of a Metagenome-Derived l-Cysteine Sulfinate Decarboxylase

Abstract

l-Cysteine sulfinate decarboxylase (CSD, EC 4.1.1.29), the rate-limiting enzyme in taurine synthesis pathway, catalyzes l-cysteine sulfinic acid to form hypotaurine. Identification of the novel CSD that could improve the biosynthetic efficiency of taurine is important. An unexplored decarboxylase gene named undec1A was identified in a previous work through sequence-based screening of uncultured soil microorganisms. Random mutagenesis through sequential error-prone polymerase chain reaction was used in Undec1A. A mutant Undec1A-1180, which was obtained from mutagenesis library, had 5.62-fold higher specific activity than Undec1A at 35 °C and pH=7.0. Molecular docking results indicated that amino acid residues Ala235, Val237, Asp239, Ile267, Ala268, and Lys298 in the Undec1A-1180 protein helped recognize and catalyze the substrate molecules of l-cysteine sulfinic acid. These results could serve as a basis for elucidating the characteristics of the Undec1A-1180. Directed evolution technology is a convenient way to improve the biotechnological applications of metagenome-derived genes.

Keywords: l-cysteine sulfinate decarboxylase; metagenomic library; sequential error-prone PCR; taurine.

Fig. 1

Amino acid substitutions in the variant Undec1A-1180 and the original Undec1A protein. The differential amino acids are in red boxes

Fig. 2

Homology modelling structure (a) and docking result (b) of Undec1A-1180 with l-cysteine sulfinic acid

Fig. 3

The product of recombinant Undec1A-1180 with CSA tested by HPLC

Fig. 4

Thermostability of recombinant Undec1A and Undec1A-1180 proteins