L-DOPA (L-dihydroxyphenylalanine) is a promising drug for Parkinson's disease and thereby has a growing annual demand. Tyrosine phenol-lyase (TPL)-based catalysis is considered to be a low-cost yet efficient route for biosynthesis of L-DOPA. TPL is a tetrameric enzyme that catalyzes the synthesis of L-DOPA from pyrocatechol, sodium pyruvate, and ammonium acetate. The implementation of TPL for L-DOPA production has been hampered and the need for the most efficient TPL source with higher L-DOPA production and substrate conversion rate is prevailing. This study involves identifying a novel TPL from Kluyvera intermedia (Ki-TPL) and displayed a robust expression in Escherichia coli. The recombinant strain YW000 carrying Ki-TPL proved strong catalytic activity with a highest L-DOPA yield compared with 16 other TPLs from different organisms. With a further aim to improve this efficiency, random mutagenesis of Ki-TPL was performed and a mutant namely YW021 was obtained. The whole cells of YW021 as biocatalyst yielded 150.4 g L-1 of L-DOPA with a 99.99 % of pyrocatechol conversion at the optimum condition of pH 8.0 at 25 degrees C, which is the highest level reported to date. Further, the homology modeling and structural analysis revealed the mutant residues responsible for the extensive L-DOPA biosynthesis.