Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides

Abstract

The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.

Optimal culture condition for the production of phenyalanine ammonia lyase from E. coli

“…To improve PAL activity and production, the entire uninterrupted pal gene from R. toruloides was inserted into the E. coli expression vector pKK223-3. E. coli cells containing this vector synthesized a protein of pal-like activity [5]. The pal gene from R. toruloides was expressed in Saccharomyces cerevisiae (S. cerevisiae) and E. coli by using a bifunctional expression system.…”

“…A recombinant strain capable of producing a large amount of PAL is therefore highly desirable in order to improve the production of L-phenylalanine from trans-cinnamic acids. Although many efforts have been made to construct recombinant strains with high PAL activity [5][6][7], few results have been reported that the effects of culturing conditions on production of PAL in recombinant E. coli. A recombinant E. coli strain producing a significant amount of PAL was constructed in our earlier report [7].…”

Optimization of medium for phenylalanine ammonia lyase production in E. coli using response surface methodology

“…Some reports showed that PAL gene from R. toruloides was expressed in E. coli [5,6]. Furthermore, a recombinant E. coli strain producing a significant amount of PAL has been constructed in our earlier report; recombinant PAL activity reached 35 U/g [7].…”

“…A recombinant strain capable of producing a large amount of PAL is therefore highly desirable in order to improve L-phenylalanine from trans-cinnamic acids. Although some efforts have been made to construct recombinant strains with high PAL activity [5,6], the yields of recombinant enzyme obtained were disappointingly low. In terms of large-scale production of PAL for industrial and medical uses, recombinant PAL production needs to be improved substantially.…”

A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase

“…(i) We used a construct of the PAL gene from Rhodosporidium toruloides (21) under the control of a high-expression promoter and expressed it in a strain of Escherichia coli to obtain large amounts of PAL (22). (ii) We used existing (23) and new strains (C.N.S., J. D. McDonald, and C.R.S.…”