Recombinant plasmid for the expression of L-phenylalanine ammonia-lyase and transformed strain carrying same

Claim 2

A recombinant plasmid comprising: (a) a vector capable of replicating in Escherichia coli; (b) a combined promoter comprising the tac promoter and, the P L promoter of the lambda phage, said P L promoter being connected downstream of said tac promoter; and (c) a DNA sequence derived from Rhodosporidium toruloides coding for L-phenylalanine ammonia-lyase, said DNA sequence being operably linked to and inserted downstream of said combined promoter, whereby the two promoters constituting said combined promoter have the same directional property and the P L promoter is located upstream of said DNA sequence so as to have the directional property which permits transcription of said DNA sequence.

Claim 3

The recombinant plasmid of claim 1 wherein said L-phenylalanine ammonia-lyase has the following amino acid sequence: ______________________________________ 1 10 Met Ala Pro Ser Leu Asp Ser Ile Ser His 11 20 Ser Phe Ala Asn Gly Val Ala Ser Ala Lys 21 30 Gln Ala Val Asn Gly Ala Ser Thr Asn Leu 31 40 Ala Val Ala Gly Ser His Leu Pro Thr Thr 41 50 Gln Val Thr Gln Val Asp Ile Val Glu Lys 51 60 Met Leu Ala Ala Pro Thr Asp Ser Thr Leu 61 70 Glu Leu Asp Gly Tyr Ser Leu Asn Leu Gly 71 80 Asp Val Val Ser Ala Ala Arg Lys Gly Arg 81 90 Pro Val Arg Val Lys Asp Ser Asp Glu Ile 91 100 Arg Ser Lys Ile Asp Lys Ser Val Glu Phe 101 110 Leu Arg Ser Gln Leu Ser Met Ser Val Tyr 111 120 Gly Val Thr Thr Gly Phe Gly Gly Ser Ala 121 130 Asp Thr Arg Thr Glu Asp Ala Ile Ser Leu 131 140 Gln Lys Ala Leu Leu Glu His Gln Leu Cys 141 150 Gly Val Leu Pro Ser Ser Phe Asp Ser Phe 151 160 Arg Leu Gly Arg Gly Leu Glu Asn Ser Leu 161 170 Pro Leu Glu Val Val Arg Gly Ala Met Thr 171 180 Ile Arg Val Asn Ser Leu Thr Arg Gly His 181 190 Ser Ala Val Arg Leu Val Val Leu Glu Ala 191 200 Leu Thr Asn Phe Leu Asn His Gly Ile Thr 201 210 Pro Ile Val Pro Leu Arg Gly Thr Ile Ser 211 220 Ala Ser Gly Asp Leu Ser Pro Leu Ser Tyr 221 230 Ile Ala Ala Ala Ile Ser Gly His Pro Asp 231 240 Ser Lys Val His Val Val His Glu Gly Lys 241 250 Glu Lys Ile Leu Tyr Ala Arg Glu Ala Met 251 260 Ala Leu Phe Asn Leu Glu Pro Val Val Leu 261 270 Gly Pro Lys Glu Gly Leu Gly Leu Val Asn 271 280 Gly Thr Ala Val Ser Ala Ser Met Ala Thr 281 290 Leu Ala Leu His Asp Ala His Met Leu Ser 291 300 Leu Leu Ser Gln Ser Leu Thr Ala Met Thr 301 310 Val Glu Ala Met Val Gly His Ala Gly Ser 311 320 Phe His Pro Phe Leu His Asp Val Thr Arg 321 330 Pro His Pro Thr Gln Ile Glu Val Ala Gly 331 340 Asn Ile Arg Lys Leu Leu Glu Gly Ser Arg 341 350 Phe Ala Val His His Glu Glu Glu Val Lys 351 360 Val Lys Asp Asp Glu Gly Ile Leu Arg Gln 361 370 Asp Arg Tyr Pro Leu Arg Thr Ser Pro Gln 371 380 Trp Leu Gly Pro Leu Val Ser Asp Leu Ile 381 390 His Ala His Ala Val Leu Thr Ile Glu Ala 391 400 Gly Gln Ser Thr Thr Asp Asn Pro Leu Ile 401 410 Asp Val Glu Asn Lys Thr Ser His His Gly 411 420 Gly Asn Phe Gln Ala Ala Ala Val Ala Asn 421 430 Thr Met Glu Lys Thr Arg Leu Gly Leu Ala 431 440 Gln Ile Gly Lys Leu Asn Phe Thr Gln Leu 441 450 Thr Glu Met Leu Asn Ala Gly Met Asn Arg 451 460 Gly Leu Pro Ser Cys Leu Ala Ala Glu Asp 461 470 Pro Ser Leu Ser Tyr His Cys Lys Gly Leu 471 480 Asp Ile Ala Ala Ala Ala Tyr Thr Ser Glu 481 490 Leu Gly His Leu Ala Asn Pro Val Thr Thr 491 500 His Val Gln Pro Ala Glu Met Ala Asn Gln 501 510 Ala Val Asn Ser Leu Ala Leu Ile Ser Ala 511 520 Arg Arg Thr Thr Glu Ser Asn Asp Val Leu 521 530 Ser Leu Leu Leu Ala Thr His Leu Tyr Cys 531 540 Val Leu Gln Ala Ile Asp Leu Arg Ala Ile 541 550 Glu Phe Glu Phe Lys Lys Gln Phe Gly Pro 551 560 Ala Ile Val Ser Leu Ile Asp Gln His Phe 561 570 Gly Ser Ala Met Thr Gly Ser Asn Leu Arg 571 580 Asp Glu Leu Val Glu Lys Val Asn Lys Thr 581 590 Leu Ala Lys Arg Leu Glu Gln Thr Asn Ser 591 600 Tyr Asp Leu Val Pro Arg Trp His Asp Ala 601 610 Phe Ser Phe Ala Ala Gly Thr Val Val Glu 611 620 Val Leu Ser Ser Thr Ser Leu Ser Leu Ala 621 630 Ala Val Asn Ala Trp Lys Val Ala Ala Ala 631 640 Glu Ser Ala Ile Ser Leu Thr Arg Gln Val 641 650 Arg Glu Thr Phe Trp Ser Ala Ala Ser Thr 651 660 Ser Ser Pro Ala Leu Ser Tyr Leu Ser Pro 661 670 Arg Thr Gln Ile Leu Tyr Ala Phe Val Arg 671 680 Glu Glu Leu Gly Val Lys Ala Arg Arg Gly 681 690 Asp Val Phe Leu Gly Lys Gln Glu Val Thr 691 700 Ile Gly Ser Asn Val Ser Lys Ile Tyr Glu 701 710 Ala Ile Lys Ser Gly Arg Ile Asn Asn Val 711 716 Leu Leu Lys Met Leu Ala ______________________________________

Claim 4

A strain of Escherichia coli which has been transformed with the recombinant plasmid of claim 2.