Breeding Of Phenylalanine Ammonia Lyase-Producing Strain And Cloning Of Full Length CDNA

L-Phenylalanine(L-Phe)is aromatic amino acid with physiological activity.It is one of the essential amino acids in human being.L-Phe has been applied as raw materials in pharmaceutical,food and feedstuff industries.L-Phe is the material for some anticancer drug. As a vehicle,it conveys the antitumor molecule or group to the target,inhibits the growth of tumor,and also reduces the poisoness of antitumor drug.L-Phe can also inhibit the overgrowth of heart muscle fibroblast cell,which will provide a new method to prevent and treat the hypertension.Studies have shown that L-Phe exhibits high activity on antitumor, anticancer and so on.The main use of L-Phe is to product aspartame(L-Asp-L-Phe-Me, APM).Aspartame was synthesized with two different essential amino acids,L-Phe and L-Asp.The sweetness of aspartame is 200times of sucrose,the heat energy that aspartame produced is only 1/200 as compared with sucrose.Aspartame is widely used in pharmaceutical and food industries.In human body,aspartame is digested to amino acids, then amino acids are taken in by the body,aspartame performs nutritional and healthy function accordingly.In addition,when people take in aspartame,the residue can not be transformed into lactic acid,and aspartame has no corruption on teeth.It is beneficial to prevent decay teeth,especially for the tooth health of children.The most advantage of aspartame is that diabetic can also eat it,because its digestion is not concerned with insulin, and then blood sugar will not increased.In a word,aspartame is suitable for patient suffering from hypertention,heart disease,adiposity.Phenylalanine ammonia lyase(PAL)catalyzes the transamination of trans-cinnamic acid to produce L-Phe.PAL can be used as useful biological tool to prepare L-Phe,and also be used in treatment of certain mouse neoplastic tumors,quantitative analysis of serum L-Phe in monitoring patients with phenylketonuia.Studies have shown that PAL can obviously inhibit the growth of some tumor cell.PAL can rapidly restrain the synthesis of DNA and protein, the division and growth of some tumor cell.However,it has little effect on normal cells.The mechanism may be that PAL decomposed L-Phe,reduce the essential amino acid necessary for tumor's growth.The tumor cell ceased growing for "hunger",the growth of the tumor was accordingly inhibited.In clinic,PAL was entrapped in liposomes,which increased the catalytic efficiency of PAL,elongated the half-time of PAL,decreased immunogenicity of antitumor drugs,and then improved the therapeutic effects.PAL is widely distributed in higher plants,some fungi,yeasts and in a single prokaryote, Streptomyces.However,it is absent in animal tissues.PAL is the first and key enzyme of the phenyl propanoid sequence,a secondary metabolic pathway operative in higher plants and is mainly involved in defense mechanisms.In mocroorgnisms,it has a catabolic role,allowing them to utilize L-Phe as a sole source of carbon.Among the microorganisms,PAL occurs abundantly in yeast,especially in the red yeast basidiomycetes family.In this paper, Rhodosporidium paludigenum was used as the research material,its morphological and cultural characteristics,physiological characteristics,phylogenetic tree based on intergenic spacer region sequences(ITS),production of PAL,and mutagenesis of JM432 have been examined.Futhermore,full length cDNA sequences of PAL from JM432 has been cloned.PAL production strain JM432 was classified and identified by the method of classical morphological characteristics,physiological characteristics and molecular biological taxonomy.The study of morphology,physiological and physiological characteristics showed that it was kind of red yeast,and can not ferment any sugar,not synthesize starch kind compound,but utilize potassium nitrate fast and strong.The sequence of 18S-26S rRNA intergenic spacer region of strain JM432 was sequenced and analysed.Homologous sequence analysis was conducted by using the BLAST software and compared with DNA sequences in the GenBank.Phylogenetic tree was constructed by neighbor-joining method using the MEGA package.The 18S-26S rRNA ITS of JM432 showed 99%similarity with Rhodosporidium paludigenum.Results showed that JM432 belongs to Rhodosporidium genus, named as Rhodosporidium paludigenum JM432. By single factor experiments,the effects of L-Phe,L-isoleucine(L-Ile),L-tyrosine(L-Tyr), NH₄⁺,inorganic salt,trace element on PAL activity were studied.Results were as follows:the effect of L-Phe-analogue on the PAL activity was not remarkable.Among the inducers,0.1% L-Phe and 0.1%L-Ile were more effective for PAL activity than 0.1%L-Tyr,enzyme activity was up to 123.04%,121.25%(as compared with the control)respectively.Inorganic salt-Fe²⁺ can activate the production of PAL,enzyme activity was of 192.17%.The use of 0.1%(NH₄)₂SO₄ increased the synthesis of PAL,enzyme activity was 258.56%of the control. The effect of trace element-Cu²⁺was very effective,PAL activity reached 384.56%.JM432 with PAL activity was used as original strain,treated with 8- methoxy-psoralen (8-MOP)in darkness for 30 minutes.8-MOP,a kind of photosensitive reagent,can increase strain sensitivity to ultraviolet(UV)light.In combination with UV light,8-MOP can generate effect of mutagenesis and was mainly used in treating some skin disease.8-MOP used in treating yeast has not been reported before.Through UV irradiation,cell suspension was spread on selected plate under red light.L-Tyr and L-Phe selection regime was used to screen the mutants.L-Phe is the substrate of PAL,can induce the syntheses of the enzyme.L-Tyr is analogue of L-Phe and also poor substrate for PAL.It has been shown that the affinity of yeast PAL enzyme for L-Tyr is low(high Km Value),and rapid growth on this amino acid can be accommodated by synthesizing more PAL enzyme.Through the use of analogue selected regime,a large numbers of mutants were obtained.ZW115,a mutant with higher PAL activity(the enzyme activity of ZW115 was more than 90%higher than the original strain) and genetic stability was obtained.The mutant strain NR128 showed good growth at 37℃as compared with the original strain.Compared with the original strain at 37℃,the activity of PAL activity of NR128 was 2 times,and the characteristics can be inherited stably.The PAL activities of mutant ZW115 and NR128 were surveyed with crude enzyme,the highest enzyme activities yielding were found in 72h.The PAL activitives of ZW115,NR128 were 0.94,2.23 times of the original strain.Although some progress has been made on the molecule biology of PAL from yeast,and the complete PAL genes was already obtained and sequenced from yeast.The complete cDNA of Rhodosporidium paludigenum is still unknown.In order to investigate PAL produced by JM432,it is necessary to obtain the full length cDNA sequence.Firstly, according to the Rhodosporidium toruloides',Rhodotorula mucilaginosa's,and Rhodotorula glutinis' PAL amino acid sequence homological contrast sequence published in Genbank,the higher homological contrast domain was selected to design the degenerate primers.In the course of designing degenerate primers,the traditional homogene cloning method was modified on two aspects:first,homo-primers were designed at the sites with higher degeneracy instead of the highest homology.Secondly,one "N" base was replaced with inosine in degenerate codes,the degeneracy of primers was decreased to 96/64,and annealing temperature is got close.A pair of degenerate primers were designed as Forward:5' -ATGACNATHMGIGTNAA-3',Reward:5'-YTYTCCATNGTRTTNSC-3'.In the experiment,two methods were applied:heat starting procedure and step up PCR technology, and a 650bp-long cDNA fragment of JM432 was obtained.Genetic specific primer(GSP)was designed against the acquired JM432 PAL cDNA fragment for 3'-rapid amplification of cDNA ends(RACE)-PCR(GSP:5' -TACTGTAACGCACGACTCGAGCAA-3').For 3' RACE-PCR,cDNA was transcribed from total RNA using the oligo-dT adaptor primer(5' -CTGATCTAGAGGTACCGGATCCT-3').PCR was performed with the GSP and the adaptor primer(5'-CTGATCTAGAGGTACCGGATCC-3').3' RACE-PCR extended the JM432 PAL cDNA sequence and gave a product of 650bo that contained 3'-ends.5' RACE primers were designed against the initial JM432 PAL cDNA fragment.For 5' RACE-PCR,cDNA was transcribed from total RNA using the RT-primer 5'-(P) GAAATTCGATCGATC-3' with AMV Reverse Transcriptase XL,treated with RNase H(analyze Hydrid RNA),and circled with T4 RNA Ligase.PCR was performed initially with genetic specific primers:antisense primer A1(5'-TGAGCAGGGCTCTTGCGACTA-3'), sense primer S1(5'-GACGCTCCTGTGCGAGTTCAA-3'),and then nested with antisense primer A2(5'-TACTGTAACGCACGACTCGAGCAA-3'),sense primer S2 (5'-GTCAGCGTACTAGTACTTCGCGGA-3').5' RACE-PCR gave a product of 1.8kb.Through the reconstruction of DNA sequence from RACE-PCR,2.3kb sequence of the full length cDNA sequence was obtained.Primers were designed against the full length cDNA sequence.By long distance PCR(LD PCR),a PCR product of 2.3kb was acquired.It is primarily proved that the DNA sequence was the full length cDNA sequence of PAL from JM432.