COMPOSITE PLASMIDS FOR AMINO ACID SYNTHESIS

Abstract: Composite plasmids containing multiple genes in transcriptional units. These composite plasmids are useful for the production of amino acids, particularly aromatic amino acids.

Description: "Composite Plasmids for Amino Acid Synthesis" BACKGROUND OF THE INVENTION 1. Field of the Invention ' Described are methods for the production of composite plasmids containing multiple genes that have been individually mutated then assembled into transcrip- tional units. These composite plasmids are useful in the production of amino acids and other metabolites and in screening for superior host cells. 2. Description of the Prior Art Bacterial fermentations are extensively employed for the industrial production of both primary metabolites such as amino acids and secondary metabo¬ lites such as antibiotics. The production of these metabolites is an industrial process of great economic significance. Naturally occurring microorganisms do not in general overproduce any metabolites since the relevant biosynthetic pathways are tightly regulated to avoid any waste of resources. The development of industrially useful production strains requires an extensive program of mutagenesis followed by selection of improved strains. Such a selection process may become extremely laborious in the later stages of strain development and even involve the screening of individual isolates by fermentation. Recombinant DNA technology has greatly facili¬ tated strain development since it is now possible to isolate the important genes in a biosynthetic pathway and to deregulate them in vitro. In addition, the clon¬ ing of genes onto multicopy plasmids is an efficient way of boosting the expression of a gene and hence the level of the relevant protein within the cell. Many biosynthetic pathways are complex, how¬ ever, involving upwards of ten individual steps. In such cases the choice of genes and their organization on a vector becomes of prime importance when considering a recombinant DNA approach to strain development. This invention describes a process for organizing the genes from a biosynthetic pathway on a vector in a manner which maximizes the utility of the recombinant DNA approach to strain optimization and thereby greatly accelerates strain development. The products of this process are termed composite plasmids. Plasmids containing individual genes or natural operons have been described, for example in L- phenylalanine production FR2486961-A, EP85958-A, GB2053906A, EP77196-A; L-proline production GB2075056-A, EP85958-A; in interferon production, ED126338-A;- In tryptophan production, J59125892-A, EP80378A, J57080398-A, US4,371,614, EP124048A. These references do not disclose the composite plasmids described in this document. An amino acid Biosynthesis overview is des- cribed in the text "Amino Acids: Biosynthesis and Genetic Regulation", edited by K.M. Herrmann and R.L. Somerville, published by Addison Wesley Pub. Co. (1983). A review of recombinant DNA procedures can be found in "Molecular Cloning: A Laboratory Manual" by T. Maniatis, E.T. Fritsch and J. Sambroo , published by Cold Spring Harbor Laboratory, NY (1982). U.S. Patent 4,514,502 describes plasmids containing drug resistance genes and a drive-unit region from a Coryneform glutamic acid-producing bacteria capable of propagating in E.coli or B.subtilis. SUMMARY OF THE INVENTION As a composition of matter, a composite plasmid comprising: (a) a first DNA segment containing a replicon covalently joined to a second DNA segment containing one or more transcriptional units; (b) if only one trans- criptional unit, then containing two or more feedback resistant " genes coding for two or more enzymes active in the synthesis of an amino acid; (c) if two or more transcriptional units, then a first transcriptional unit containing one or more feedback resistant genes coding for one or more enzymes active in the synthesis of an amino acid and one or more additional transcriptional units containing one or more genes coding for enzymes useful in the production of an amino acid. Among the amino acids preferred are aromatic amino acids, particularly phenylalanine, tryptophan and tyrosine. A preferred embodiment of the invention is a composite plasmid wherein an additional transcriptional unit contains a gene for a catabolic enzyme or a trans¬ port protein. A more preferred embodiment is a compo¬ site plasmid containing 2 or more feedback resistant genes selected from mutants of the following genes: pheA, aroF, tyrA, pheR, tyrR, tyrB, aroL, aroH, aroG, trpE, aspC, or trp BAD or C. Composite plasmids are capable of replication in procaryotic or eukaryotic cells, given transcription units specific for each cell type. One object of the invention is a transcrip¬ tional unit containing a catabolic enzyme or a permease useful in amino acid synthesis. A preferred embodiment of the invention is the utilization of an amylase on one of the transcriptional units of the composite plasmid. Another object of the invention is a method of screening for microorganisms useful in amino acid production wherein a composite plasmid containing a gene coding for a feedback resistant enzyme catalyzing a rate-^limiting step in amino acid production is used to transform a cell to be screened, then quantitating the production of the desired amino acid. The composite plasmids of the invention can be used to screen any Gram-negative organism. Similarly, any Gram negative microorganism can be utilized in amino acid production using the composite plasmids. The com¬ posite plasmids exemplified can be used in Gram-negative microorganisms to produce aromatic amino acids, par¬ ticularly L-phenylalanine and L-tyrosine. Among the Gram negative microorganisms that can be used are the following genera: Neisseria, Veillonella, Brucella-, Bordetella, Pasteurella, Haemophilus, Escherichia, Erwinia, Shigella, Salmonella, Proteus, Yersinia, Enterobacter, Serratia, Azotobacter, Rhizobium, Nitro- somonas, Nitrobacter, Thiobacillus, Pseudomonas, Aceto- bacter, Photobacterium, Zymomonas, Aeromonas, Vibrio, Desulfovibrio, and Spirillum. Also described is a method for producing a composite plasmid by mutating individual genes to a feedback resistant condition such that the enzymes pro¬ duced remain active in amino acid synthesis even in the presence of surplus amino acid product, then connecting the mutated genes to a transcriptional unit. Preferred embodiments of composite plasmids are pME202, pME219, pME214, and pPT112. BRIEF DESCRIPTION OF THE FIGURES Figure 1 — Schematic of aromatic amino acid biosynthetic pathway Figure 2 — Growth curves of HW77/pPT112 on glucose and starch Figure 3 — Fermentation of HW77/pPT112 on soluble starch Figure 4 — Screening strains for phenyl- alanine production using composite plasmids for L- phenylalanine production (pME202) with zone size mea¬ sured after 48 hr. Figure 5 — Comparison of phenylalanine pro¬ duction by HW77 and HW77/pME202 during fermentation DESCRIPTION OF THE PREFERRED EMBODIMENTS One object of the invention is to combine on a suitable vector the genes coding for the enzymes that are of greatest importance in influencing the flux of carbon down the biosynthetic pathway of interest. These enzymes are usually the points in the pathway at which regulation principally occurs. In addition to these regulated genes, it is .also necessary to include the genes for other enzymes which modulate the regulated steps. These genes usually are in the biosynthetic pathway of interest but they also occur within intermediary metabolism and consequently affect the supply of precursors to the pathway. Other genes may be of more indirect importance. For example, it may be necessary to include: the genes of pathway regulators, either to enhance the flux of interest or to decrease flux through an undesired competing pathway; genes that influence permeability of the cells, to either substrates or products; genes that allow growth of the organism on a more desirable substrate; genes involved in regulating the cloned genes in such a manner that they can be turned on or off in a manner that facilitates the fermentation; genes that can be used to influence the stability of the plasmid or to select for 5 the plasmid. The genes of interest for a composite plasmid may optimally be organized as a synthetic or semi- synthetic operon in which all the genes are transcribed from a promoter of the desired regulatory character- 11T istics (see chart 1). Ideally the genes are modified such that they are simply assembled by the use of inter¬ vening restriction sites. Use was made of the restric¬ tion enzyme BamHI which allows for overlap of both the useful restriction site and a functional ribosome bind- 15. ing site. Finally, it was best to include a transcrip¬ tional terminator sequence downstream of the final gene in the operon to limit the extent of the transcript. The whole operon ideally is flanked by restriction sites so that it can be treated as a "cassette", and contain 20 as little redundant DNA as possible to minimize its size. Such an operon was constructed by conventional recombinant DNA techniques, involving initial cloning, the genes followed by the determination of their DNA sequence, directed mutagenesis to introduce the desired 25 restriction sites, and construction of optimal linkers, promoters and terminators by the use of synthetic oligo-. nucleotides. These recombinant DNA techniques are well known to those skilled in the art of genetic engineerng. A transcription unit is defined schematically 30 in Chart 1. It contains assembled components not found together in nature and consists of a regulatory region containing a promoter/operator region, one or more genes mutated to feedback resistance, one or more restriction sites situated between components of the transcription 35 unit and a transcription terminator after the last gene. Chart 1 General Structure of an Operon for the Optimum Organization of Genes __/****gene 2****/_/****qene ****/T/ p = promoter o = operator (may overlap promoter) Site of desired transcriptional regulation (positive or • negative) (gene) 1 - (gene) n = genes of interest to relevant biosynthetic pathway. T = Transcriptional terminator. R n 1 - Rn = Restriction sites to facilitate construction and/or manipulation of operon. ADVANTAGES OF COMPOSITE PLASMIDS A composite plasmid is exceedingly versatile and increases the flexibility of the recombinant DNA approach to strain development in the following ways. 1. If the cloned genes for the regulated steps in the pathway have not already been mutated to yield feedback inhibition resistant (FBI ) derivatives then mutagenesis of the plasmid and screening for pro- ducers will quickly yield the desired mutants. This method has the advantage that the recipient background is not subject to mutagenesis and therefore the accumu¬ lation of unknown and possibly deleterious mutations that occurs with conventional strain development is avoided. 2. The operon can be simply switched between vectors with different copy numbers to assess the level of expression for optimum productivity. 3. Similarly, the operon can be switched between different vectors to optimize stability. If necessary the operon can be simply reintegrated into the host genetic background. 4. The level of expression can b_. varied at a given copy number by simple modification to one promoter. 5. The ratio of the different ger-.e products can be finely tuned by altering the efficiency of the ribosome binding sites (RBS) . This in turn is greatly facilitated by the presence of restriction sites within the ribosome binding sequences. If necessary a wide disparity in required levels of expression could be accommodated by the use of an attenuator or by splitting the construct into two operons with promoters of dif¬ ferent strengths. 6. Since transformation with a plasmid carry¬ ing such a construct effectively deregulates a strain immediately it is a very rapid method of screening for undefined differences between strains that influence productivity and thus choosing the best background for a producing strain. 7. Since such a plasmid can embody all that is known about a pathway it can be used in conjunction with mutagenesis to screen for mutants of a particular strain that offer increased productivity. A strain can be mutagenized followed by transformation and then screened for even more productive strains. This avoids firstly the many rounds of mutagenesis that would be required to deregulate the pathway to the same extent as the plasmid and secondly the potentially deleterious effects of mutagenesis on the cloned genes. 8. The operon can be cloned into plasmids with different or extended host ranges to allow for production in different species or genera. The plasmid will be relatively simple to optimize in these back¬ grounds by virtue of its single promoter and the presence of restriction sites in the RBS sequences. 9. Additional genes of importance can readily be incorporated as the requirements of the fermentation- alter or as understanding of the system evolves. 10. Since the complexity of competing " trans- cription/expression is avoided the construction ani optimization of plasmids carrying many genes involved with the biosynthesis of a product is made much easier. DESCRIPTION OF THE EXAMPLES As examples of the application of this inven¬ tion we now describe how the biosynthesis of the amino acids phenylalanine and tyrosine can be significantly deregulated and how synthetic operon plasmids can be usefully employed in the development of strains that produce aromatic amino acids, such a L-phenylalanine or L-tyrosine. The biosynthesis of the aromatic amino acids is an example of a complex pathway that proceeds via a shared common pathway to chorismate where it branches to yield phenylalanine (ten steps), tyrosine (ten steps), tryptophan (twelve steps), the aromatic vitamins and enterochelins, see figure 1. In E.coli (and other enteric organisms) the crucial step in the regulation of the common pathway is the first reaction catalyzed by the three DAHP synthe- tase isozymes. The rate of this reaction determines the rate at which carbon is committed to the pathway from the precursors phosphoenolpyruvate (PEP) and erythrose- 4-phosphate (E4P). The three isozymes are each inhibited by one of the end products of aromatic amino acid biosynthesis and are coded for by the genes aroF (tyrosine sensitive) aroG (phenylalanine sensitive) and aroH (tryptophan sensitive). In the phenylalanine branch the regulated enzyme is chorismate mutase- prephenate dehydratase, the product of the pheA gene, a bifunctional enzyme in which the prephenate dehydratase activity is very sensitive to feedback inhibition by phenylalanine. Analogously in ttie t i-oHn branch, the tyrA gene product chorismate mut^ s-prephenate dehydro- genase is sensitive to feedback inhibition by tyrosine. In addition to feedback inhibition which is the main control operating to recαlate carbon flux through the pathway, the levels of the above-mentioned enzymes and shikimate kinase (aroL) are. all subject to regulation by control at the transcriptional level. The pheA expression is controlled by the phenylalanine repressor, the product of the pheR gene, and by attenua¬ tion control. The aroF and tyrA genes together con¬ stitute a natural operon and their expression is coordinately regulated by the tyrosine repressor, the product of the tyrR gene. This tyrR also regulates the expression of aroG. Finally, the aroH gene expression is regulated by the tryptophan repressor, the product of the trpR gene. As examples of amino acid production by compo¬ site plasmids we constructed plasmids that deregulate either phenylalanine or tyrosine production by suitably engineering the aroF, pheA and tyrA genes from E.coli K12. We used as a source of these genes a 6Kb EcoRI fragment isolated from a gene library by its ability to complement lesions in pheA and aroF G H. This fragment is essentially identical to that described by Zurawski et al. and was cloned into the multicopy vector pAT153 to yield pME65. The pheA and aroF genes were located and sequenced by established methods and deregulated as described in the Examples. Preferred composite plasmids and the host HW77 E.coli were deposited and are listed in the table of deposited plasmids. Table of Deposited Plasmids HW 77 (Host Cell) ATCC 13281 pME 202 ATCC 53136 pME 214 ATCC 53137 pME 219 . ATCC 53138 pPT 112 ATCC 53139 The American Type Culture Collection is located at 12301 Parktown Drive, Rockville, MD 20852, USA. Example 1 - Preparation of .PheA, AroF, AspC and Amylase Genes 1. Preparation of pheA gene The sequence of the pheA gene is described in Chart 2. Insertion of a BamHI-Bglll linker at the Ncol site was found to render the prephenate dehydratase activity of the enzyme substantially resistant to feed¬ back inhibition by L-phe. In addition, both the choris¬ mate mutase and prephenate dehydratase activities of the enzyme were enhanced. Transcriptional deregulation was effected by replacing the promoter and attenuator sequences with a synthetic promoter based on the natural pheA promoter but lacking the dyad symmetry overlapping the pribnow box sequence (-10). This replacement was made between the EcoRI site before the gene and a Haell site lying in the N-terminus of the natural pheA gene. Finally, a BamHI site was introduced 3' to the pheA gene by directed mutagenesis. The resulting optimized pheA sequence is illustrated in Chart 3. 2. Preparation of aroF gene The sequence of the aroF gene is described in Chart 4. A feedback inhibition resistant derivative was isolated in the following way: The aroF gene was first subcloned on a Hindlll-Ba HI fragment into the inter¬ mediate copy number vector pLG338. This was transformed into a strain (HW295) lacking all three DAHP synthetase isozymes. This resultant : strain was then able to grow on minimal medium in the '-. scΛce of exogenous aromatic amino acids and shikimaie by virtue of the wild type aroF gene product carried on the plasmid. In the presence of lOOmcg tyrosine ml -1 , however, the aroF gene product is inhibited and consequently the strain is unable to synthesize sufficient Phe and Trp for growth. Mutants capable of growth on minimal media were isolated, pooled and retransform HW295 and the resulting transformants were screened for their ability to grow on minimal medium supplemented with tyrosine. Transformants capable of growth on this medium were assumed to have received a mutant plasmid and were I ECORI G AATTCACCAAGACGGGAACACAAGACGTAAAARRTATGACAATGAACATTACCAGCAAA S I CAMTGGAAATTACTCQSCCCATCQGCCAACATCTCSCAGACCBTCTOGCCAAACΓGGAA 12* AAATGGCAAA(_ACATCTCATTAATCCA(_ATAT ATRCTGTCCAAAGAGCCACAAGGGTTT 188 GTTCCTGACCCCACAATCAATACACCRAACGGOGTTCTGGTTGCCAGTGGTAAACATGAA 248 GATATGTACACCCCAARTAACGAATTCATCAACAAGCTGGAACGGCAGCRCAATAAACTG 3 ΒΒ CAGCACAAACGCGAAGACGTCGTGCaSCAACATCSGTG AAAG ACGCCAA'CTTCGTCG AAG 36 • AAGTTCAACAAGAGTAGTCCMrTATATTGAGTGTATCGCCAACGCGCCrTCGGGCGCGTT 42i TTTOTTCA ( -AGCGTX_AAAACAGTACEGGTACTGTACTAA-CTCACTTAA-_GAAACAAAC 4 βf H K H I P P P P A P P P T P P ATCAAACA<»τAC05TTirT CrτCGCATTCTTTrτrACCrτCCCCTGAATCGGACGOGTT 54* TCGTCGTG G AAACAGAATOCGAAGACG AACAATAMGCCTCCCAAATCGGGGGGCCTTT β • 8 H T S E N P L L A R TTTATIG ATAACAAAAAGGCAACACTATG ACATSGAAAACCOGTTACTGGCGCTGCGA-- 6 β • E K I S A D E K L A L L A E R E L AGAAAATCAGt.ca.arGGATGAA.UlATTATTAGCGTTACTGGCAGAACGGCGCGAACrGG 728 A V E V G K A K I. L S H R P V R D I D R Ca_TCGAGGTGGGAAA«CCAAACTGCTCTCGCATCGCCa-GTACGTG ATATTCATCGTG 781 E R D L B H _, I T I_ G K. A H H L D A H AACGCGATTTCCTGGAA-CATTAATTAOGCTCGGTAAA.OGCACCATCTGGACaCCCATT 841 T I T R L P Q _. I I B D S V T Q Q A I. A<_ATTACTOGC(_ GTTCCAGCT<-AS_ArTCAAGArrCCGTArrAACrCAGCAGGCTT GC 988 L Q Q a H K I H P H S A R I A P L G P TCCAA<_AACATCTCAATAAAAtTAATCCGCACTCAGCACGCATCGCTTTTCrCGGCCCCA 968 JΓ Q S Y S H L A A R O Y A A R R _* E O 7 AAGGTTCTTATTCCCATCrrcCGGCαGGCCAGTATGCτGCCOGTCACTTroAGCΛATTCA 1828 I E S G C A R P A D I P N Q V E T G Q A TTCAA_CTCGCRGCGCCAAATTTGCA_ATATTRT AATCAGG GGAAACO_IGCCAGGCCA 1888 D R A V V P I E H T S S G A I N O V Y O ACTATCCA3TCGTAC03ATTCAAAATAC(_AGCRCCGGTGCCATAAACGACGTRRACGATC 1141 -, l3 H T S t. S t V- G e H T _. T I D H C TGCTGCAA(_ATAC(_AGCTT3TCGATTCTTCGCC-rtGATGACGTTAACTATCGACCAT GTT 1288 _, -. V S G T T D t. S T t N T V ϊ S H P O TGTTGGTCTCOGGCACTACTGATTTATCCACCATCAATACGGTCTACAGCCAT CGCAGC 1261 P P Q Q C S K P -. N R Y P H W I E Y T CATTCCAGCAATGCAGCAAATTCCrTAATCGTTATCCGCACTGGAAfiATTGAATATACa. 1321 E S T S A A M E K V A Q A K S P H V A A AAAGTACGTCTGCGGCAA GGAAAAGGTTCCACAGGCAAAATCACCSCATGT GCTGCGT 1388 L G S E A G β T y G O V L E R I B A TGGGAAGCC AAGCTGGOSGCACTTTGTACGGTTTCCAGGTACTGGAGCGTAT GAACCAA 1 41 H Q R Q H P T R P V V A K A I H V S ATCAGCX5ACAAAACTTCACCCGATTTGTGGTGT GGCG03TAAA_-CCATTAACGTGTCTC 1518 D Q. V P A K T T M A T G Q Q A G A L ATCAGGTTCCGGCπAAAACaCGTWTTAATCGCGACCGGGCAACAAGCaSG'IGCGCTGG 1568 V E A t. t. V I. R N H N t. I T R L E S R TTCAAGCGTTGCTCGTACreCGCAACCACAATCTGATTATCACCCGTCTGGAATCACGCC 1628 P I I G H P M B B N P r i. O I Q A M L E CGATTCACGGTAΛTCCATOGGA*. ACATGTTCTATCTGGATATTCAGGCCAATCΓIG AAT 16 8 8 A 8 N 0 K A X B G B I T R S N K V CAGCGGAAATCCAAAAAGO.TTGAA_CAGTTAGGGGAAAT ACC GTTCAAroAAGGTAT 1748 L G C Y P S B H V V P V D P T *** TGGGCTβTTACCCA_U_raACλA(XiTAGTGCC_TGriGATCCλACCTGλTGAAAACGTSCα3 1888 GAlGAtGTGAATCAtCa!GCACTCGATTATTACrGGCGATTGTCATTCGCCrG ACGCAAT 186 • AACAOGCGGCTTTttCTC GAAAACSCTG GCGTAATCGCCGAACCAGTGCTCCACCr : 1928 CGGAAACTGTCAATAAACnCCTGCT ATCGCCCTGCTCCAGCAACrCAATCGCCrCGCa: 1988 AAAOGCrrATAGTAACGTTKATTAACGCCAGArrACGCTCTGACGACATAATOATGTCG 2848 GCATAAAGCTGCGGATCC 2858 BuBI 4' Sequence of the EcoRI-BaaHI Praaent Encoapeβslng The s aft Gen*. _K£T Tha Haell site used in the promoter swap %%%ttt The Ncol site used Cor the Bglll linker autagenesis $S$$SS EcoRI -35 PROMOTER -18 SO GAATTCWTTTTGT1GACAGCGTGAAAACAGTA05GGTATAATACTAAΛGTCAΛAAAAG 68 — iti M T S E H P L L A L R E R X S A GCAACACTATGACATCGGAAAACCCGTTACTGGCGC GCSAGAGAAAAT AGCGCGCrGG 28 6 %%%% % O E K L IT A L A B R R B L A V E V G K ATGAAAAATTATTMCGTTACTGGCAGAACGGOGCGAACKGCΣ-TCGAGGTGGGAAAΛΪ 188 A -. S H R P V R O I D R B R O L B CCAAA<_TGCTCTCGCATCOCCΣ_GTACGTGATATTCATCOΪΑAACACGATTR-CTGGAAA 248 R I T G K A H B L D A H Y I T R L P GATTAATTACGCTCGGTAAAGCGCACCATCTGGACGCCCATTACATTACTCGCCΓGTTCC 88 Q L X I E O S V T Q Q λ l. L Q Q H L N AGCTCATCAnGAAGATTCCGTATTAACTCAGCAGGCTTTGCTCCAACAACAT rCAATA 368 K I N P H S A R I A P L G P JC G S Y S H AAATTAATCσ5CACTCAGC_ACGCATα_CITTTCTOGGCCCαAAGGTT rTATTCCCAT 28 A A R Q Y A A R H P E Q P I E S G C λ TTGCGGCGCGCCAGTATGCKCCCTTCACTTIOAGCAATTCATTGAAAGTGGCTGCGCCA 88 K P A D I P N Q V B T G Q A D Y A V V P AATTTGCCGATATTrrrAATAGGTGGAAACCGGCCAGGCO_lACTATGCCGTCGTACOGA S48 I E H T S S G A I N D V Y D L Q H T S TTGAAAATACCAGCTCCGGTGCCATAAACGACGTTTACSATCrGCTGCAACATACCAGCT Sββ L S I V G E H T T I O H C L V S G T TGT03ATTGTTGGCGAGATGACGTTAACTATCaACCATTCTr_GTTGGTCrC03GCACTA 668 T O L S T I N T V 'Ϊ S a P Q P P Q Q C S CTGATTTATCCACCATCAATACGGTCTACAGCCATCOSCAGCCATTCCAGCAATGCAGCA 729 K F N R Y P H W K I E Y T E S T S A A AATTCCrTAATCGTTATCCGCACTGGAAGATTGAATATACOGAAAGTACGTCTGCGGCAA 788 M E K V A Q A K S P R V A A L G S B A G TGGAAAAGGTTGCACAGGCAAAAT \CCGCATGTTOCTGCGTTGGGAAGCGAAGCTGGCG 848 G T Y G L Q V r, E R I B A H Q R Q H P GCACrTTGTACGGTTTCCAGGTACrGGAGCaTATTGAASCAAATCAGCGACAAAACrTa. 988 T R P V V A R K A I H V S D Q V P A K CCCGATTTOTGGTGTTGGCGCGTAAAGCa_TTAACGTGTCTGATCAGGTTC03GCGAAAA 968 T T L M A T G Q Q A G A L V B A L L V CCAOGTTGTTAATGGCGACCGGGCAACAAGCCGGTGCGCTGGTTCAAGCGTTCcrGGTAC 1929 L R N H N L ^ rt ^ R L E S R P H G N P TGCGCAACCACAATCTGATTATGACCCGTCTGaAATCACOCCOGATTCACGGTAATCCAT 1988 W R S P W E E M F Y L D T O A N L E S A '_ GGMATCTC»TGGGAAGAGATGTTCTATCTGGATATT AGGCCAATCTTGAATCAGCGG 1149 E M Q. A' U K E L G E I T R S M K V L G AAATGCAAAAAGCATTGAAAGAGTTAGGGGAAATCACCCGTTCAATGAAGGTATTGGGCT 12B8 C Y P S E N V V P V D P T •*«*•* GTTACCCAAGTGAGAACGTAGTGCCTGTTGATCCAAC<_TGAIGAAAAGGATCC 1253 BamHI Ch rt 3 Optimised Bits A Sequence. The sequence of the EcoRl-BaaHt fragment carrying the ieA gene from pHE198 Is presented. The Haell site used in the promoter swap ._%_%% The 12 bp insertion conferring PBI-R .-.. — - , --.-..-.-. The Shine i Dalgarno sequence in the RBS. an The -19 and -35 regions of the promoter — — — HinDIII AAGCTTTTACOGGAAMTCCrCGGGCATA_«TAAAATCGGTCTGGCAAGTTrT CTrTGT (8 CCTTCCGCATATCCrrrAAGGTATAGACCQCGATCCBCCrβTGAATCGCrAAATGCATCα 128 TCATCTTTTATGGCGCTGCCOGATATCGCATCTTCCATACQSACATCATACCAGTCGGTG 188 CTGGTC-UGGTTGGCGCGTGMTATAAGGATCAATerGACATCCGCTAACCAGTAAAGCC 248 AACAAGGGaGCGATAAACTTTTTCATCAirrTCrTTCTCCrTTTTCAAA--CATAGCGGACT 388 GTTTTCAAAGGGAG^GTAAATTTATCTATACAGAGGTAAGGGTTG AAAGCGCGACTAAAT 368 TGCCTOTGTAAATAAAAATGTACGAAATATGGAr_GAAAACtTTA<_TTTATGTGTTATCG 28 H TTAOGTCATCCTCGCTGAGGATCAACTATCGCAAACaAGCATAAACAGGATσSCCATCAT 488 Q. K D A L H M V a i T D B Q V L M T P B GCAAAAAGACSCGCTGAATAACGTACATATTACOSACGAACAGGTTTTAATGACTCOGGA 5 8 Q L X A A P P L S L Q Q B A Q I A D S R ACAACTGAAGGCCSCTTTTCCATTGAGCCTGCAACAAGAAGCCCAGATTGCTGACTOSOS 688 K S I S D I I A G R O P R L I. V V C G P TAAAACCATTTCAGATATTATCGCOGGGCGCGATCCΓCGTCTGCTGGTXΪTATG GGTCC 668 C S I H D P E T A L S Y Λ R R P K A Λ TTGTTCCATTCATCATCCGGAAACTGCTCTGGAATATGCΓCGTCGATTTAAAGCCCTTGC 728 A E V S D S L Y V H R V Y P S L- P R T CGCλGAGGTCλGCGATAGCCTCTATCTGGTAAroCGCGTCTATTτTGAAAAACCCa_TAC 788 T V G W K G L I N D P H H D G S P D V B OCTGTCGGCTGGAAAGGGTTAATTAACGATCCCCATATGGATGGCTCTTTTGAIGTAGA 848 A G L Q I A R X L L B V M M G L P AGCCSGGCTGCAGATCGCGCGTAAATTGCTGCTTGAGCTGGTGAATATGGGACTGCCACT 998 A T E A L D P N S P Q Y L G D P S W S GC^Aa.GAAGOGTTAGATCOSAATAGCCCTCAATACCTGGGCGATCTGTTTMCTGGTC 969 •I* A I G A R T T E S Q T H R B M A S G S AGCAATTGGTGCTCGTACAACGGAATO3CAAACTCACCGTGAAATGGCCTC05GGCrTTC 1928 K P V G P K H G T D G S L A T A I N A M CATGCCGGTTGGTTTTAAAAACGGCACCEACGGCAGTCTGGCAACAGCAATTAACGCrAT 1888 R A A A Q P H R P V G I N Q A G Q V A I, GCGCGCCGCCGCCCAGCCnCACOGTTTTGTTGGCATTAACCAGGCAGGGCAGGTTGCGTT 1148 _. Q T Q G N P D G H V i r. R G G K A P H GCTACAAACTO_GGGGAATCO_GACGGCCATGTGATCCrGCGCGGTGGTAAAGCGCCGAA 1288 Y S P A D V A Q C E K E M B Q A G L R P CTATAGCCCTGCGGATGTTGCGCAATGTGAAAAAGAGATGGAACAGGCGGGACTGCGCCC 1268 S H V D C S H G N S H K D Y R R Q P A GTCTCKATGGTAGATTGCAGCCACGGTAATTCCAATAAAGATTATCGCCaTCAGCCTGC 1328 V A E S V V A Q I K D G N R S I I G M GGTGGCλGλATCσ.TGGTTGCTCλλATCλλΛGATGGCAΛTCGCTCλΛTTλTTGGTCTGλT 1388 I E S M i a i S H Q S S B Q P R S B H K GATOSAAASTAATAT σ.OSA-iGGCλATCλβTCrrcα.MαU CBCQCλGTiAAAtGλλ 1448 Y G V S V T O A C I S M B H T D A L X ATλCGGΪGTλTCCTTλACα.ATGCCTGCλCTMCTGGGλAArcλCCTλTOCCTWCTGCα 1588 E i a Q O Ϊ. M G Q -. T A R V A *** TGAAATTCATCAGGATCTGAACSGGCAGCTGACGGCZCGCSTGGCTTAAGAGGTTrA'rrA 1568 TGGTTGCTQAATTGACOBCATTACGCGATCAAATTGASGAAGTCGATAAAGCGCTGCXGA 1628 ATTTATTAGCGAAGCGTCTGGAACTGGTTSCTGAACTGGGCGAflGTGAAAAGCOGCrTTG 1688 CACTβCCTATTTATGTTCαϊGCAGCGCGAGGCATCTATβTTGGCCrCGCGTCGTβCAGAG 1748 GCGGAA__CTCTGGGTGTACa.CCAGAT<-T 1769 Bglll Chart 4 Sequence of the HlnOIII-Bglll Fragment Encompassing the Λ fiP Gene. _U_T Sequence altered to a BamHI site In RBC ----- » Co don altered in m-R mutant (CAAaβn-CGAarg) *1* PvuII site uaed in terminator swap underlined %% tt% assayed for DAHP synthetase activity. One mutant that carried a substantially feedback inhibition resistant aroF gene was kept for further analysis. Restriction fragment switching experiments revealed that the 5 feedback lesion was located on a BstXI to PvuII fragment. Subsequent DNA sequencing revealed a single base pair alteration that changed glutamine 152 to an arginine residue. The promoter and tyrR binding site were 10 removed by introducing a BamHI site into the aroF ribo¬ some binding site. Sequences downstream of the aroF gene were removed by replacing a PvuII-Sall fragment with a synthetic terminator also carried on a PvuII-Sall fragment. The sequence of the optimized aroF is illus- 15 trated in Chart 5. In addition to pheA,. aroF and tyrA genes, the aspC gene from E.coli K12 and the alpha amylase gene from B.lichenoformis were prepared for use in composite plasmids. rs 4. — si 3. Preparation of aspC gene The cloning of the aspC gene has been des¬ cribed in European Patent Application 84100521.8 (Pub. 23 o * 116860). For the purposes of the present invention it was necessary to sequence the gene and introduce convenient restriction sites. The DNA sequence is pre¬ sented in Chart 6. A BamHI site was introduced into the ribosome binding site by directed mutagenesis. A Bglll 30- site was introduced downstream of the gene by making use of a convenient StuI site to insert a Bglll linker. This enabled the aspC gene to be isolated on a con¬ venient BamHI to Bglll fragment. The optimized aspC gene is shown with ribosome binding site in Chart 7. 5 4. Preparation of Amylase gene The cloning of the B.lichenoformis gene is described in European Patent Application 84308.868.3. 5 The nucleic acid sequence for the amylase gene is shown in Chart 8. Into the ribosome binding region we intro¬ duced a BamHI site by directed mutagenesis. A second BamHI site was introduced downstream of the gene by inserting BamHI linkers into a convenient Hindlll 0 site. Details of these modifications are described in Chart 9. Examples of Composite plasmids and their applications 5 Example 2 Constructs in which the genes are transcriptionally deregulated by virtue of their high copy number. 0 The plasmid pME157 carries a FBI R pheA gene transcribed from its wild type promoter cloned into the high copy number vector pAT153. The aroF gene along with a portion of the tyrA gene on a Hindlll-Bglll frag¬ ment was first modified by the addition of a Bglll ~ linker to the Hindlll end and then cloned into the BamHI ■ site of pME157. Two classes of plasmid resulted from this procedure. In the first (pME171) the pheA and aroF gene are transcribed colinearly. The second class (pME170) carries the two genes transcribed conver- p 0 gently. pME171 was then modified to encode a FBI Λ aroF product by switching a BstXI fragment with the FBI R aroF plasmid pMX13 to yield the deregulated plasmid pST35. The effect of these plasmids on the levels of DAHP syn- thetase and prephenate dehydratase are summarized in 5 Table 1. BarnBI « Q f O A _. » » v a i T D B Q V GGATC C λTCATeCλAAAA-_λCGCGCTGλATAΛOGTλCλ?ATTλC0GλCGλACλGβrR 68 L M T P B Q L X A A P P L S L Q Q E A Q TAATGACTCaGAACAACTOAAGGCCGCTTTT CATTGAGCCrGCAAO.AGAAGCCCAGA 128 I A D S R X S I S D I I A G R D P R L L TTGCrGACrCGαiTAAA-ttCATTtCAGATArrATCGCCaGGCGOGAT CTCGTCrGCTGG 188 V V C G P C S I H D P E T A B Y A R R TACTATGTGGTCC:TGTT a.TTCATGATC03G--AACTGCTCrGGAATATGCTCGTOGAT 248 P X A L A A E V S D S L Y L V K R V Y F TTAAAGCCCXTGCCSCAGAGGTCAGCGATAGCCrCTATCrGGTAATGCGCGTCrATTTlG 388 E K P R T T V G W K G I H O P H M O G AAAAACCCQSTACCACTGTCSGCrGGAAAGGGTTAATTAACaATCCCCATATGGATGGCT 369 S P O V E A G L Q I A R X L L I. B _. V H CTTTTCATGTKAAGCCGGGCTGCAGAT QCGCGTAAATTGCTGCTTGAGCTGGTGAATA 428 H G L P L A T B A L O P N S P R Y L G O TGGGXCTGCCλCrGGCGλCSGAAGCGTTMSATCα-AλTKCCOGCSλTλCCTGGGCSλTC 488 •I* L P S H S A I G A R T T B 3 Q T H R B M TGTTTA__CTGGTCAGCAATTGGTGCTCGTACAACGGAATCGCAAACTCAC .TGAAATGe .5 8 A S G L S H P V G P X M G T D G S L A T CCTCCGGGCTTTCCATGCCGGTTGGTTTTAAAAACGGCACOSACGGCAGTCrσGCAACAG 688 A I H A H R A A A Q P H R P V G I M Q A CAATTAACGCTATGCSCGCCGCCGCCCAGCGSCACCGTTTTGTTGGCATTAACCAGGCAG 668 G Q V A L L Q T Q G H P O G B V I L R G GGCAGGTTGCGTTG TACAAACTCAGGGGAATCCSGACGGCCATGTGATCCrGCGCGGTG 728 G X A P N Y S P A D V A Q C E X E H E Q GTAAA-<CGCa:AACTATAGCCC7GCGGATGTTGCGCAATGTGAAAA_>__AGATGGAACAGG 738 A β L R P S L H V D C S B β H S H X D Y CGGGACTGCGCCO_TCTCTGAlGGTAGλTTGCλGCCλCSGTλATTCCλλTλλΛ__λTTλTC 848 R R Q P A V A B S V V A Q I X D G M R S GCCGTCAGCCTGCGGTGGCAGAATCσ.TGGTτGCTCAAATα.AAGATGGCAATCSCTCλA 998 I I G L H I B S N i a E G H Q S S E Q P TTATTGGTCTGATOATCaAAAGTAATATCCACGAGGGCAATCAGTCTTCCGAGCAACCGC 9S9 R S B- M X Y G V S V T O A C X S W S H T . GCAGTGAAATGAAATACGGTGTATC03TAAC03AB_CCtGCATTMCTGGGAAATGACQ3 1929 O A L L R E I H Q O L H G Q L T A R V A » ATGCCTTGCTGCGTGAAATT ATCAGGATCrGAACGGGCAGCTGACGGCTCGCGTGGCTT 1889 ♦ ••••Baaϋl Sail AATGACGATCCaSGGCGATTCTACGCCO-GGTTTTTTATGTOSAG 1125 $$$889 $$$$$$ Smal Smal C a3Tt 5 Optimised &χ_T Clone. JS-Kf Site of PBI-R mutation *l« Smal sites in terminator SSSSSS Dyad symmetry in terminator ------- PvuI site uaed in terminator swap underlined ttt%%% τte« yA-acAC CCTCTitoϊf *AWcegAA-uiA eΛ--aA -τGcτecitfτfτιι rr «O TATACCTTCeACAaCAATCTCACSr TWCAAAAA AaCCTGCUfl -TCArCAGTAATAG 120 TTGSAAI t n TAAATCTCCCGTTACCCTgAgAflCgGACrTCCCTTCTCIAACCATAATC 180 N P B M I T A A P A O P I L G L A ffl CCrCCTCATGTTTCAGAACATTACCl-CCqCTCeTKCGACCCGATTCTqCCCCTGgC 240 O L P R A O C R P β K t M b β l G V Y X CGλTCTβrrrCGTGCCβATβAACβT CCGGαkAAATTλACCTeβGβλTTβGTGTCTATAA 300 O B T G K T P V t. T S V X K A E O Y t t AβλTGAUCGGG AAAACCCCSGTACTGACCA-KSTGλAA.UU.βCl^.-VAUβTλTCTGCr 360 C N E T T X M Y L G I O β l P E P G R C CGAA_W.TβAAACCACCAAAAATTACCTCGGCArrGACGGCATCCCT JkTTTGβTCGCTβ 420 T O B L P S K β S A L X M O X X A R T CACTCAW_AACTGCTGTTTt-OTAAAGGTAGCG CCTβATCAATGACAAACGTGCTCGCAC 480 A Q T P G G r β A _, R V Λ A O F t. A K -l GG»CAOACT CGβG<XiGCλCTCGCGCλCTACGCβTGG TGCCGλTTTCCIGGαUUAAA 540 T S V X R V H V S N P S W P M I X S V P TACCAGCGrrAAGCGTGTβTGGGT(_ACrCAACC λAβCTCGCCβ»ACCλrAAβλβ βTCTT 600 M S A S L S V K C Y A Y Y D A B M I T L TAACTCTGα-GGTCTG-AAgrrCGTGAATACGCTTATTATGATGCGGAAAATeACACTCT 660 O P O A L I M S L M C A Q A G O V V -. P Tt_ACTrCGATGCACTαλTTAACAG CT_AATGAAGCrCAGGCraG GλCβTλβTCCTGTT 720 a G C C B M P T G I O P T L E Q W Q T t. CCATGGCTGCTGCCATAACCCAACCGGTATCGλCCCrACGCTGGλACAATGβCAAACACr 790 A Q t. s v ι κ a t» _, p t. F o r A Y Q G r GGCACAACTCTCCGTrQuakAAGGCPWTTACCGCTβTrτGACTTCGCTTACCAβCβTTT 840 A R G t. E C O A E G l. X λ r λ A H B R C TGCCCGTGGTCTGGAAGAAβATrCTGAAGCUCTGCKCrCrrrCGCGGCTATGCATAAAGλ 900 C I V A S S Y S K M r G L Y N C R V G A GCTGATTGTTGCαGTTCCTACTCTAAAAACrCTGG CTβTACAACGAG GTβTTGβ GC 960 C T L V A A O S E T V O X A r S Q I- X A rreTACTCTGGTTGCT(κCGACAGTβAAACCGTπ_ATCG G Arri_AGCCAAATGAAA<_C 1020 A I R A M Y S M P P A H G A S V V A T I GGCGATTCGCGCTAACTACrCTAACCCACCAGCACACGGCGCTTCTβTTβrrGCCACCAT 1080 S H O A L R A I M E O C C T O H R Q X CCTGAGCAACGATGCGTTACGTβCGATTTGGGAACAAGAGCTGACTGATATGCGCCAGCG 1140 l O R R Q t- P H g ε X G A N K O TATTCAGCGTATGCGTCAβTTGTrCGTCAATACGCTGCAGGAAAAAGGCGCAAACCGCGA 1200 P S P t I X Q H G H P S r S G _. T K E Q CTTCAGCTTTATCATCAAACAβAACGβCATGTTCTCCTTαkGTGGCCTGACAAAAGAACA 1260 V L R t. R E E F G V Y A V A S G R V .I V AGTGCTGCGTCTGCGCGAAGAGTtTGCCGTATATGCG β TTGCTTCTGGTCGCGTAAATβT 1 20 A G N T P D M H A P t. C E A r V A V t. "-' GGCCGGGATGACACCAGATAACATGGCTCCGCTGTGCGAAGCGATTGTGGCAGTG TGTA 1 80 AGCATTAΛΛAΛCΛΛTβAΛGCCCGCTGΛΛAΛGCGGGCTGΛGACTGATGACAAΛCG AΛCΛT 1440 TGCCTCATGCGCTACGCTTATCAGGCCT 1 68 C art 6 Natural _UffC Gene Sequence. ____! Site of BamHI site Introduced in RB3 Table 1 DAHP Synthetase and Prephenate Dehydratase Activities of HW77 Carrying Various Phe Plasmids. PREPHENATE DAHP PLASMID DEHYDRATASE SYNTHASE H 77 alone ND ND pME214 19 65 PME208 36 49 pMH19 141 324 pME202 705 1145 pME219 491 694 pME170 290 4505 pME171 372 3570 pST35 546 7433 pME237 240 3146 pMΞ238 209 3420 pAT153 3 34 H 77 was transformed with the plasmids indicated. Each strain was grown to ODggg 1.0 in minimal medium supplemented with 0.5% casamino acids. Cell extracts were then prepared and used to assay the activities of prephenate dehydratase and DAHP synthase. Prephenate dehydratase activity was measured in the presence of L-phenylalanine. DAHP synthase activity was measured in the absence of feedback inhibitors and so represents the total DAHP synthase activity. Enzyme activities are expressed in mϋ min "1 mg protein -1 . The three plasmids pME171, pME170 and pST35 were introduced into the tyrA organism HW77. Cross- feeding experiments on these strains (Table 2) showed that the plasmids pME170 and pME171 caused a significant production of L-phenylalanine. H 77 carrying pST35, however, grew very badly on minimal medium and as a consequence showed very little cross-feeding. It p appears that a high level of FBI aroF is deleterious to the cell and that lower levels of the enzyme are required for optimum L-phenylalanine production under these conditions. Example 3 Composite plasmids which include a transcriptionally deregulated pheA gene. The plasmid pJ 2 carries the FBI pheA gene transcribed from the synthetic promoter. Plasmid pME227 p carries a FBI aroF gene transcribed from its own promoter and ending with the synthetic terminator sequence described above. Both plasmids are based on the multicopy plasmid pAT153. An EcoRI fragment carry¬ ing pheA was constructed by the addition of EcoRI linkers to an EcoRI-BamHI fragment of pJ 2 carrying pheA. This fragment was then cloned into the EcoRI site of pME227. The resulting plasmids carrying pheA trans¬ cribed in both orientations relative to aroF were desig¬ nated pME238 (divergent) and pME237 (colinear). The DAHP synthetase and prephenate dehydratase activities in strains carrying these plasmids are given in Table 2. Table 2 Cross-Feeding Experiments on HW77 Carrying Cloned Genes Pertaining to Phenylalanine Production (Phe Plasmids) . HW77 was transformed with all the plasmids described in the examples section to give the strains listed below. The relative extent of cross-feeding after 24 hr and 48 hr is indicated. PLASMID CROSS-FEEDING OBSERVED 24 hr 48 hr H 77 alone (+) + pME214 ++ ++++ PME208 ++ ++++ pMH19 +++ +++++ pME202 +++ +++++ pME219 ++ ++++ pME170 ++ ++++ pME171 ++ ++++ pST35 NG (+)* pME237 NG (+)* pME238 NG (+)* pPT112 ++ +++ 4 0 pAT153 (+) * Some cross-feeding observed but from what were obviously a few mutant colonies that had managed to grow. NG = no growth Both plasmids when introduced into HW77 (ATCC 13281) prevented the strain from growing on minimal media. Again this is probably due to the excessive p expression of FBI aroF. As a result of this neither ■a-UIX M P B M I T A A F λ O P I L G L GGATCCTCGTCATCTTTαkβλACATTACCβ CGCrCCrβCCβACCCGλTTCTβGβC rGC CO A D L P R Λ O C R P G X I H L β X S V Y eeGATCTGIfTCGTGCC--ATGAACGTeCCGGCAAAA'rTAACeTCGGGArrGGTGTCTATA 120 K D B T G K T F V L T S V X X A E Q Y L AAGATGAGλCGβGCλλAACCCCGGTACTGACC-λGCGTβAJUAAGβCTβλACλGTATCrGC 180 L B N B T T X H Y L G t D G I F B P G R TCGAΛΛATGAΛACCACC-VAAΛATTΛCCTCGGCATrGACGGCATCCCTGAATTTGGTCGCT 240 C T Q I L L F G X G S A L I N D X R A R GCλCTCAGGAACTG TGTTTGGTAAAGGTAG GCCCTGATCAATGACAAACGTGCTCGCλ 300 T A Q T P G G T G A L R V A A O P L A X CGGCACAGACTCCGGGGGGCACTGGCGCACTACGCGTGGCTβCCGArrTCCTGGCAAAAA 360 N T S V K X V M V S H P S lf P N a K S V ATACCAGCGTTAAGCGTGTGTGGGTGAGCAACCCAAGCTGG CGAACCATAAGAGCGTCT 420 F H S A G L E V R E Y A Y Y O A E M H T TTAACTCTGCAGGTCTGGAAGTTCGTGAATACGCRRATTATΒATGCGGAAAATCACACTC 480 L D F D A L I M S L N E A Q A G D V V L TTGACTIXGATGCACIGATTAACAGCCTGAATGAAGCTCAGGCTΒGCGACGTAΒTΒCTΒT S40 F B G C C B N P T G I D P T L E Q TI Q T TCCATGGCTGCTGCCATAACCCAACCGGTATCGACCCTACGCTGGAACAATGGCAAACAC 600 L A Q L S V E K G W L P L F O F A Y Q G TGGCACAACTCRCCGTTGAGAAAGGCTGGTTACCGCTGTTTGACTRCGCTTACCAGGGTT 660 F A X G L E E O A E G L X A F A A .- H K TTGCCCGTGGTCTGGAAGAAGATGCTGAAGGACTGCGCGCTTTCGCGGCTATGCATAAAG 7 J 0 E L I V A S S Y S X N P G L Y H E R V 3 AGCTGATTGTTGCCAGTTCCTACTCTAAAAACTITGGCCTGTACAACGAGCGTGTTGGCG 80 A C T _, V A A O S E T V D R A R S Q H K CRTGTACTCTGGTTGCTGCCGACAGTGAAACCGTTGATCGCGCATTCAGCCAAATGAAAG 3 0 A Λ I R A N Y 3 N P P A H G A S V V A T CGGCGATTCGCGCTAACTACTCTAΛCCCACCΛGCACACGGCGCTTCTGTTGTTGCCΛCCΛ 900 I L S H O A L X A I H E Q E L T D H R G TCCTGAGCAACGATGCGTTACGTGCGATTTGGGAACAAGAGCTGACTGATATGCGCCAGC 60 R I C R .I R Q L F V N T L Q E K G A N R GTATTCAGCGTATGCGTCAGΓΓGTTCGTCAATACGCTGCAGGAAAAAGGCGCAAACCGCG 1020 D F 3 F I I X Q N G -- F S ' F S G L T X T ACTTCAGC_TTATCATCAAA<_AGAACGGCATGTTCTC -TCAGTGGCCTGACAAAAGAAC 1080 Q V L X L R E E F G V Y A V A S G R V M AAGTGCTGCGTCTGCGCGAAGAGTTTGGCGTATATGCGGTTGCTTCTGGTCGCGTAAATG 1140 V A β lf T P O N H A P L C B A I V A V L * TGGCCGGGATGACACCAGATAACATGGCTCCGCTGTG GAAGCGATTGTGGCAGTGCrGT 1200 _ • AAGCATTAAAAAC_MTGAAGCCCGCTGAAAAGCGGGCn_AGACTGATGACAAACGCAACA 1260 Bglll TTGCCTGATGCGCTACGCTTATCAGGGAGATCT 1293 Chart 7 Optimised &αpC Sequence. CCORX ^TTOkGATTT_1-TGAACAGGArrrcAAβCATrβCCTATαUM^ 60 TCCeτeTOCTGACATGGCrrTATTAAAAGAAGGGACGAGCCCTeAGGAAGAACATTrCAG 120 CCCXΛTrGCGCrrerrGAAAACGAGGTGGAATTTTATTTTGAGAAAAAACAAACCGGTCT 180 TGAACAGTCTGTAAAAATAAAAAAAGAATGGGTAAAAGATATTTTAAAAGACCGATATCA 240 GGATATGAAAAAGAATCGTCTTαW_GCCAAACCTGATCAGGAGCCTGTTCCGCTrCCGAA 300 GCAAGCGAAAATTAATCCCGATGAAAAAGTGATTGCCCTCACATTTGATGACGGTCCGAA 360 TCCCGCTACAACGAATAAAATATTAAACGCTTTACAGAAGCATGAAGCGCATGCGACCTT 20 CTrTGTGCTTGGAAGCAGAGCCCAATATTATCCCGAAACGATAAAACGGATGCTGAAGGA 480 AGGAAACGAAGTCGGCAACCATTCCTGGGACCATCCGTTATTGACAAGGCTGTCAAACGA 540 AAAAGCGTATCAGGAGATTAACGACACGCAAGAAATGATCGAAAAAAT ACCGGACACCT 600 GCCTGTACACTTGCGTCCTCCATACGGCGGGATCAATGATTCCGTCCGCTCGCrTTCCAA 660 TCTGAAGGTTirArrGTGGGATGTTGATCCGGAAGATTGGAAGTACAAAAATAAGCAAAA 720 - GλTTGTCAATCλTGTCλTβλGCCλTGCGGGAGACGGAAAAATCGTCTTλλTGCACGATAT 780 TTλTGCλACGTCCGCAGλTCCTGCTGλAGλGAπATTλAAAAGCTβAAAGCAAAACiGCTλ 840. TCΛATTGGTAACTGTATCTCλGCrKAAGMGTGMGλAGCλGλGAGGCTλTTGAAΪAAA 900 TGAGTAGAAAGCGCCATATCGGCGCTTTTCTTTTGGAAGAAAATATAGGGAAAATGGTAC 960 TTGTTΛΛΛΛΛTTCGGλATΛTTTATACAACATCATATGTTrCACATTGΛΛΛGGGGAGG-VGΛ 1020 n X O Q K X L Y A X L L T L L F A L X ATCATGAAACAACAAAAACGGCTTTACGCCCGATTGCTGACGCTGTTATTTGCGCTCATC 1080 r L L P H S A A A A A N L H G T L M Q Y TTCTTGCTGCCTCATrCTGCAGCAGCGGCGGCAAATCTTAATGGGACGCTGATGCAGTAT 1140 r E W Y M P M D G Q H W X R L Q N D S A TTTGAATGGTACATGCCCAATGACGGCCAACATTGGAAGCGTTTGCAAAACGACTCGGCA 1200 Y L A E H G I T A V W I P P A Y X G T S TATTTGGCTGAACACGGTATTACTGCCGTCTCGATTCCCCCGGCATATAAGGr^CGAGC „._:60 Q A D V G Y G A Y D L Y D L G E F H Q X CAAGCGGATGTGGGCTACGGTGCTTACGACCTTTATGATTrAGGGGAGTTTCAT -AAAA 1 20 G T V R T X Y G T X G E L Q S A I X 3 L GGGACGGTTCGGACAAAGTACGGCACAAAAGGAGAGCTGCAATCTGCGATCAy-AGTCTT 1380 H S R O I N V Y G D V V I N H X G G A O CATTCCCGCGACATTAACGTTTACGGGGATGTGGTCATCAACCACAAAGGCGGCG'Λ'GAT 1440 A T E D V T A V E V D P A D R N R '. I S GCGACCGAAGATGTAACCGCGGTTGAAGTCGATCCCGCTGACCGCAACCGCGTAATTT A 1 S00 G E H L I X A W T H F H F P G R G S T Y GGAGAACACCTAATTAAAGCCTGGACACArrTTCATnTCCGGGGCGCGGCAGCACATAC 1566 S O F X H H H Y H P D G T D M O E S R K AGCGATTTTAAATGGCATTGGTACCATTTTGACGGAACCGATTGGGACGAGTCCCGAAAG 1620 L N R I Y K F O G K A H D E V S N E N CTGAACCGCATCTATAAGTTTα^AGGAAAGGCTrGGGATTGGGAAGTTTCCAATGAAAAC 1680 G M Y O Y N Y A D I D Y D H P O V A A GGCAACTATGAtTATTTGATGTATGCCGACATCGATTATGACCATCCTGATGTCGCAGCA 1740 E X K R W β T W Y A N C L Q L O G F R L GAAATTAA-_AβATGGGGCλCr-»TλTSCOUTGAACTGCAATTGGAC∞TTTCCGTCTT 1800 D A V K H I X F S P L R D V N M V R E GATGCTCTCAAACACATTAA t ItTZTlllt IGCGGGATTCGGTTAATCATGTCAGGGAA 1860 X T G K E .- F T V A E Y W Q N D t. G A L AAAACGGGGAAGGAAATGTTTACGGTAGCTGAATArKG AGAATGACTTGGGCGCGCrG 1920 E M ¥ M K T N P M I S V _T D V F L I1 Y GAAAACTATTTGAACAAAACAAATTTTAATCATΪCAG ill Π H-ACOTGCCGCTTCATTAT 1980 Q P H A A S T Q G G G Y O M R K L L N G CAGTTCCATGCTGCATCGACACAGGGAGGCGGCTATGATATGAGGAAATτGCTGAACGGT 2040 T V V S X H P L K S V T P V O M H D T O ACGGTCGTrTCCAAGCATCCGrrGAAATCGGTTACATTrGTCGATAACCATGATACACAG 2100 P G Q S L E S T V Q T W F K P L A Y A F CCOGGCCAATCGCTTGAGTCGACrGTCCAAACATCβTTTAAGCCGCTrGCTrACGCTrrr 2160 I L T R E S G Y P Q V F Y β O N Y β T X ATTCTCλCAAGGGAAT TGGλTλCCCT λGGTrrrCTACGββGλTATGTACGGβλCGAAA 2220 G D S Q R E X P A L X R X I E P I L K λ GGAGACTCCCAGCGCGAAATTCCTGCCTTGAAACACAAAATTGAACCGATCTTAAAA<_CG 2280 R X Q Y A Y G A O B D Y F D B H O I V G AGAAAACAGTATOCGTACGGAGCACAGCATGATTATTTCGACCACCATGACATTCTCGGC 2340 H T R C G D S S V A M S G L A A L X T D TGGλCλAGGGAAGGCGλCAGCrCGβTTGCλAArrCλGGTTTGGCGGCλTTλATAACλβAC 2400 G P G G A K R N Y V G R Q K A G C T M H GGACCCGGTGGGGOAAGCGAATGTATGT GGCCGGCAAAACGCCGGTGAGACATGGCAT 2460 O X T G N X S E P V V I N S E G W G E r GACATTACCGGAAACCGTTCGGAGCCGGTTGTCATCΛΛTTCGGAAGGCTCCGGAGAGTTT 2 S 20 H V N G G S V S I Y V O R *** CACGTAAACGGCGGGTCGβTTTCAATTTATGrrCAAΛGATAGAAGAGCλGAGAGGACGGA 2 S 80 τTTCeTGAAGGAAATeCGl"rrirTTArϊtrgCCCGTCTTATAAATTTCTτTGATTACATT 26 O TTATAATTAATTrrAACAAAGTGTCATCAGCCCTCAGGAAGGACrrGCTGACAGTTTGAA 2700 TCGCATAGGTAAGGCGGGGATGAAATGGCAACGTTATCTGATGTAGCAAAGAAAGCAAAT 2760 GTGTCGAAAATGACGGTATCGCGGGTGATCAATCATCCTGAGACTGTGACGGATGAATTG 2820 HinOIII AAAAAGCrr 2829 Chart 8. Natural amylase gene sequence. Ba-UX H K Q Q K R L Y A R L L T L L F GCATcet-AATCATGAAAC-UkαuuuuCccciT CCcecg T Ccrc cac qT rr c to A L X F L L P H S A A A A A K L K G T L CβCTCATCrTCTrGCTGCCTCATTCTGCAGCAGCGG GGCAAAT rtAATGGGACGCTβA 120 H Q Y P E W Y H F N D G Q H W K R L Q N TCCAGTATTTTGAATGGTACATGCCCAATGACGGCCAACATTGGAAGCGTTTGCAAAACG 180 O S A Y L A B H G I T A V W I P P A Y X ACTCGGCATATTTGGCTGAACACGGTATTACTGCCGTCrGGATTCCCCCGGCATATAAGβ 240 G T S Q A O V G Y G A Y O L Y O L G E P GAACGAGCCAAGCGGATCTGGGCTACGGTGCTTACGACCTTTATGATTTAGGGGAGTTTC 300 R Q K G T V X T X Y G T K G E L Q S A X ATCAAAAAGGGACGGTTCGGACAAAGTACGGCACAAAAGGAGAGCTGCAATCTGCGATCA 360 K S L H S X D I H V Y G O V V I M a X G AAAGTCTTCATTCCCGCGACATTAACGTTTACGGGGATGTGGTCATCAACCACAAAGGCG 420 G A O A T E D V T A V B V ff P A D R M R GCGCTGATGCGACCGAAGATGTAACCGCGGTTGAAGTCGATCCCGCTGACCGCAACCGCG 480 V r S G E H L I X A H T H F H P P G R G TAATTTCAGGAGAACACCTAATTAAAGCCTGGACACATTrTCATTTTCCGGGGCGCGGCA 540 S T Y S O F K W H H Y H F O G T O H O B GCACATACAGCGATrrrAAATGGCATTGGTACCATTTTGACGGAACCGATTGGGACGAGT 600 S R K L H R I Y K F Q G X λ '«. O H E V S CCCGAAAGCTGAACCGCATCTATAAGTTTCAAGGAAAGGCTTGGSATTGGGAAGTTTCCA 660 N E N G N Y D Y L 1- Y A D I D Y D H P D ATGAAAACGGCAACTATGATTATTTGATGTATGCCGACATCGATTATGACCATCCTGATβ 720 V A A E I K R W G T W Y A N E L. Q L D G TCGCAGCAGAJUkrrAAGAGATGGGGCACTTGGTATGCCAATGAACTGCAATTGGACGGTT 780 F R L D A V X a i X r S F L X O W V H H TCCGTCTrGATGCTGTCAAACACATTAAATTTTCTITTTTGCGGGATTGGGTTAATCATG 840 V R E X T G X E H F T V A E t-' Q -r D L TCACWGAAAAAACGGGGAAGGAAATGTrrACGGTAGCTGAATATTGGCA AA-KACTTGG 00 G A L E M Y L M X T H Γ K Γ. S V F D V P GCGCGCTGGAAΛACTATTTGAACAΛAΛCAΛATTTTΛΛTCATTCASTtSTTT β ACGTGCCGC 960 H Y O F H A A S T Q G C G Y O M R X L TTCATTATCAGTTCCATOCTGCATCGACACAGGGAGGCGGCT/ . G * t__.τ GA«iΛΛΛTTGC 1020 L N G T V V S X H P L X S '' T F V D N H TGAACGGTACGGTCGTTTCCAAGCATCCGTTGAAATCFOTTACATTTGTCGATAACCATG 080 O T Q P G Q S L E S T V Q T M F X P A ATACACAGCCGGGGCAATCGCrrGAGTCGACTGTCCAAACATGGTTTAAGCCGCTTGCTT 11 0 Y A F I L T R E S G Y P O V F Y G O W Y ACGCTTTTATTCTCACAAGGGAATCTGGATACCCTCAGGTTTTCTACGGGGATATGTACG 1200 G T K G O S Q X C I P A L K H K I E P I GGACGAAAGGACACTCCCAGCGCGAAATT CTGCCTTGAAACACAAAATTGAACCGATCT 1260 L K A R R Q Y A Y G A Q H D Y F D H H D TAAAAGCGAGAAAACAGTATGCGTACGGAGCACAGCATGATTATTTCGACCACCATGACA 1 20 I V G W T X E G D S S V A N S G L A A L TTGTCGGCTGCACAAGGGAAGGCGACAGCTCGGTTGCAAATTCAGGTTrGGCGG ATTAA 1380 I T O G P G G A X R R Y V G R Q H A G E TAACΛβACGGACCCGβTGGGOCAAΛCCGAATGTATGTCGGCCGGCAAAACGCCGGTGACA 1440 T M H O X T G N R S E P V V I H S E G H CATGGCATGACATTACCGGAAACCGTTCGGAGCCGGrrGTCATCAATTCGGAAGGCTGGG 1500 G E F H V N G G S V S I Y V g R "* GAβAGTTTCλCGTAAACGGCGGGTCGGTTTCAATTTATGTT λλAGATAGAλGAGCλGAG 1560 AGCACGGATTTCCTOAAGGAAATCCOτnmtATTTTGCCCGTCTTATAAATTTCTTrC 1620 ArrACATTTTATAATTAATTTTAACAAAGTGTCATCAGCCCTCAGGAAGGACTrGCTβAC 1(90 AβrXTGAATCGCATλGGTAAGGCGGGGλTCAAATGGCAACGTTAT rGλTGTAGCλAAGλ 1740 AAGCAAΛTGTGTCGAΛΛATGACGGTATCGCGGGTGATαUttCATCCTGAGACTGTGACGG 1900 Beaut ATGAATTβAAAAAGCTCCGGATCC 1824 Chart 9. Optimised amylase sequence. strain gave significant zones of cross-feeding on minimal plates seeded with a phenylalanine auxotroph. Example 4 A semi-synthetic operon construct. In this example the pheA and aroF genes are combined into a single transcriptional unit. The plasmid pJW2 carries the pheA gene transcribed from the synthetic promoter. The mutant BamHI site downstream of pheA was used to clone the aroF gene carrying a BamHI site in its ribosome binding site. Finally, remaining tyrA sequence was removed by the addition of a synthetic terminator. This plasmid pME202 carries a semi- synthetic operon on an EcoRI-Sall fragment of 2,400 bp. To further increase, the utility of this fragment a derivative of pME202 was constructed that carries an EcoRI linker at the Sail. site (pME204). Table 3 indi- cates the approximate numbers found. The plasmid pME202 (ATCC 53136) was introduced into a number of strains and the levels of DAHP &ynth _ase and prephenate dehydratase determined during growth on a number of media. One example of these levels is indicated in Table 1. It is clear that the plasmid ■ ,- >«es overproduction of the two activities and a comparison of enzyme levels during growth in the presence and absence of phenylalanine confirms that expression of the two genes has been sub¬ stantially deregulated. Table 3 Plasmid Constructs using the Phe Operon from p E202 at various copy numbers. Rl_p_****pheA****_****aroF****_T_Sl pME202 Phe operon now carried on EcoRI fragment "cas¬ sette". This fragment was recloned into the following vectors: pBR322 to give pMH19. Copy number approximately 50. pLG388 to give pME208. Copy number approximately 10. RP4 to give pME214. Copy number approximately 2. (pAT153 to give pME204). Copy number approximately 100. E.coli HW77 carrying pME202 was tested for L- phe production in cross-feeding experiments (Table 2). Extremely large zones were obtained in contrast to the untransformed controls. This demonstrates the advantage of being able to coordinate the expression of the two genes in the operon construct. The level of FBI aroF product is now acceptable to HW77 growing on minimal medium. Example 5 Utility of the semi-synthetic phenylalanine operon con¬ struct in assessing the effect of varying plasmid copy number. In order to ascertain the effect of copy num¬ ber on expression the EcoRI fragment from pME204 that carried the aroF pheA operon was recloned into the fol¬ lowing vectors: pBR322 (copy number 50) to give pMH19, pLG338 (copy number 10) to give pME208 and RP4 (copy number 1-3) to give pME214. These plasmids were intro¬ duced into.- the tyrA strains H 77 and HW760 and the resultant transformants tested for their growth on minimal medium and L-phe productivity in cross-feeding experiments. The results of these experiments are sum¬ marized in Table 4. It was found that while the high copy number derivatives were clearly deleterious to the growth of HW760, H 77 could tolerate all the plasmids. Furthermore, the lower copy derivatives pME208 and pME214 did not prevent the growth of H 760. HW760 carrying_pME21* or pME208 gave very large zones of cross-feeding. This example serves to indicate how the optimum expression level can be attained by varying the copy number of the pheA aroF operon through judicious choice of vector. Example 6 Extending the host range of the semi-synthetic phenyl¬ alanine operon. The EcoRI fragment from pME204 that encom- passes the pheA aroF operon was recloned into a number of broad host range vectors namely: RP4 to give pME214 pCT460 to give pME211 pKT231 to give pME212 Table 4 Growth of HW77 and H 760 Carrying Various Phe Plasmids. PLASMID GROWTH 24 hr 48 hr ++ ++++ pME214 ++ ++++ PME208 ++ ++++ pMH19 ++ ++++ pME202 ++ ++++ pME219 ++ ++++ pME170 ++ ++++ pME171 ++ ++++ pST35 (+)* pME237 (+)* pME238 (+)* pAT153 ++ ++++ ++ ++++ pME214 ++ ++++ pME208 + +++ pMH19 + pME2Q2 (+) * Essentially no growth but a few mutant colonies emerging. The strains were grown overnight in L-broth supplemented with suitable antibiotic where appro¬ priate. One microlitre of the overnight culture was then streaked onto minimal agar supplemented with 50 ug ml "1 tyrosine and any relevant antibiotic. Growth was scored after 24 hr and 48 hr. These plasmids were then introduced by trans¬ formation, conjugation or mobilization into a variety of strains and L-phe production detected by cross-feeding of an E.coli L-phe auxotroph. Table 5 details the various organism/plasmid combinations and the cross- feeding observed. Significant cross-feeding was observed with a number of organisms including pseudomonads and facultative methylotrophs. In principle this approach to deregulating a pathway by simply introducing such an artificial operon •on a suitable plasmid could be extended to almost any microorganism that had beneficial characteristics. Expression would probably have to be reoptimized, e.g., for Gram positive organisms and this would entail alterations to the synthetic promoter and the various ribosome binding sites. Such an undertaking would be greatly facilitated by the modular structure of the operon and the presence of- restriction sites in the Ribosome binding sequences. Example 7 Ex ^ e-naion of the operon - incorporation of the gene for alpha amylase. The modified alpha amylase gene is flanked by a BamHI site in the Ribosome binding sequence and a BamHI linker in the Hindlll site downstream of the gene. The BamHI fragment carrying amy was cloned into the downstream BamHI site in pME202 to give an artifi¬ cial three " gene operon (pPT112) deposited as ATCC 53139, see Chart 11. This plasmid was transformed into HW77 and its growth on starch investigated. A comparison of the growth of HW77/pPT112 on starch and glucose revealed that there was little difference between growth rates in starch and glucose, but growth on starch was subject to a much longer lag phase, see figure 2. A fermentation performed on HW77/pPT112 grown on 50g 1~ ~ - soluble starch revealed significant accumula¬ tion of L-phe (figure 3). Fermentation conditions were as described in example 10 except that glucose was not included in the initial medium and a sugar feed was not used. The soluble starch was batched into the fermenter at 50g 1 prior to autoclaving. Table 5 Use of Phe Operon on broad host range plasmids to enhance L-phe production in other genera. Plasmids: pME202 Phe operon in pAT153 - see text. pME204 As pME204 but with EcoRI linker at Sail site. pME211 Phe operon from pME204 cloned at EcoRI site of pCT460. pME212 Phe operon from pME204 cloned at EcoRI site of pKT231, transcribed - Hindlll pME213 Phe operon from pME204 cloned at EcoRI site of pKT231, transcribed - str. Phe operon from pME204 cloned at EcoRI site of RP4, transcribed towards Hindlll site. HOST pME202 pME214 pME211 pME212 pME213 E.coli + + + + + E.intermedia - S.marcesens - S.typhimurium + Ps.fluorescens + + + Ps.putida + + + Facultative Methylot :rophs: (grown on methanol) Mycoplana rubra + Pseudomonad spp. + Methylobacterium organophilum - Ps.extorquens - Vibrio extorquens — + Production of L-Phe caused by introduction of plasmid as detected by cross-feeding experiments or by TLC analysis of shake flask media. - No L-Phe production detected in response to plasmid. Remaining host/plasmid combinations were not tested. Example 8 Extension of the operon - Incorporation of the aspC gene. The artificial pheA aroF operon was further modified to include the gene for aspartate amino trans- ferase (aspC) as follows: pIF18 which carries the aspC gene flanked by a BamHI site in the ribosome binding site and a Bglll site downstream of the gene was used as a source of the BamHI-Bglll fragment encompassing aspC. This fragment was then cloned into pME202 that had been partially cleaved with BamHI to give a deriva¬ tive pME219 (deposited as ATCC 53138) that carries the aspC gene downstream of aroF so as to yield a three gene artificial operon (chart 10). When introduced into HW77 this plasmid caused L-phe production as detected by cross-feeding. Analysis of cell free extracts revealed elevated levels of aspC activity (Table 6). Table 6 Phenotypic Effect of pME219. pME219 was transformed into HW77 to give HW1058. Strain Plasmid Aspartate Aminotransferase Activity U mg HW77 - 604 HW1058 pME219 1913 The strains were grown to O.D. QQQ in glucose minimal medium supplemented with 0.5% casamino acids. The benefit of this construct is that the plasmid not.only deregulates the phenylalanine pathway but also yields elevated aspC levels. This has utility in that an L-phe fermentation can be supplemented with chemically synthesized PPA along with aspartate or glutamate to increase L-phenylalanine titres. Such a hybrid fermentation/bioconversion has advantages that include greater ease of L-phe isolation and higher L-phe productivity. flAAI 1_ Jl 11 mm ;ι«ACAOCSTGAAAAα^AC0GaTATAAtACTAAAgTCACAAAAAa (0 N T S I M F L L A l R t X X S A L gCAACACtATGACATCG-AAAACCei-T-*CTSaCSCTg SA-_A--^^ 120 D E X L L A L L A E I R X L A V E V β X ATgAAAAATTATTA0CCTTACTCGCAGAACGOCS GAACTqqCCOTC0A_MTGC<--iAAAO 180 A K L L S I R P V R O X O B S O L L I ccxA λ cτβcrcτeβcλτeβeccβατλcβτGλZλττβλτcoτβλACβcβλRτβcτβGAAA 240 R L I T L O X A I I L O A R T I T R L F GATTAλrrACGCTCGGTAAAGCGCλCCATCrβGACG CCλTZλCλnλCTCGCCTβTTCC 300 Q L t X X O S V L T Q Q λ l. L Q O R L M AGCTCλTCλrTGλAGATTCCGTATTAACTCAβCAGGCmGCTCCAACAACATCtCλλTA 3(0 K X M P I S A X I A F L G P K G S Y S a AAATTΛATCCGCAeTCλGCλCGCλT βCTTrrCTCGGCCCCλλAGGTTCTTArrCCCATC 420 L A A X O Y A A X a P E O P X E S β C A TTGCGCCGCGCCAΒTATΒCTGCCCGTCACTTTGAGCAATTCATTGAAAΒTΒGCTGCΒCCA 490 X P A D : P M Q V B T O Q A O Y A V V P AATTTGCCGATAN 11 LAATCAGATSGAAACCSGCCACOCCSACTATOCCOTCGTACCGA S40 X C M T f S β λ X M D V Y O L L Q H T S TTGAλAATACCλG TCCGGTGCCλTAAACSλCGTRACSλSCrβCTG λλCλTAeCAβCT 600 L S X V G B N T L T X O a C L L V S β T TGTCGATTGTTGGCGA-_ATGACGTTAA tATCβACCA«GTTTβTTGGTCTCCGGCACTA «0 T O L S T I K T V Y S R P Q P P Q Q C S CTGλrrrATCCλCCATCλλSλCGβTCTACλGCCλTCCGCλβCCATTCCAGCλλTGCλGCλ 720 K P L N R Y P R W K X E Y T S 3 T S A A AATTCCrrAATCGTTATCCGCACTGGAAGATTGAATATACCSAAAGTACGTCTGCGGCAA 790 H E X V A Q A K S P H V A A L G S E A Q TGSAAAAGGTTGCACAGG AAAATCACCGCATGTTGCTG GTrGGGAAGCGAAGCTGGCS 840 G T L Y G L Q V L t R X E A N Q R Q H F GCACTTTGTACSGrrKCλGGTACTGGλGCGTλTTβλAG(_AAATUGCGλ(_AAAλ rrCλ 900 T R f V V L A R X A I M V S D O V P A X CCCGATTTGTGGTGTTββCGCGTAAAGCCATTAACGTGTCTGATCλGGTTCCGGCGAAΛA 960 T T L L M A T O Q Q A G A L V E A L L V CCACGTTGTTAATGGCaACCGGGOU.aU.GCCGGTGCG TGGTTGAAGCGTTGCtGGTAC 1020 L X H H M L X X T X L E S R P X B G N P TGCGCAACCACAATeT β ATTATGACCCGTCTGGAATCACGCCCGATTCACGGTAATCCAT 1080 H R S P S E N F Y L B X Q A H L E S λ GGAGATCTCCATGGGAAGA β AT β TTCTATCTGGATATTCA ββ CCAATCrrGAATCAGCGG 1140 E H Q X A L K E L G S X T R S H X V L G AAATGCλAAAAGCATTGλAAGλ β TTAGG β GAAATCλCCCGTTCAλTGAAGGTλTTGGGCT 1200 C Y F S E M V V P V O P T *••••• n GTTACC λACTαAβλACGTAGTGCCTGTTGATCCAACCTGλTGA_VAAGGλTCCCCATCλT 1260 Q X O A L M M V H X T O C Q V L K T P E GCAAAAAβACGCGCTGAATAACGTACATATTACCGACGAACAββTrrrAATGACTCCGGA 1320 Q L R A A P P L S L Q Q 8 A Q I A 0 8 R ACAACTCAAβGCCGCTTTTCCATWAGCCTGCAACAAGAACCCCACATTGCTCACTCGCG 1380 K S X S O X X A G R O P R L L V V C G F TAAAAGCATTTCACATATTATCCCCGCCCGCGATCCTCGTCTCCTGGTAGTATGTGGTCC 1 40 C S i a O F S T A L t Y A X R P K A L A TTGrrCCATTCATGAT CGGAAACTGCTCTGGAATATG rCGT GATTTAAAGCCCTTCC 1500 A E V S O S L Y L V K X V Y F E X P I T CGCAGAgGTgAGCCATAOCCTCTATCTGGTAATGCOCGTCTA. I ϊ tώAΛAAACCCCGTAC 1560 T V G H K Q L X M D P B II S β S F D V t CACTSTCGCCTGaAAAGaGTTAATtAACaATCCCCATATGGATCCCΪ t i I -l-ATGTASA 1620 A β L Q I A R X L L L X L V H H β L P L AGCeGGGCTGCAGATCGCOCSTAAAWGCTGCrtqAGCTGGTCAATATqGGACTGCCACT 1680 A T S A L D F N S P Q Y L β O L P S H S GG GACGGAAGCGTrAGATCCGAATAGCCCGCAATACCTGGGCGATCiarT'JAGCTGGTC 1740 A X G A X T T I S g T B R X M A S G L S λGCλATTGCTGCTCSTλCAλCGβAλTCGCλAACTOkCCSTβAλATGG CTCCGGGCTTK 1800 n P V G F X N G T O β S L A T A I H λ H CλTGCCGGTTGGTrnAAAλACGGα.CCGλCGβCAGTCTGβCAACAGαλnAλCGCTAT 1860 X λ λ λ O P I R F V G X M Q λ β g V A L GCGCGCCGCCGCCC_U_CCGCACCGTrrτGTTGG ATTAACCAGG AGGGCλGGrrGCGTr 1920 L Q T Q G M F O G R V X L R β G K A P H eCTACAAACTCAGGGGAATCCGGACGGCCATGTGATCCTGCGCGGTGGTAAAGCG CGAA 1980 Y S P Λ D V A Q C E K S K K Q A G L X P CTATAGCCCTGCGGATGTtGCGCAATGTGAAAAAGAGATGGAACAGGCGGGACTGCGCCC 2040 S L N V D C S a G N S N K D Y X R Q P λ GTCTCTGATGGTAGATTGCAGCCACGβTAATTCCAATAAAGATTATCGCCGTCAGCCTGC 2100 V A S S V V A Q I K O G M X S X X G L H GGTGGCAGAATCCGTGGTTGCTCAMTC_-AAGATGG«ATCGCTCAATTATTGGTCTGAT 2160 X E S N X H E G M Q 3 S E Q F X S X -. K GATCGAAAGTAATATCCACGAGGGCAATCAGTCTT CGAGCAACCGCGCAGTGAAATGAA 2220 Y G V S V T D A C X S M E M T D A L L R ATACGGTGTATCCβTAACCGATGCCTGCATTAGCTGGGAAATGACCGATGCCTTβCTGCG 2280 E I H Q D L N G Q L T A X V A *•»**« TGAΛATTCATCAGGATCTGAACGGGCAGCTGACGGCTCGCGTCX.CTTAATGAGGATCCTC 2340 H P E M X T A A P A D P I L G L A D L GTCATGTTTGAGAACATTACCGCCGCTCCTGCCGACCCGATTCTGGGCCTGGCCGATCTG 2400 F R A O E X P G K I N L G I G V Y X D E TTICGTGCCGATGAACGTCCCGGCAAAAITAACCTCGGGATTGGTGTCTATAAAGATGAG 2460 T G X T F V L T S V X X A B Q Y L L E H ACGGGCAAAACCCCGGTACTGACCAGCGTGAAAAAGGCTGAACAGTATCTGCTCGAAAAT 2520 E T T R H Y L G I O G I P E F G R C T Q GAAACCACCAAAAATTACCTCGGCATTGACGGCATCCCTGAATTTGGTCGCTGCACTCAG 2580 E L L P G X G S A L X H O K R A R T A Q GAACTGCTGTTTGGTAAAGGTAGCGCCCTGATCAATGACAAACGTGCTCGCACGGCACAG 2640 T P G G T G A L X V A A O F L A X N T S ACTCCGGGGGGCACTGGCGCACTACGCGTGGCTGCCGATTTCCTGGCAAAAAATACCAGC 2700 v K R V W V S H P S W P H H K S V Γ N S GTTAAGCGTGTGTGGGTGAGCAACCCAAGCTGGCCGAACCATAAGAGCGTCTTTAACTCT 2760 A G L E V X E Y A Y Y D A E H H T L O F GCAGGTCTGGAAGTTCGTGAATACGCTTATTATGATGCGGAAAATCACACTCTTGACTrC 2820 D A L I N S L N B A Q A G D V V L F H G GATGCACTGATTAACAGCCTGAATGAAGCTCAGGCTGGCGACGTAGTGCTGTTCCATGGC 2880 C C a N P T G X D P T L E Q K g T L A Q TGCTGCCATAACCC_-ACCGGTATCGACCCTACGCTGGAACAATGGCAAACACTGGCACAA~2940 L S V E K G P L F O F A Y Q G F A R CTCTCCGTTGAGAAAGGCTGGTTACCGCTGTTTGACTTCGCTTACCAGGGTTTTGCCCGT 3000 G L E E D A E G L R Λ F A A H H X E L I GGTCTGGAAGAAGATGCTGAAGGACTGCGCGCTTTCGCGGCTATGCATAAAGAGCTGATT 3060 V A S S Y S R M F G L Y H E R V G A C T GTTGCCAGTTCCTACTCTAAAAACTTTGGCCTGTACAACGAGCGTGTTGGCGCTTGTACT 3120 L V A A D S E T V D X A P S Q K K A A I CT(_GTTGCIGCC»CλGTβ-_AλCC6TTGATCGCG»TTCλβCCλAATGλAλGCGGCGArr 3180 R λ M Y I M P P A H β λ S V V λ T X L S CGCGCTAACTACTCTAACCCACCAGCACACGGCGCTKTCTTGTTG CACCATCCTGAGC 3240 H D A L R A I W E Q B L Ϊ D 1. R Q R I Q AACGATGCGTTACGTGCGATTTGGGAACAAGAGCTGACTGATATGCGCCAG GTATTCAG 3300 X H R Q L F V H T L O B X G A N X D F S CGTATGCGTCAGTTGTTCGTCAATACGCTGCAGGAAAAAGGCGCAAACCGCGACTTCAGC 3360 F I I X Q N β -I P S F S G L T X E Q V L TTTATCATCAAACAGAACGGCATGncrCCTrCAGTGGCCTGACAAAAGAACAAGTGCTG 3420 X L R B B P G V Y A V A S G X V M V A G CGTCTGCGCGλλGλGTTTGGCGTATATGCGGTTGCTTCTG β TCGCGTAAATGTGGCCGGG 3480 N T P D M H A P L C B A X V A V L *** ATGACACCAGATAACATGGCTCCGCTGTGCGAAGCGATTGTGGCAGTGCTGTAAGCATTA 3S40 AAAACAATGAAGCCCGCTGAAAAGCGGGCTGAGACTGATGACAAACGCAACATTGCCTGA 3600 TGCGCTACGCTTATαuSGGAGATCCCGGGCGATTCTACGCCCGGGTTTTTTATGTCβAC 3659 ChaJTt 10. Sequence of the pheA aroF aspC operon in pME219. ___ Hybrid Bglll/Bu_J_X titt downatrβaa of __s£ GAA11'C.11111_._GACAGCOTGAAAACAOTACGCOTATAATACTAAAOTCACAAAAAG 60 H T S S M P L B A L X B K X S A L D GCAACACTATGACATCGGAAAACCCGTTACTGGCG TGCGAGAGAAAATCAGCGCGCTGG 120 E X L L A L L A E R X E L A V E V G K A ATGAAAAATTATTAGCGTTACTGGCAGAACGGCGCGAACTGGCCGTCGAGGTGGGAAAAG 180 K L L S H R P V X O I O X E X D L L E R CCAAACTGCTCTCGCATCGCCC-KJTACGTGATATTGATCGTGAACGCGATTTGCTGGAAA 240 L I T L G X A H H L D A B Y I T R L F O GATTAATTACGCTCGGTAAAGCGCACCATCTGGACGCCCATTACATTACTCGCCTGTTCC 300 L I X E D S V L T Q Q A L L Q Q B L H X AGCTCATCATTGAAGATTCCGTATTAACTCAGCAGGCTTTGCTCCAACAACATCTCAATA 360 X N P H S A R I A F L G F K G 3 Y S H L AAATTAATCCGCACTCAGCACGCATCGCTTTTCTCGGCCCCAAAGGTTCRTARRECCATC 420 A A R Q Y Λ A X H F E O F I E S G C A K TTGCGGCGCGCCAGTATGCTGCCCGTCACTTTGAGCAATTCATTGAAAGTGGCTGCGCCA 480 F A D I F N Q V E T G Q A D Y A V V P I AATTTGCCGATATTTTTAATCAGGTGGAAACCGGCCAGGCCGACTATGCCGTCGTACCGA 540 E N T S S G A I M O V Y D L L Q H T S L TTGAAAATACCAGCTCCSGTGCCATAAACGACGTΤTACGATCTGCTGCAACATACCAGCR 600 S I V G E M T L T I O H C L L V S G T T TGTCGATTGTTGCCGAGATGACGTTAACTATCGACCATTGTTTGTTGGTCTCCGGCACTA 660 D L S T I N T V Y S H P Q P F Q Q C S X CTGATTTATCCACCATOU.TACGGTCTACAGCCATCCGCAGCCATTCCAGCAATGCAGCA 720 Γ L N R Y P H H X I E Y T E S T S A A N AATTCCTTAATCGTTATCCGCACTGGAAGATTGAATATACCGAAAGTACGTCTGCGGCAA 780 E K V A Q A X S P H V A A L G S E A G G TGGAAAAGGTTGCACAGGCAAAATCACCGCATGTTGCTGCGTTGGGAAGCGAAGCTGGCG 940 T L Y G L Q V L E R I E A N Q R Q N F T GCACTTTGTACGGTTΓGCAGGTACTGGAGCGTATTGAAGCAAATCAGCGACAAAACTTCA 900 R P V V L A R X A I N V S O O V P A X T CCCGATTTGTGGTGTTGGCGCGTAAAGCCATTAACGTGTCTGATCAGGTTCCGGCGAAAA 960 T L L M A T G Q Q A G A L V E A L L V L CCACGTTGTTAATCGCGACCGGGCAACAAGCCGGTGCGCTGGTTGAAGCGTTGCTGGTAC 1020 R N H N L X H T R L E S R F X H G H F W TGCGCAACCACAATCTGATTATGACCCGTCTGGAATCACGCCCGATTCACGGTAATCCAT 1080 R S P E E H F Y L D I Q A N L E S Λ E OGAGATCTCCATGGGAAGAGATGTTCTATCTGGATATTCAGGCCAATCTTGAATCAGCGG 1140 H Q X A L X E L G E I T R S H X V L G C AAATGCAAAAΛGCATTGAAAGAGTTAGGGGAAATCACCCGTTCΛATGAAGGTATTGGGCT 1200 Y P S E M V V P V D P T * N GTTACCCAAGTGAGAACGTAGTGCCTGTTGATCCAACCTGATGAAAAGGATCCCCATCAT 1260 O X O A L H N V M I T O E Q V L H T P E GCAAAAAGACGCGCTGAATAACGTACATATTACCGACGAACAGGTTTTAATGACTCCGGA 1 20 Q L K A A F P L S L Q Q E A Q I A O S X ACAACTGAAGGCCGCTTTTCCATTGAGCCTGCAACAAGAACCCCAGATTGCTGACTCGCG 1380 X S I S O I I A G R O P R L L V V C G P TAAAAGCATTTCAGATATTATCGCCGGGCGCGATCCTCGTCTGCTGGTAGTATGTGGTCC 1440 C S I H D P E T A L E Y A R R P R A L A TTGTTCCATTCATGATCCGGAAACTGCTCTGGAATATGCTCGTCGATTTAAAGCCCTTGC 1500 A E V S D S L Y L V H R V Y F E X P R T CGCAGAGGTCAGCGATAGCCTCTATCTGGTAATGCGCGTCTATTTTGAAAAACCCCGTAC 1560 T V β W X G L I M D P B H D G S F D V E CA tGTCGβCTGGAAAGGGTTAATTAACGATCCCCATATGGATGG l'C1i IKATGTAGA 1620 A G L Q I A R X L L L E L V H B G L P L AGCCGGGCTGCAβATCGCGCGTAAATTGCTGCTTGAGCTGGTβAATATGGGACTGCCACT 1680 A T E A L D P M S P Q Y L G D L F S W S GGCGACGGAAGCGTTAGATCCGAATAGCCCGCAATACCTGGGCβATCtGTTTAGCtGGTC 1740 A X G A R T T E S Q T H R E H A S G L S AGCAATTGGTGCTeGTACAACGGAATCGCAAACTCACCGTGAAATGCCCTCCGGGCTITC 1800 N P V G F K M G T D G S L A T A I H A N CATGCCGGTTGGTTrrAAAAACGGO.CCGACGGCAGT TGGCAACAGCAArrAACGCTAT 1860 R A Λ A Q P H K F V G I N Q A G Q V A L GCGCGCCGCCGCCCAGCCGCACCGTTTTGTTGGCATTAACCAGGCAGGGCAGGTTGCGTr 1920 L Q T Q G N P O G B V I L R G G X A P N GCTACAAACTCAGGGGAATCCGGACGGCCATGTGATCCTGCGCGGTGGTAAAGCGCCGAA 1980 Y S P A D V A Q C B X E H E Q A G L X P CTATAGCCCTGCGGATGTTGCGCAATGTGAAAAAGAGATGGAACAGGCGGGACTGCGCCC 2040 S L M V O C S H G M S N K D Y X R g P A GTCTCTGATGGTAGATTGCAGCCACGGTAATTCCAATAΛAGATTATCGCCGTCAGCCTGC 2100 V A E S V V A Q X X D G H X S X I G L M GGTGGCAGAATCCGTGGTTσcrCAAATCAAAGATGGCAATCβCTCAArrATTGGTCTβAT 2160 I E S H I B E G N g S S B Q P X S B H K GATCGAAAGTAATATCCACGAGGGCAATCAGTCTKCGAGCAACCCCGCAGTGAAATSAA 2220 Y G V S V T O A C X S W B R T O A L L R ATACGGTGTATCCGTAACCGATGCCTGCATTAGCTGGGAAATGACCGATGCCrrGCTGCG 2280 E X B Q O L M G Q L T A X V A * * TβAAATTCλTCλGGλTCTGAACGGGCλGCTGλCGGCTCGCGTGGCTTAATGAGGλT CCλ 2340 M K Q Q K X L Y A X L L T L L P A L I AT ATGAAACAACAAAAACGGCTrrACGCCCGATTCCrGACGCTGTTATTTGCGCTCATC 2400 P L L P a S A A A A A N L M G T L N Q Y TTCTTCCrGCCTCATTCTGCAGCAGCGGCGGCAAATCTTAATGGGACGCTGATGCAGTAT 2460 P E tf Y H P M D G Q H H K X L Q M D S λ TTrGAATGGTACATGCCCAATGACGGCαkACATTGGAAGCGTTTGCAAAACGACTCGGCA 2520 Y L A E H G X T A V H X P P A Y X G T S TATTTGGCTGΛΛCACGGTATTΛCTCCCGTCTGGATTCCCCCGGCATATAAGGGΛΛCGAGC 2580 Q A D V G Y G A Y D L Y D L G E F H Q X CAAGCGGATGTGGGCTACGGTGCTTACGACCTTTATGATTTAGGGGAG1TTCATCAAAAA 2640 G T V R T X Y G T X G B L Q S A I K S L GGGACGGTTCGGACAAAGTACGGCACAAAAGGAGAGCTGCAATCTGCGATCAAAAGTCrT 2700 H S R O I M V Y G D V V I N H X G G A O CATTCCCGCGACATTAACGTTTACGGGGATGTGGTCATCAACCACAAAGGCGGCGCTGAT 276v A T E O V T A V E V O P A O R M R V I S GCGACCGAAGATGTAACCGCGGTTGAAGTCGATCCCGCTGACCGCAACCGCGTAATTTCA 2820 G E H L I X A W T H F H F P G R G S T Y GCAGAACACCTAATTAAAGCCTGGACACATTTTCATTTTCCGGGGCGCGGCAGCACATAC 2880 S O F R H H Y H P O G T O M O E S X X AGCGATTTTAAATGGCATΓOGTACCATTTTGACGGAACCGATTGGGACGAGTCCCGAAAG 29 0 L N X I Y K F Q G K A O H E V S N E N CTGAACCGCATCTATAAGTTTCAAGGAAAGGCTTGGGArrK3GAAGTTTCCAATGAAAAC 3000 G N Y D Y L H Y A D I O Y D H P D V A A GGCAACTATGATTATTTGATGTATGCCGACATCGATTATGACCATCCTGATGTCGCAGCA 3060 E I X R G T H Y A N E L Q L O G F R L GAAATTAAGAGATGGGGCACTTGGTATGCCAATGAACTGCAATTGGACGGTTrCCGTCTT 3120 O A V X a i X P S F L X D H V N B V X B GATGCTGTCAAACACATTA A111 ICI 1 1.11 rGCGGGATTGGOTTAATCATGTCAGGCAA 3180 X T G K E It F T V A E Y M Q N D L G A L AAAACGGGGAλGGAAATGTTTλCGGTλGCTGAATATTβGCλβλATGACTTGGGCGCGCrG 32 0 C M y N X M F M H ___ _ D F H Y GAAAACTATTTG_U_αuWU.αυΛTrrTAATαkTTCAGTGTTTGACGTGCCGCTTCATTAT 3300 Q P B A A S T 0 G G G Y O H X K L L H G CAGTTCCATGCTGCATCGACACAGGGAGGCGG TATGATATGAGGAAATTGCTGAACGGT 3360 T V V S X H P L K S V T P V O M H D T Q ACCGTCGTTTCCAAGCATCCGTTGAAATCGGTTACATTTCTCGATAACCATGATACACAG 3420 P G Q S L B S T V Q T W F X P L A Y A P CCGGGGCAATCGCTTGAGTCGACTGTCCAAACATGGTrrAAGCCGCTTGCTTACGCTTTT 3480 I L T R B S G Y P Q V P Y G D B Y G T X ATTCTCACAAGGGAATCTGGATACCCTCAGGTTXTCTACGGGGATATGTACGGGACGAAA 3 S 40 G D S Q R E I P Λ L X B X I E P I L R A GGAGACTCCCAGCGCGAAATTCCTGCCTTGAAACACAAAATTGAACCGATCTTAAAAGCG 600 R X Q Y A Y G A Q H D Y F D B H D I V G AGAAAACAGTATGCGTACGGAGCACAGCATGATTATTTCGACCACCATGACATTGTCGGC 3660 W T R E G D S S V A N S G L A A L I T D TGGACAAGGGAAGGCGACAGCTCGGTTGCAAATTCAGGTTTGGCGGCATTAATAACAGAC 3720 G P G G A X R H Y V G R Q N A G E T H GGACCCGGTGGGGCAAAGCGAATGTATGTCGGCCGGCAAAACGCCGGTGAGACATGGCAT 3780 O I T G N R S E P V V I N S E G H G E F GACATTACCGGAAACCGTrCGGAGCCGGrrGTCATCAATTCGGAAGGCTGGGGAGAGTTT 3840 H V N G G S V S I Y V Q R * CACGTAAACGGCGGGTCGGTTT AATTrATGTTCAAAGATAGAAGAGCAGAGAGGACGGA 3900 ΓΓTCCTGAAGGAΛATCCGITTTIITATTTTGCCCGTCTTATAAATTΓCTTTGATTACATT 3960 TTATAATTAATrTTAACAAAGTGTCATCAGCCCTCAGGAAGGACTTGCTGACAGTTTGAA 4020 TCGCATAGGTΛAGGCGGGGATGAΛATGGCAACGTTΛTCTGATGTAGCAAAGAAAGCAAAT 4080 GTGTCGAAAATGACGGTATCGCGGGTGATCAATCATCCTGAGACTGTGACGGATGAATTG 4160 AAAAAGCTCCGGATCCCGGGCGATTCTACGCCCGGG t T T 11 f TGTCGAC 4150 Chart 11. Sequence of the pheA aroF amy operon in pPTl.12. Example 9 Application of phenylalanine operon plasmids to strain screening. A total of 31 strains of E.coli, along with some other enteric species, were transformed with pME202 and screened for both growth on minimal medium and cross-feeding of an L-phe auxotroph. The results are summarized in figure 4. Since the complete deregulation of pheA and aroF would take 5-6 rounds of conventional mutagenesis it can be seen that the composite plasmid approach has a very great advantage in terms of speed. The mutagenesis of 45 strains to this extent would con- servatively take ten man years to accomplish, and there would be no guarantee that the lesions would be directly comparable. Using the composite plasmid approach strains can easily be deregulated with identical lesions and put through initial screens in under two weeks. Example 10 Utility of Phe Plasmids in Fermentation.. , To examine the potential of strain^ carrying Phe plasmids for L-phenylalanine production we compared the productivity of HW1057 (HW77pME202) with HW77 ir_ 10 liter fermenters. Both strains were grown from a single colony in L-broth for 15 hr. The HW1057 culture was supple¬ mented with 100 μg ampicillin ml "1 . These overnight cultures were used to inoculate a shake flask containing the following minimal medium: Glucose 19.4 ( H 4 ) 2 HP0 4 2.0 K 2 PO 4 6.6 FeAramCit 0.1 Tyrosine 0.225 MgS0 4 .7H 2 0 2.25 Rates of addition are given in grams per liter. Trace element solution was also added (1 ml per liter). Ampicillin was added to the medium for the growth of HW77pME202. This seed culture was grown for 12 hr at 33° and then used to inoculate a 10 liter stirred tank fer- menter containing the same medium. The fermentation- was continued until the tyrosine was exhausted, i.e., about . 11 hr at which point the glucose feed was commenced. Growth was limited by the tyrosine starvation to an A 670 of about 20. A 70% glucose feed was used at a rate of 0.63 g 1 hr . L-phenylalanine concentrations were monitored throughout the fermentation. Figure 5 gives details of a typical comparison between HW77 and HW77 carrying the Phe plasmid pME202. It is clear that the presence of the plasmiά has enhanced both the rate of accumulation and the final broth titre of L-phenylalanine. The details of ' ^ productivity and final broth titre are summari7e<5~- ι che following table: STRAIN PRODUCTIVITY FINAL TITRE (g /Liter, hr) (gm/Liter, hr) HW77 0.11 6.07 HW77+pME202 0.43 11.6 Analysis of the fermenter broths also revealed that whereas HW77 accumulated significant quantities of both prephenate and chorismate HW77pME202 did not. This indicates that the deregulated pheA gene on pME202 is having the desired effect of increasing the flux down the L-phenylalanine branch of the pathway by elevating prephenate dehydratase activity in vivo.

Claims: CLAIMS : 1. As a composition of matter, a composite plasmid comprising: (a) a first DNA segment containing a replicon covalently joined to a second DNA segment containing one or more transcriptional units; (b) if only one trans¬ criptional unit, then an artificial operon containing two or more genes coding for two or more polypeptides active in the synthesis of an amino acid; (c) if two or more transcriptional units, then a first transcriptional unit comprising an artificial operon containing one or more genes coding for one or more polypeptides active in the synthesis of an amino acid and one or more addi¬ tional transcriptional units containing one or more genes coding for polypeptides usefμl in the production of an amino acid. 2. The composite plasmid of Claim 1(c) wherein an additional transcriptional unit contains one or more genes coding for one or more catabolic enzymes or a transport protein. 3. The composite plasmid.of Claim 2 wherein a catabolic enzyme is an amylase. 4. The composite plasmid of Claim 1 wherein the polypeptides active in the synthesis of an amino acid are enzymes. 5. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of phenylalanine is pheA. 6. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of tryptophan, phenylalanine or tyrosine is aroF. 7. The composite plasmid of Claim ' 4 wherein one of the genes coding for an enzyme active in the synthesis of tyrosine is tyrA. 8. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of phenylalanine or tyrosine is tyrB. 9. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of tryptophan, phenylalanine or tyrosine is aroL. 10. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of tryptophan, phenylalanine or tyrosine is aroH. 11. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of tryptophan, phenylalanine or tyrosine is aroG. 12. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of tryptophan is trpE. 13. The composite plasmid of Claim 4 wherein one of the genes coding for an enzyme active in the synthesis of phenylalanine or tyrosine is aspC. 14. The composite plasmid of Claim 4 wherein one of the genes coding for enzymes active in the syn¬ thesis of tryptophan is trpB, trpA, trpD, or trpC. 15. The op'.rc- plasmid of Claim 1 wherein the replicon is capable of replication in a prokaryotic cell. 16. A method of producing a composite plasmid comprising: (a) mutating individually to feedback inhibi¬ tion resistance one or more genes coding for an enzyme active in the synthesis of an amino acid; (b) connecting the mutated genes to a transcriptional unit; (c) expressing the transcriptional unit resulting in enzyme synthesis thereby increasing the synthesis of the amino acid. 17. The method of Claim 16 wherein an addi¬ tional step comprises: (a) inserting an additional transcriptional unit containing one or more genes coding for one or more enzymes useful in the production of amino acids. 18. A method of screening for microorganisms useful in amino acid production comprising: (a) transforming a bacterial cell with a plasmid containing an artificial operon containing a gene coding for the synthesis of a feedback resistant enzyme that is rate-limiting in amino acid synthesis; (b) selecting the transformed cells.; (c) isolating a pure culture; (d) quantitating the production of the desired amino acid or an amino acid precursor from the pure culture in (c). 19. The method of Claim 18 wherein an addi¬ tional step comprises: - (e) cross-feeding to determine one or more of the following: (1) amino acid concentration, (2) amino acid precursor concentration, or (3) growth in the presence of an amino acid analog. 20. A composite plasmid of Claim 1 comprising plasmid pME202. 21. A composite plasmid of Claim 1 comprising plasmid pME214. 22. A composite plasmid of Claim 1 comprising plasmid pME219. 23. A composite plasmid of Claim 1 comprising plasmid pPT112. 24. A method for the production of an amino acid comprising: (a) fermenting a bacterial cell con¬ taining the composite plasmid of claim 1; (b) isolating the amino acid. 25. The method of Claim 24 wherein the amino acid is an aromatic amino acid. 26. The method of Claim 25 wherein the aromatic amino acid is L-phenylalanine. 27. The method of Claim 25 wherein the aromatic amino acid is tyrosine. 28. The method of Claim 25 wherein the aromatic amino acid is tryptophan. 29. A composition of matter comprising a DNA sequence selected from the following: (a) chart 3; (b) chart 5; (c) chart 7; (d) chart 9; (e) chart 10; (f) chart 11. .30. A plasmid containing a DNA sequence of claim 29. 31. A method for the production of an aromatic amino acid comprising: (a) fermenting a bacterial cell containing a plasmid of cl^im 30; (b) isolating the amino acid. 32. The method of claim 32 wherein the aromatic amino acid is L-phenylalanine. 33. The method of claim 32 wherein the aromatic a i ^o acid is L-tyrosine. 34. The method of claim 32 wherein the aromatic amino acid is L-tryptophan.

Inventors: EDWARDS MARK RICHARD, FOTHERINGHAM IAN GRAHAM, HUNTER MICHAEL GEORGE, TAYLOR PAUL PHILLIP, EDWARDS, MARK, RICHARD, FOTHERINGHAM, IAN, GRAHAM, HUNTER, MICHAEL, GEORGE, TAYLOR, PAUL, PHILLIP
Applicants: NUTRASWEET CO, THE NUTRASWEET COMPANY
Application year: 1986
Main classification: C12P13
IPC codes: C12N1, C12N9, C12N15, C12P13, C12R1