Process for the production of hyaluronic acid in Escherichia coli or Bacillus subtilis

Abstract

The present invention relates to a method for the production of hyaluronic acid (HA) in Bacillus subtilis and Escherichia coli through plasmid vectors wherein the gene is under the control of strong promoter Pgrac, and a system for the selection of stable bacterial strains for the production of high levels of hyaluronic acid.

Claims

1. A process for the production of hyaluronic acid in Bacillus subtilis, wherein said process comprises: (a) culturing Bacillus subtilis cells transformed with a grac-lac system, in a medium under conditions suitable for producing hyaluronic acid, and in the presence of isopropyl--thiogalactopyranoside (IPTG) as an inducer, wherein said Bacillus subtilis cells are transformed with: (i) at least one episomal plasmid vector comprising a sequence coding for a hyaluronan synthase, and a sequence coding for a UDP-glucose dehydrogenase in tandem, under the control of a single strong inducible promoter Pgrac which uses the lac repressor, or (ii) at least one episomal plasmid vector comprising a sequence coding for a hyaluronan synthase, a sequence coding for a UDP-glucose dehydrogenase, a sequence coding for a UDP-glucose pyrophosphorylase and a sequence coding for a glucose 6 phosphate isomerase, under the control of a single strong inducible promoter Pgrac which uses the lac repressor; and (b) recovering hyaluronic acid from the culture medium; wherein said Bacillus subtilis cells transformed with the plasmid vector of (i) or (ii) are preselected on an IPTG gradient; and wherein hyaluronic acid having a weight average molecular weight in the range 100-500 KD is produced when the fermentation time is 80-160 hours; hyaluronic acid having a weight average molecular weight in the range 500-1000 KD is produced when the fermentation time is 40-80 hours; or hyaluronic acid having a weight average molecular weight in the range 110.sup.6-210.sup.6 D is produced when the fermentation time is 12-40 hours.

2. The process according to claim 1, wherein the episomal plasmid vector (i) or (ii) further comprises a sequence coding for the lac repressor.

3. The process according to claim 1, wherein the IPTG inducer is added in step a) in quantities of between 0.01 and 10 mM in solution.

4. The process according to claim 1, wherein said Bacillus subtilis cells are B. subtilis WB800N or B. subtilis 1012 cells.

5. The process according to claim 1, wherein the hyaluronan synthase (hasA) is obtained from a strain of Streptococcus and the UDP-glucose dehydrogenase (hasB or tuaD), UDP-glucose pyrophosphorylase (hasC or gtaB) and glucose 6 phosphate isomerase (hasE or pgi) are obtained from Bacillus subtilis.

6. The process according to claim 1, wherein the sequences coding for the hyaluronan synthase, UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase and glucose 6 phosphate isomerase comprise an upstream Shine-Dalgarno sequence.

7. The process according to claim 1, wherein said plasmid vector of (i) comprises SEQ ID NO:1.

8. The process according to claim 1, wherein hyaluronic acid having a weight average molecular weight in the range 100-500 KD is produced when the fermentation time is 80-160 hours.

9. The process according to claim 1, wherein hyaluronic acid having a weight average molecular weight comprised in the range 500-1000 KD is produced when the fermentation time is 40-80 hours.

10. The process according to claim 1, wherein hyaluronic acid having a weight average molecular weight comprised in the range 110.sup.6-210.sup.6 D is produced when the fermentation time is 12-40 hours.

11. The process according to claim 3, wherein the IPTG inducer is added in step (a) in quantities of between 0.01 and 5 mM or between 0.4 mM and 1 mM in solution.

12. The process according to claim 5, wherein the hyaluronan synthase (hasA) is obtained from a strain of Streptococcus zooepidemicus.

Description

(1) The present invention will be now disclosed by way of example but not of limitation, according to preferred embodiments with particular reference to the attached figures, wherein:

(2) FIG. 1 shows a comparison in plates between the growth of cells E. coli TOP10, incorporating plasmid pHT01 (control) and cells E. coli TOP10, incorporating pBS5 (hasA+tuaD);

(3) FIG. 2 shows the gel analysis of the expression of gene tuaD in E. coli BL21 DE3;

(4) FIG. 3 (SEQ ID NO: 12) illustrates the vector map pHT01 comprising Pgrac promoter consisting of the groE promoter, the lacO operator and the gsB SD sequence; the replication origin ColE1; Amp.sup.R ampicillin resistance gene; lad gene (lad repressor); and Cm.sup.R chloramphenicol resistance gene;

(5) FIG. 4 shows the analysis in gel electrophoresis of the constitutive expression of hyaluronan synthase (Strept) in E. coli; the encoded protein designated SeHAS is 417 amino acids long (calculated molecular weight 47,778; calculated PI 9.1) and is the smallest member of the HAS family identified thus far; the enzyme migrates anomalously fast in SDS polyacrylamide gel electrophoresis (about 42000 Da);

(6) FIG. 5 shows the comparison between the profiles of expression of HA in strains of E. Coli TOP10+pBS5 (hasA+tuaD) and TOP10+pHT01 (control) through carbazole analysis of glucuronic acid at 530 nm;

(7) FIG. 6 shows the comparison in plates among the expression of glucuronic acid in strains of Bacillus subtilis WB800N and 1012 transformed with pBS5; when bacteria are seeded in presence of IPTG 1 mM, they die because tuaD expressed at high amounts in B. subtilis is toxic;

(8) FIG. 7 shows the expression of glucuronic acid in Bacillus subtilis in plates wherein large and translucent colonies produce HA;

(9) FIG. 8 shows the results of plating assays to verify the stability of plasmid after 24 hours of cells growth in presence of IPTG and saccharose and in presence or absence of chloramphenicol.

(10) The following examples describe the various steps required for the embodiment of the process of production of HA according to the present invention, by way of example but not of limitation.

Example 1

Cloning of the tuaD Gene (UDP-Glucose Dehydrogenase) from Bacillus Subtilis

(11) The sequence of the tuaD gene, which is 9300 bp long in B. subtilis, is present in the databases as access number AF015609; it codes for the operon which leads to teichuronic acid synthesis and comprises 8 genes, tuaABCDEFGH. In our case the gene of interest tuaD falls between the bases 3582-4984 bp. Software analysis for restriction enzymes indicates that the restriction sites ClaI, EcoRI, PstI, HindIII and SphI are present, and therefore cannot be used for cloning. The start codon is not a methionine but a valine; in the present invention it was replaced with the codon for methionine, which translates the protein much more efficiently. Two oligonucleotide primers synthesised with the following sequence were used to recover this sequence:

(12) TABLE-US-00001 (SEQIDNO:2) 5 atgaaaaaatagctgtcattggaacag3 and (SEQIDNO:3) 5 ttataaattgtcgttcccaagtct3

(13) The genomic DNA from B. subtilis (strain 168) was obtained with the Qiagen extraction kit. With 32 cycles of PCR, using DNA from B. subtilis as template and the two said oligonucleotides, an amplificate of the expected molecular weight was obtained. The amplificate obtained was tested for the presence of restriction enzyme EcoRI. After cutting with this enzyme in 1% agarose gel, two bands of DNA weighing 470 bp and 920 bp are present, which correspond to those expected. To clone the tuaD gene in an expression vector, two other oligonucleotides with the following sequence were synthesised:

(14) TABLE-US-00002 (SEQIDNO:4) 5 gctggatccatgaaaaaatagctgtcattgg3 and (SEQIDNO:5) 5 ctcgctagcttataaattgacgcttcccaag3

(15) so as to insert said sequence between the restriction sites BamHI and NheI in the expression vector, plasmid pRSETB (INVITROGEN).

(16) A Shine-Dalgarno (SD) sequence needs to be introduced into the tuaD gene upstream of the 5 end of the gene to allow efficient recognition by the bacterial RNA polymerase. For this purpose the DNA was amplified with the following oligonucleotides:

(17) TABLE-US-00003 (SEQIDNO:6) 5 cgacatatgaaaaaatagctgtcattgg3 and (SEQIDNO:7) 5 ctcgctagcttataaattgacgcttcccaag3.

(18) They contain in 5 two restriction sites NdeI and NheI which allow its cloning in vector pRSET B between the same sites. In this way, a particularly efficient sequence SD, which is necessary for RNA polymerase in order to synthesise the protein, is present upstream of the NdeI restriction site of plasmid pRSET B. Restriction site XbaI, which will be required for the subsequent clonings, is also present even before said sequence. The vector created, pRSET B, was therefore called pRSEtuaD.

(19) Thus in this plasmid, the sequence coding for tuaD falls between the restriction sites NdeI and NheI; restriction site XbaI, which is necessary for the subsequent cloning, is present before and upstream of said plasmid, and other restriction sites, including BamHI--BglII--XhoI, are present behind the tuaD gene.

(20) The diagram below summarises the sites of interest present in plasmid pRSEtuaD

(21) XbaI--NdeI----------------tuaD----------------NheI-BamHI--BglI-XhoI

(22) The plasmid described is an expression vector which also functions in E. coli, because the gene is under the control of the T7 promoter; if it is transformed to bacterial cells BL21 DE3, which are able to transcribe T7 RNA polymerase, it therefore enables them to express the tuaD gene. After induction with 1 mM of IPTG the transfected cells are able to produce the protein of the expected molecular weight, but not hyaluronic acid. The construction is particularly efficient because the level of expression is very high. The sizes of the colonies which carry plasmid pRSEtuaD are very small compared with the control cells (FIG. 1), which demonstrates the toxicity of the tuaD gene. This cloning is difficult precisely because it is apparently difficult for the colonies to grow; the particularly high level of expression of this protein probably drains the glucose available for uncontrolled synthesis of D-glucuronic acid, thus depriving the cell of its main energy source. The cells in which the tuaD synthesis is induced with IPTG are unable to survive for a long time, so the gene product is toxic.

(23) In conclusion, the tuaD gene was isolated and cloned in a plasmid and the sequence proved correct. The gene expressed in E. coli is able to produce a protein of the expected molecular weight corresponding to that described for tuaD (54 kDa, FIG. 2); however, in the absence of hyaluronan synthase, these cells are unable to produce significant amounts of hyaluronic acid, and the consequent accumulation of glucuronate is toxic to the cell.

Example 2

Cloning of the hasA (Hyaluronan Synthase) Gene from Streptococcus zooepidemicus

(24) The gene sequence for hyaluronan synthase is present in the databases with access number AY173078, and is 3552 bp long; the sequence coding for the protein is between bases 1 and 1254. The restriction sites HindIII and StuI are present in this sequence, and therefore cannot be used for cloning, but can be used to verify the cloning. Two oligonucleotides for use with PCR were designed and synthesised to recover the coding sequence:

(25) TABLE-US-00004 (SEQIDNO:8) 5 atgagaacattaaaaaacctcataac3 e (SEQIDNO:9) 5 taataattttttacgtgttccccag3

(26) The genomic DNA from the bacterium Streptococcus zooepidemicus was recovered with the Qiagen extraction kit. The 1254 bp coding sequence was recovered with PCR. The expected amplificate of the correct dimensions was controlled with restriction enzyme HindIII, and gave rise to two bands of approx. 100 bp and 1150 bp which correspond to the expected cut.

Example 3

Construction of Expression Plasmid pHT01hasA for Bacillus subtilis

(27) To clone said gene in expression vector pHT01 (MobitecFIG. 3) containing the gene promoter-repressor system grac-lac, the above-mentioned sequence must be cloned between restriction sites BamHI and XbaI. Two other oligonucleotides with the following sequence were created for this purpose:

(28) TABLE-US-00005 (SEQIDNO:10) 5 ggaggatccatgagaacattaaaaaacctcat3 e (SEQIDNO:11) 5 cagtctagattataataatttttacgtgtcc3

(29) In the first oligonucleotide, restriction site BamHI was created near 5, while in the second oligonucleotide, restriction site XbaI was created, again at 5. The amplificate obtained through these two oligonucleotides was cloned in plasmid pGEM4Z (PROMEGA) between restriction sites BamHI and XbaI to give plasmid pGEM4hasA.

(30) The DNA sequence between said two restriction sites was analysed with an ABI 7000 sequencer, and proved correct.

(31) HindIII-BamHI----------------hasA----------------XbaI-SalI

(32) The plasmid was checked for expression of the recombinant protein in E. coli, and presented a molecular weight of approx. 42 kDa (which agrees with the weight reported for that protein in the literature, although it has a theoretical molecular weight of 47.778 kDa, FIG. 4).

(33) To clone said sequence between restriction sites BamHI and XbaI of vector pHT01, plasmid pGEM4hasA was cut in sites BamHI and XbaI, and the 1240 bp band was cloned in the same sites as plasmid pHT01 to obtain plasmid pHT01hasA. This plasmid is unable to produce significant quantities of hyaluronic acid because it lacks the tuaD gene. This proves that the presence of hasA alone is not sufficient to express significant amounts of HA.

Example 4

Construction of Expression Plasmid pHT01hasA-tuaD for Bacillus subtilis

(34) With this construction, the hasA gene is placed in tandem with the tuaD gene under the control of inducible promoter Pgrac present in plasmid pHT01 (Mobitec). Plasmid pGEM4hasA (described in the previous example) was used as vector for this purpose, as it already contains the hasA gene. Said plasmid was cut in sites XbaI and SalI, while the sequence of the tuaD gene was cut by plasmid pRESEtuaD in sites XbaI and XhoI and then cloned in the same sites (XhoI and SalI are compatible).

(35) pGEM4hasA

(36) HindIII-BamHI----------------hasA----------------XbaI-SalI

(37) pRSEtuaD

(38) XbaI--NdeI----------------tuaD----------------NheI-BamHI--BglI-XhoI

(39) obtaining this sequence:

(40) HindIII-BamHI---------hasA---------XbaI--NdeI---------tuaD-----------NheI-BamHI--BglI-XhoI

(41) At this point the hasA gene is in tandem with the tuaD gene; fragment BamHI----NheI, which is obtained from the plasmid by cutting with said restriction enzymes, contains the hasA gene and the tuaD gene in tandem. The fragment was then cloned in vector pHT01 between restriction sites BamHI and XbaI (XbaI is compatible with NheI), giving rise to plasmid pBS5, the complete, controlled sequence of which is set out below:

(42) TABLE-US-00006 (SEQIDNO:1) 0 TTAAGTTATTGGTATGACTGGTTTTAAGCGCAAAAAAAGTTGCTTTTTCGTACCTATTAA 60 TGTATCGTTTTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAA 120 GCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCATGAT 180 AACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTT 240 TATTAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATAT 300 TTAAGTTAAACCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAG 360 GTATAGGTGTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGT 420 ATAAATCATAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTT 480 TAGATACACCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAATAACCTAACTCTC 540 CGTCGCTATTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCCTTGTCACTAAGA 600 AAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTATGTTTCGGTATAAAACACTAA 660 TATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCT 720 CTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCCTAAA 780 TTTTTATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCC 840 TTTTTTAAAAGTCAATATTACTGTAACATAAATATATATTTTAAAAATATCCCACTTTAT 900 CCAATTTTCGTTTGTTGAACTAATGGGTGCTTTAGTTGAAGAATAAAGACCACATTAAAA 960 AATGTGGTCTTTTGTGTTTTTTTAAAGGATTTGAGCGTAGCGAAAAATCCTTTTCTTTCT 1020 TATCTTGATAATAAGGGTAACTATTGCCGATCGTCCATTCCGACAGCATCGCCAGTCACT 1080 ATGGCGTGCTGCTAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCG 1140 GTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTA EcoRI 1200 AGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTC 1260 GAGCTCAGGCCTTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGA 1320 AACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT 1380 ATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTT 1440 CACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCG 1500 AAAATCCTGTTTGATGGTGGTTGACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTC 1560 GTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCAT 1620 TGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT 1680 CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGC 1740 TATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGC 1800 CGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAG 1860 ATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGT 1920 CTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAAT 1980 GGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAG 2040 ATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCAC 2100 GCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTG 2160 CAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTG 2220 TGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGT 2280 TTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACC 2340 GGCATACTCTGCGACATCGTATAACGTTACTGGTTTCATCAAAATCGTCTCCCTCCGTTT 2400 GAATATTTGATTGATCGTAACCAGATGAAGCACTCTTTCCACTATCCCTACAGTGTTATG 2460 GCTTGAACAATCACGAAACAATAATTGGTACGTACGATCTTTCAGCCGACTCAAACATCA 2520 AATCTTACAAATGTAGTCTTTGAAAGTATTACATATGTAAGATTTAAATGCAACCGTTTT 2580 TTCGGAAGGAAATGATGACCTCGTTTCCACCGGAATTAGCTTGGTACCAGCTATTGTAAC 2640 ATAATCGGTACGGGGGTGAAAAAGCTAACGGAAAAGGGAGCGGAAAAGAATGATGTAAGC 2700 GTGAAAAATTTTTTATCTTATCACTTGAAATTGGAAGGGAGATTCTTTATTATAAGAATT BamHI 2760 GTGGAATTGTGAGCGGATAACAATTCCCAATTAAAGGAGGAAGGATCCATGAGAACATTA 1 MRTL 2820 AAAAACCTCATAACTGTTGTGGCCTTTAGTATTTTTTGGGTACTGTTGATTTACGTCAAT 1 KNLITVVAFSIFWVLLIYVN HindIII 2880 GTTTATCTCTTTGGTGCTAAAGGAAGCTTGTCAATTTATGGCTTTTTGCTGATAGCTTAC 1 VYLFGAKGSLSIYGFLLIAY 2940 CTATTAGTCAAAATGTCCTTATCCTTTTTTTACAAGCCATTTAAGGGAAGGGCTGGGCAA 1 LLVKMSLSFFYKPFKGRAGQ 3000 TATAAGGTTGCAGCCATTATTCCCTCTTATAACGAAGATGCTGAGTCATTGCTAGAGACC 1 YKVAAIIPSYNEDAESLLET 3060 TTAAAAAGTGTTCAGCAGCAAACCTATCCCCTAGCAGAAATTTATGTTGTTGACGATGGA 1 LKSVQQQTYPLAEIYVVDDG 3120 AGTGCTGATGAGACAGGTATTAAGCGCATTGAAGACTATGTGCGTGACACTGGTGACCTA 1 SADETGIKRIEDYVRDTGDL 3180 TCAAGCAATGTCATTGTTCACCGGTCAGAAAAAAATCAAGGAAAGCGTCATGCACAGGCC 1 SSNVIVHRSEKNQGKRHAQA 3240 TGGGCCTTTGAAAGATCAGACGCTGATGTCTTTTTGACCGTTGACTCAGATACTTATATC 1 WAFERSDADVFLTVDSDTYI 3300 TACCCTGATGCTTTAGAGGAGTTGTTAAAAACCTTTAATGACCCAACTGTTTTTGCTGCG 1 YPDALEELLKTFNDPTVFAA 3360 ACGGGTCACCTTAATGTCAGAAATAGACAAACCAATCTCTTAACACGCTTGACAGATATT 1 TGHLNVRNRQTNLLTRLTDI 3420 CGCTATGATAATGCTTTTGGCGTTGAACGAGCTGCCCAATCCGTTACAGGTAATATTCTC 1 RYDNAFVERAAAQSVTGNIL 3480 GTTTGCTCAGGCCCGCTTAGCGTTTACAGACGCGAGGTGGTTGTTCCTAACATAGATAGA 1 VCSGPLSVYRREVVVPNIDR 3540 TACATCAACCAGACCTTCCTGGGTATTCCTGTAAGTATCGGTGATGACAGGTGCTTGACC 1 YINQTFLGIPVSIGDDRCLT 3600 AACTATGCAACTGATTTAGGAAAGACTGTTTATCAATCCACTGCTAAATGTATTACAGAT 1 NYATDLGKTVYQSTAKCITD 3660 GTTCCTGACAAGATGTCTACTTACTTGAAGCAGCAAAACCGCTGGAACAAGTCCTTCTTT 1 VPDKMSTYLKQQNRWNKSFF 3720 AGAGAGTCCATTATTTCTGTTAAGAAAATCATGAACAATCCTTTTGTAGCCCTATGGACC 1 RESTISVKKIMNNPFVALWT 3780 ATACTTGAGGTGTCTATGTTTATGATGCTTGTTTATTCTGTGGTGGATTTCTTTGTAGGC 1 ILEVSMFMMLVYSVVDFFVG 3840 AATGTCAGAGAATTTGATTGGCTCAGGGTTTTGGCCTTTCTGGTGATTATCTTCATTGTT 1 NVREFDWLRVLAFLVIIFIV 3900 GCTCTTTGTCGTAATATTCACTATATGCTTAAGCACCCGCTGTCCTTCTTGTTATCTCCG 1 ALCRNIHYMLKHPLSFLLSP 3960 TTTTATGGGGTACTGCATTTGTTTGTCCTACAGCCCTTGAAATTGTATTCTCTTTTTACT 1 FYGVLHLFVLQPLKLYSLFT XbaI 4020 ATTAGAAATGCTGACTGGGGAACACGTAAAAAATTATTATAATCTAGAAATAATTTTGTT 1 IRNADWGTRKKLL 4080 TAACTTTAAGAAGGAGATATACATATGAAAAAAATAGCTGTCATTGGAACAGGTTATGTA 1 MKKIAVIGTGYV 4140 GGACTCGTATCAGGCACTTGCTTTGCGGAGATCGGCAATAAAGTTGTTTGCTGTGATATC 1 GLVSGTCFSIGNNKVVCCDI 4200 GATGAATCAAAAATCAGAAGCCTGAAAAATGGGGTAATCCCAATCTATGAACCAGGGCTT 1 DESKIRSLKNGVIPIYEPGL 4260 GCAGACTTAGTTGAAAAAAATGTGCTGGATCAGCGCCTGACCTTTACGAACGATATCCCG 1 ADLVEKNVLDQRLTFTNDIP 4320 TCTGCCATTCGGGCCTCAGATATTATTTATATTGCAGTCGGAACGCCTATGTCCAAAACA 1 SAIRASDIIYIAVGTPMSKT 4380 GGTGAAGCTGATTTAACGTACGTCAAAGCGGCGGCGAAAACAATCGGTGAGCATCTTAAC 1 GEADLTYVKAAAKTIGEHLN 4440 GGCTACAAAGTGATCGTAAATAAAAGCACAGTCCCGGTTGGAACAGGGAAACTGGTGCAA 1 GYKVIVNKSTVPVGTGKLVQ EcoRI 4500 TCTATCGTTCAAAAAGCCTCAAAGGGGAGATACTCATTTGATGTTGTATCTAACCCTGAA 1 SIVQKSKGGRYSFDVVSNPE 4560 TTCCTTCGGGAAGGGTCAGCGATTCATGACACGATGAATATGGAGCGTGCCGTGATTGGT 1 FLREGSAIHDTMNMERAVIG 4620 TCAACAAGTCATAAAGCCGCTGCCATCATTGAGGAACTTCATCAGCCATTCCATGCTCCT 1 STSHKAAAIIEELHQPFHAP 4680 GTCATTAAAACAAACCTAGAAAGTGCAGAAATGATTAAATACGCCGCGAATGCATTTCTG 1 VIKTNLESAEMIKYAANAFL 4740 GCGACAAAGATTTCCTTTATCAACGATATCGCAAACATTTGTGAGCGAGTCGGCGCAGAC 1 ATKISFINDIANICERVGAD 4800 GTTTCAAAAGTTGCTGATGGTGTTGGTCTTGACAGCCGTATCGGCAGAAAGTTCCTTAAA 1 VSKVADGVGLSRIIGRKFLK 4860 GCTGGTATTGGATTCGGCGGTTCATGTTTTCCAAAGGATACAACCGCGCTGCTTCAAATC 1 AGIGFGGSCFPKDTTALLQI 4920 GCAAAATCGGCAGGCTATCCATTCAAGCTCATCGAAGCTGTCATTGAAACGAACGAAAAG 1 AKSAGYPFKLIEAVIETNEK 4980 CAGCGTGTTCATATTGTAGATAAACTTTTGACTGTTATGGGAAGCGTCAAAGGGAGAACC 1 QRVHIVDKLLTVMGSVKGRT 5040 ATTTCAGTCCTGGGATTAGCCTTCAAACCGAATACGAACGATGTGAGATCCGCTCCAGCG 1 ISVLGLAFKPNTNDVRSAPA 5100 CTTGATATTATCCCAATGCTGCAGCAGCTGGGCGCCCATGTAAAAGCATACGATCCGATT 1 LDIIPMLQQLGAHVKAYDPI HindIII 5160 GCTATTCCTGAAGCTTCAGCGATCCTTGGCGAACAGGTCGAGTATTACACAGATGTGTAT 1 AIPEASAILGEQVEYYTDVY 5220 GCTGCGATGGAAGACACTGATGCATGCCTGATTTTAACGGATTGGCCGGAAGTGAAAGAA 1 AAMEDTDACLILTDWPEVKE 5280 ATGGAGCTTGTAAAAGTGAAAACCCTCTTAAAACAGCCAGTCATCATTGACGGCAGAAAT 1 MELVKVKTLLKQPVIIDGRN 5340 TTATTTTCACTTGAAGAGATGCAGGCAGCCGGATACATTTATCACTCTATCGGCCGTCCC 1 LFSLEEMQAAGYIYHSIGRP 5400 GCTGTTCGGGGAACGGAACCCTCTGACAAGTATTTTCCGGGCTTGCCGCTTGAAGAATTG 1 AVRGTEPSDKYFPGLPLEEL Nhe/XbaISmaI 5460 GCTAAAGACTTGGGAAGCGTCAATTTATAAGCTAGAGTCGACGTCCCCGGGGCAGCCCGC 1 AKDLGSVNL 5520 CTAATGAGCGGGCTTTTTTCACGTCACGCGTCCATGGAGATCTTTGTCTGCAACTGAAAA 5580 GTTTATACCTTACCTGGAACAAATGGTTGAAACATACGAGGCTAATATCGGCTTATTAGG 5640 AATAGTCCCTGTACTAATAAAATCAGGTGGATCAGTTGATCAGTATATTTTGGACGAAGC 5700 TCGGAAAGAATTTGGAGATGACTTGCTTAATTCCACAATTAAATTAAGGGAAAGAATAAA 5760 GCGATTTGATGTTCAAGGAATCACGGAAGAAGATACTCATGATAAAGAAGCTCTAAAACT 5820 ATTCAATAACCTTACAATGGAATTGATCGAAAGGGTGGAAGGTTAATGGTACGAAAATTA HindIII 5880 GGGGATCTACCTAGAAAGCCACAAGGCGATAGGTCAAGCTTAAAGAACCCTTACATGGAT 5940 CTTACAGATTCTGAAAGTAAAGAAACAACAGAGGTTAAACAAACAGAACCAAAAAGAAAA 6000 AAAGCATTGTTGAAAACAATGAAAGTTGATGTTTCAATCCATAATAAGATTAAATCGCTG EcoRI 6060 CACGAAATTCTGGCAGCATCCGAAGGGAATTCATATTACTTAGAGGATACTATTGAGAGA 6120 GCTATTGATAAGATGGTTGAGACATTACCTGAGAGCCAAAAAACTTTTTATGAATATGAA 6180 TTAAAAAAAAGAACCAACAAAGGCTGAGACAGACTCCAAACGAGTCTGTTTTTTTAAAAA 6240 AAATATTAGGAGCATTGAATATATATTAGAGAATTAAGAAAGACATGGGAATAAAAATAT 6300 TTTAAATCCAGTAAAAATATGATAAGATTATTTCAGAATATGAAGAACTCTGTTTGTTTT 6360 TGATGAAAAAACAAACAAAAAAAATCCACCTAACGGAATCTCAATTTAACTAACAGCGGC 6420 CAAACTGAGAAGTTAAATTTGAGAAGGGGAAAAGGCGGATTTATACTTGTATTTAACTAT 6480 CTCCATTTTAACATTTTATTAAACCCCATACAAGTGAAAATCCTCTTTTACACTGTTCCT 6540 TTAGGTGATCGCGGAGGGACATTATGAGTGAAGTAAACCTAAAAGGAAATACAGATGAAT 6600 TAGTGTATTATCGACAGCAAACCACTGGAAATAAAATCGCCAGGAAGAGAATCAAAAAAG 6660 GGAAAGAAGAAGTTTATTATGTTGCTGAAACGGAAGAGAAGATATGGACAGAAGAGCAAA 6720 TAAAAAACTTTTCTTTAGACAAATTTGGTACGCATATACCTTACATAGAAGGTCATTATA 6780 CAATCTTAAATAATTACTTCTTTGATTTTTGGGGCTATTTTTTAGGTGCTGAAGGAATTG 6840 CGCTCTATGCTCACCTAACTCGTTATGCATACGGCAGCAAAGACTTTTGCTTTCCTAGTC 6900 TACAAACAATCGCTAAAAAAATGGACAAGACTCCTGTTACAGTTAGAGGCTACTTGAAAC 6960 TGCTTGAAAGGTACGGTTTTATTTGGAAGGTAAACGTCCGTAATAAAACCAAGGATAACA 7020 CAGAGGAATCCCCGATTTTTAAGATTAGACGTAAGGTTCCTTTGCTTTCAGAAGAACTTT 7080 TAAATGGAAACCCTAATATTGAAATTCCAGATGACGAGGAAGCACATGTAAAGAAGGCTT 7140 TAAAAAAGGAAAAAGAGGGTCTTCCAAAGGTTTTGAAAAAAGAGCACGATGAATTTGTTA 7200 AAAAAATGATGGATGAGTCAGAAACAATTAATATTCCAGAGGCCTTACAATATGACACAA 7260 TGTATGAAGATATACTCAGTAAAGGAGAAATTCGAAAAGAAATCAAAAAACAAATACCTA 7320 ATCCTACAACATCTTTTGAGAGTATATCAATGACAACTGAAGAGGAAAAAGTCGACAGTA 7380 CTTTAAAAAGCGAAATGCAAAATCGTGTCTCTAAGCCTTCTTTTGATACCTGGTTTAAAA 7440 ACACTAAGATCAAAATTGAAAATAAAAATTGTTTATTACTTGTACCGAGTGAATTTGCAT 7500 TTGAATGGATTAAGAAAAGATATTTAGAAACAATTAAAACAGTCCTTGAAGAAGCTGGAT 7560 ATGTTTTCGAAAAAATCGAACTAAGAAAAGTGCAATAAACTGCTGAAGTATTTCAGCAGT 7620 TTTTTTTATTTAGAAATAGTGAAAAAAATATAATCAGGGAGGTATCAATATTTAATGAGT 7680 ACTGATTTAAATTTATTTAGACTGGAATTAATAATTAACACGTAGACTAATTAAAATTTA 7740 ATGAGGGATAAAGAGGATACAAAAATATTAATTTCAATCCCTATTAAATTTTAACAAGGG 7800 GGGGATTAAAATTTAATTAGAGGTTTATCCACAAGAAAAGACCCTAATAAAATTTTTACT 7860 AGGGTTATAACACTGATTAATTTCTTAATGGGGGAGGGATTAAAATTTAATGACAAAGAA HindIII 7920 AACAATCTTTTAAGAAAAGCTTTTAAAAGATAATAATAAAAAGAGCTTTGCGATTAAGCA 7980 AAACTCTTTACTTTTTCATTGACATTATCAAATTCATCGATTTCAAATTGTTGTTGTATC 8040 ATAAAGTTAATTCTGTTTTGCACAACCTTTTCAGGAATATAAAACACATCTGAGGCTTGT 8100 TTTATAAACTCAGGGTCGCTAAAGTCAATGTAACGTAGCATATGATATGGTATAGCTTCC 8160 ACCCAAGTTAGCCTTTCTGCTTCTTCTGAATGTTTTTCATATACTTCCATGGGTATCTCT 8220 AAATGATTTTCCTCATGTAGCAAGGTATGAGCAAAAAGTTTATGGAATTGATAGTTCCTC 8280 TCTTTTTCTTCAACTTTTTTATCTAAAACAAACACTTTAACATCTGAGTCAATGTAAGCA 8340 TAAGATGTTTTTCCAGTCATAATTTCAATCCCAAATCTTTTAGACAGAAATTCTGGACGT 8400 AAATCTTTTGGTGAAAGAATTTTTTTATGTAGCAATATATCCGATACAGCACCTTCTAAA 8460 AGCGTTGGTGAATAGGGCATTTTACCTATCTCCTCTCATTTTGTGGAATAAAAATAGTCA 8520 TATTCGTCCATCTACCTATCCTATTATCGAACAGTTGAACTTTTTAATCAAGGATCAGTC 8580 CTTTTTTTCATTATTCTTAAACTGTGCTCTTAACTTTAACAACTCGATTTGTTTTTCCAG 8640 ATCTCGAGGGTAACTAGCCTCGCCGATCCCGCAAGAGGCCCGGCAGTCAGGTGGCACTTT 8700 TCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA 8760 TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT 8820 GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGT 8880 TTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACG 8940 AGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGA 9000 AGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCG 9060 TATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGT 9120 TGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATG 9180 CAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGG 9240 AGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGA 9300 TCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCC 9360 TGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC 9420 CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTC 9480 GGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCG 9540 CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACAC 9600 GACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC 9660 ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTT 9720 AAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC 9780 CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA 9840 AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC 9900 ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGT 9960 AACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGG 10020 CCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACC 10080 AGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTT 10140 ACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA 10200 GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT 10260 TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG 10320 CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA 10380 CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA 10440 CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT 10500 CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGA 10560 TACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA 10620 GCGCCCAATACG

(43) In this sequence the hasA gene is present between bases 2808 and 4062, and a Shine-Dalgarno sequence (GGAGGA) is correctly present before the gene to increase the efficiency of transcription. Next, the tuaD gene is present between bases 4105 bp to 5490 bp; here again, an efficient Shine-Dalgarno sequence (AGGAGA) is present before the gene. Moreover, the start codon of valine present in the tuaD gene was replaced with the more efficient methionine. The plasmid, tested for restriction enzymes HindIII and EcoRI, gives a correct restriction pattern with the following bands: 3957 bp, 1650 bp, 1522 bp, 1243 bp and 610 bp.

(44) This vector is able to express hyaluronic acid in Bacillus subtilis, and also in E. Coli; in fact, the carbazole test performed towards cells transfected with pBS5 with respect to cells containing the vector without these sequences shows the presence of glucuronic acid (FIG. 5peak around 530 nm), which is one of the constituents of hyaluronic acid, exclusively in the cells engineered with pBS5.

(45) Plasmid pHT01 is a shuttle vector able to grow in both E. coli and B. subtilis. However, it has been surprisingly found that the plasmid can be grown more efficiently in E. Coli cell strain INV-1 than in strain TOP-10, which is much more efficient in the transformation, because it contains the constitutively expressed lac repressor.

(46) Plasmid pBS5 contains the inducible promoter Pgrac which uses the lac repressor. Also in E. coli, this promoter, induced with 1 mM IPTG, allows the bacterial polymerases to code for the downstream genes for the HA synthesis. Then, the Applicant has obtained the transformation of this plasmid (to) in E. coli JM110 cells, bacterial cells lacking two genes, Dam and Dcm, which lead to DNA methylation at the level of recognition sequence GATC (Dam) and CCAGG CCTGG (Dcm). This DNA transferred to the B. subtilis cells is able to produce hyaluronic acid with a higher weight average molecular weight than that obtainable with DNA transferred in E. coli INV-1 strain.

Example 5

Bacterial Transformation in Bacillus subtilis: Media and Bacterial Strains for the Formation of Competent Cells

(47) The transfer of engineered plasmids to Bacillus subtilis uses the natural entry capacity of the plasmids during a given step of bacterial growth, and is consequently a natural effect. The transformations with pBS5 were performed with different bacterial strains, in particular WB800N (MOBITEC) or 1012 (MOBITEC). The first bacterial strain was developed for the expression of recombinant proteins because it lacks eight proteases which could degrade the proteins secreted in particular (the product of the hasA gene, hyaluronan synthase, is a transmembrane protein which could therefore undergo proteolysis). Strain 1012 was used as host cell for the expression of the plasmids of series pHT.

(48) The following media are required for the transformation:

(49) Stock Solution of Metals 1000

(50) 2 M MgCl.sub.2

(51) 0.7 M CaCl.sub.2

(52) 50 mM MnCl.sub.2

(53) 5 mM FeCl.sub.3

(54) 1 mM ZnCl.sub.2

(55) 10S-Base

(56) 10MM

(57) 2 g (NH.sub.4).sub.2SO.sub.4

(58) 14 g K.sub.2HPO.sub.4

(59) 6 g KH.sub.2PO.sub.4

(60) add distilled water to 100 ml and autoclave

(61) HS Medium

(62) For 100 ml:

(63) 10 ml 10S-base

(64) 12.5 ml 4% glucose (m/v)

(65) 5 ml 0.1% L-tryptophan (m/v)

(66) 2 ml 1% casaminoacids (m/v)

(67) 25 ml 2% yeast extract (m/v)

(68) 10 ml 8% arginine (m/v), 0.4% histidine (m/v)

(69) 10 ml 1% sodium citrate (m/v)

(70) 0.01 ml 1M MgSO.sub.4

(71) 25.49 ml distilled water

(72) LS Medium

(73) For 100 ml:

(74) 10 ml 10S-base

(75) 12.5 ml 4% glucose (m/v)

(76) 0.5 ml 0.1% L-tryptophan (m/v)

(77) 1 ml 1% casaminoacids (m/v)

(78) 5 ml 2% yeast extract (m/v)

(79) 0.5 ml 0.5M MgCl.sub.2

(80) 0.5 ml 0.1M CaCl.sub.2

(81) 10 ml 1% sodium citrate (m/v)

(82) 0.01 ml 1M MgSO.sub.4

(83) 59.990 ml distilled water

Example 6

Preparation of Competent Cells from Bacillus subtilis

(84) A single colony of Bacillus subtilis is grown overnight in 5 ml of HS medium at 37 C. The next day, 500 l of this culture is incubated with 50 ml of HS medium and again grown under vigorous stirring at 200 rpm. When the cells have reached the steady state, 10 ml aliquots are collected every 15 minutes. 1 ml of glycerol is added to each aliquot, which is left on ice for 15 minutes. 1 ml aliquots are then taken and stored at 80 C. until use. The fractions with the highest rate of transformation are used for the following transformations.

Example 7

Transformation of Competent Bacillus subtilis Cells and their Selection on IPTG Gradient

(85) The bacterial cells rendered competent are thawed rapidly in a thermostatic bath at 37 C., diluted in 20 ml of LS medium in a 250 ml Erlenmeyer flask, and placed under stirring for 2 hours at 30 C. When that time has elapsed, 1 ml aliquots are placed in 15 ml tubes to which 10 l of EDTA is added, and maintained at ambient temperature for 5 minutes. Plasmid DNA pBS5 is added to the test tube and incubated for 2 h at 37 C., under stirring, with the maximum aeration. After gentle centrifugation the cells are plated in pre-heated selective medium. The bacterial colonies are obtained after two days. The cells cannot be grown in solution because they grow very slowly, and die after the addition of IPTG; above all, the few living cells no longer contain the recombinant plasmid. To select viable bacteria able to express high levels of hyaluronic acid, the cells were plated in the presence of an IPTG gradient. As shown in FIG. 6, the cells placed near a high concentration of IPTG die (because the tuaD expressed at high levels is toxic to B. subtilis); however, the cells plated in a position where a lower dose of IPTG occurs survived.

(86) When the latter were examined, they presented as large, translucent colonies (FIG. 7), indicating the expression of hyaluronic acid; these cells, selected and grown in the presence of IPTG, also survive at higher doses of IPTG and preserve the plasmid during fermentation.

(87) Through this system of selection viable bacterial lines are obtained, which are stable and above all secrete high levels of hyaluronic acid even after many cell divisions.

(88) The stability of the plasmid was verified by growing the cells for 24 hours in the presence of IPTG and saccharose, and in the presence or absence of chloramphenicol. As clearly shown in FIG. 8, the number of colonies remains identical, demonstrating what has been stated, because plasmid contains chloramphenicol resistance gene, while the strain which has not been transfected, is devoid of said gene.

Example 8

Fermentation of Transformed, Selected B. subtilis Cells

(89) Bacillus subtilis cells transformed with pBS5 plasmid and selected on IPTG gradient were cultured in a 20 L fermenter in 5 L of MM++ medium and glucose or saccharose as carbon source.

(90) IPTG was added as inductor after the start of fermentation.

(91) In the following, some fermentation processes for the production of HA having different weight average molecular weights are illustrated, said processes mainly differing because of: the starting source of carbon; the added feed (glucose or saccharose), activated about 7 hours after induction with IPTG 0.4-0.5 mM; the occurred fermentation time and therefore the final cells mass obtained; the temperature of fermentation (the temperature of fermentation can be established in a range between 20 C. and 38 C.).

Example 8A

Production of HA Having a Weight Average MW Comprised in the Range of 100-500 KD

(92) The bacterial strain B. Subtilis 1012, transfected with the plasmid pBS5 selected in IPTG gradient as described in Example 7, was used.

(93) Procedure: a single colony resistant to IPTG was inoculated into 5 ml of sterile LB medium containing 10 M of chloramphenicol, 10 M of neomycin and 0.05 mM of IPTG. The culture was grown at 37 C., under stirring at 200 rpm.

(94) After 8 hours, 50 l of this culture were inoculated into a flask containing 50 ml of the medium mentioned above (with 0.5 mM of IPTG), and it was made to grow under the same conditions described above.

(95) Subsequently, spent further 14-16 hours, 2 ml of this culture were inoculated into a flask containing 500 ml of the medium above, and it was made to grow under the same conditions until reaching a O.D..sup.600nm of 0.6-0.8.

(96) 500 ml of the culture thus obtained were then inoculated in the fermenter and the fermentation conditions involved maintaining the culture under stirring at 1300 rpm, aeration with 10-12 liters of air/min, a temperature of 37 C. and a pH of 6.9 to 7.1. The initial source of carbon was 1% glucose.

(97) After 6 hours of fermentation, a 2% glucose supply was added. At 24 hours of fermentation, IPTG was added to a final concentration of 0.4 mM; this induction proceeded for 6 hours; at the end, 10% glucose was added in stages.

(98) At the end of fermentation (130 hours), the bacterial culture was discharged and centrifuged at 7500 rpm at 8 C. for 20 minutes.

(99) The fermentation broth thus obtained, clarified as free of the cellular component, was analyzed to determine the concentration of HA with the carbazole method (Bitter and Muir, 1962, Anal. Biochem. 4:330-334).

(100) Results: The analysis resulted in a concentration of HA of 7.5 g/l.

(101) Determination of weight average molecular weight MW:

(102) For its analysis it was used the method of the intrinsic viscosity (as described in Terbojevich et al., Carbohydr. Res. 1986, 363-377, incorporated herein by reference).

(103) Results: the analyzed HA sample showed a weight average molecular weight MW in the range of 200-400 KD.

(104) Culture media used:

(105) LB broth (Miller), pH 7

(106) MM++ (Minimal Medium Bs), containing per liter:

(107) 5 g NH.sub.4Cl; 1 g NH.sub.4NO.sub.3; 3 g K.sub.2HPO.sub.4; 1 g KH.sub.2PO.sub.4; 1 g Na.sub.2SO.sub.4

(108) to the sterile media they were added 100 ml of a sterile solution containing:

(109) 0.1 g MgSO.sub.4. 7H.sub.2O; 0.005 g CaCl.sub.2.2H.sub.2O; 2 ml biotine solution (biotine solution 1 mg/l); 1 ml Fe solution (FeCl.sub.3 solution 0.2M); yeast extract 5 g/l, 0.01% Hydrolyzed Casein; uracil 5 mg/l, DL-tryptophan 5 mg/l; Histidine 400 g/l; Arginine 400 g/l; glucose solution (1% per liter).

Example 8B

Production of HA Having a Weight Average MW Comprised in the Range of 500-1000 KD

(110) The bacterial strain B. Subtilis WB800N, transfected with the plasmid pBS5 selected in IPTG gradient as described in Example 7, was used.

(111) Procedure: a single colony resistant to IPTG was treated as above disclosed according to example 8a. The initial source of carbon was saccharose at 2%. The fermentation conditions involved maintaining the culture under stirring at 600 rpm, aeration with 22-24 liters of air/min, a temperature of 37 C. and a pH of 6.9 to 7.1.

(112) After 6 hours of fermentation, IPTG was added to a final concentration of 0.4 mM; this induction proceeded for about 4 hours; at the end, 3% saccharose was added in stages, monitoring its concentration in the culture up to the end of fermentation (ended after 62 hours).

(113) The culture media used for the fermentation were those disclosed according to example 8a.

(114) At the end of the process, the fermentation broth was analyzed to determine the concentration of HA with the carbazole method.

(115) Results: the analysis resulted in a concentration of HA of 4.0 g/l.

(116) Determination of weight average molecular weight MW:

(117) For its analysis it was used the method of the intrinsic viscosity as indicated in the previous example 8a.

(118) Results: the analyzed HA sample showed a weight average molecular weight MW in the range of 550-800 KD.

Example 8C

Production of HA Having a Weight Average MW Comprised in the Range of 1106-2106 D

(119) The bacterial strain B. Subtilis 1012, transfected with the plasmid pBS5 selected in IPTG gradient as described in Example 7, was used.

(120) Procedure: a single colony resistant to IPTG was treated as above disclosed according to example 8a. The initial source of carbon was saccharose at 2%: in this example the further supply was glucose (further experimental tests showed that it can be substituted with equal or lower amounts of saccharose). The fermentation conditions were the same as those used in example 8a, but the fermentation temperature was of 30 C.

(121) The culture media used for the fermentation were those disclosed according to example 8a.

(122) Cell mass development was of 30 g/l after 20 hours.

(123) At the end of the process (ended after 35 hours), the fermentation broth was analyzed to determine the concentration of HA with the carbazole method.

(124) Results: the analysis resulted in a concentration of HA of 3.3 g/l.

(125) Determination of weight average molecular weight MW:

(126) For its analysis it was used the method of the intrinsic viscosity as indicated in the previous example 8a.

(127) Results: the analyzed HA sample showed a weight average molecular weight MW in the range of 1.510.sup.6-210.sup.6 D.

(128) The system engineered in B. subtilis is inducible, so the fermentation process can be continued by stimulating the production of HA to obtain the desired weight average molecular weight MW; fermentation times between 80 and 160 hours result in a medium-low weight average molecular weight MW, comprised in the range between 100-500 KD, fermentation times between 40 and 80 hours result in a weight average molecular weight in the range between 500-1000 KD, fermentation times between 12 and 40 hours result in a weight average molecular weight MW in the range 110.sup.6-210.sup.6 D. With the experiments and the results obtained above, the Applicant has demonstrated to have perfected a system of production of HA in B. subtilis by plasmid vectors by: engineering of 2 genes (or 4 genes) plasmid vectors for the synthesis of enzymes needed for the production of said polysaccharide, whose gene control is placed under the control of inducible promoter Pgrac; perfecting a system of selection of these transfected strains of B. subtilis, for the production of stable, viable, replicating and HA secreting strains; creating an inducible system of HA production, thus controllable both in order to obtain high concentrations of HA and for the production of said polysaccharide at different weight average molecular weight MW.