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EP-4741416-A1 - METHOD FOR PRODUCING A TRYPSIN-LIKE PROTEASE IN BACILLUS

EP4741416A1EP 4741416 A1EP4741416 A1EP 4741416A1EP-4741416-A1

Abstract

The invention relates to a method for producing a trypsin-like protease in Bacillus, the method comprising expressing in a Bacillus host cell a gene coding for a preproenzyme comprising or consisting of (i) a trypsin-like protease having a sequence identity of at least 70% compared to the wild-type trypsin-like protease from the fungus Fusarium oxysporum, and (ii) preferably, a prosequence located upstream of the trypsin-like protease and having at its C-terminus a basic amino acid residue, and (iii) a bacterial secretion signal sequence located upstream of the trypsin-like protease and of the optionally present prosequence, wherein the bacterial secretion signal sequence is capable of directing secretion in a Bacillus host cell to a culture medium, thereby providing a culture medium comprising the mature trypsin-like protease.

Inventors

  • RZEZNICKA, Kamila
  • HOFFMANN, GREGOR

Assignees

  • Kerry Group Services International Ltd

Dates

Publication Date
20260513
Application Date
20241111

Claims (15)

  1. A method for producing a trypsin-like protease in Bacillus, the method comprising the steps of (a) expressing in a Bacillus host cell a gene coding for a preproenzyme comprising or consisting of - a trypsin-like protease having a sequence identity of at least 70% compared to SEQ ID NO: 1; - a prosequence located upstream of the trypsin-like protease and having at its C-terminus a basic amino acid residue, preferably K or R, more preferably wherein the prosequence consists of an amino acid sequence having a sequence identity of at least 70% compared to any of SEQ ID NO:7 to SEQ ID NO:37; - a bacterial secretion signal sequence located upstream of the trypsin-like protease and of the optionally present prosequence, wherein the bacterial secretion signal sequence is capable of directing secretion in a Bacillus host cell to a culture medium, preferably wherein the bacterial secretion signal sequence originates from Bacillus; thereby providing a culture medium comprising the mature trypsin-like protease; (b) optionally, working-up the cell culture obtained in step (a) thereby providing a solid cell culture residue and a liquid cell culture supernatant; (c) optionally, purifying the mature trypsin-like protease from the cell culture supernatant; (d) optionally, formulating the mature trypsin-like protease; and (e) optionally, packaging the purified mature trypsin-like protease.
  2. The method according to claim 1, wherein the prosequence has at its N-terminus an aliphatic amino acid residue, preferably A.
  3. The method according to any of the preceding claims, wherein the bacterial secretion signal sequence originates from a species within the genus Bacillus selected from the group consisting of B. amyloliquefaciens, B. paralicheniformis, B. aerius, B. aerophilus B. altitudinis, B. alvei, B. anthracis, B. aquimaris, B. atrophaeus, B. badius, B. boroniphilus, B. butanolivorans, B. circulans, B. coagulans, B. coahuilensis, B. cohnii, B. firmus, B. globisporus, B. horikoshii, B. hwajinpoensis, B. idriensi, B. infantis, B. laterosporus, B. luciferensis, B. mojavensis, B. muralis, B. mycoides, B. pasteurii, B. polymyxa, B. pseudofirmus, B. pseudomycoides, B. safensis, B. selenatarsenatis, B. shackletonii, B. simplex, B. sonorensis, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis subsp. spizizenii, B. subtilis subsp. subtilis, B. tequilensis, B. thermoleovorans, B. thuringiensis, B. vallismortis, B. velezensis, B. vietnamensis, and B. vireti; preferably B. amyloliquefaciens, B. brevis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, and B. subtilis.
  4. The method according to any of the preceding claims, wherein the bacterial secretion signal sequence is the secretion signal sequence from secretory Bacillus proteins selected from the group consisting of YjfA, YfhK, Csn, LytD, Bpr, WapA, BglC/EglS, LytB, LipA/EstA, YckD, YbdN, YobB, YhfM, BglS, YjdB, YbbE, GlpQ, SacC, YurI/Bsn, PhoB, YdjM, AbnA, YwjE/ClsB, YqgA, LipB/EstB, FliZ, DacB, SacB, YrvJ, YlaE, YqxI, NprB, YbfO, YlqB, SpoIID, YwmC, YvbX, YkvV/StoA, XynA, YbbC, YkvT, NprE, YolC, YqzG, YvcE/CwlO, YkwD, YqxM, Mpr, TasA, YwmD, DacF, LytF, YjiA, PbpD, PelB, SpoIIQ, CccA, CitH/mdh, AspB, YlbL, YoqH, YpbG, YpcP/ExoA, YpuD, YvpB, YwcI, YweA, YwgB, YwmB, YwoF, YwsB, YwtF, Pel, PenP, LytC, YfkN, YngK, YwqC, RpmG, YojL/CwlS, YncM, PhrK, Vpr, PhrC, YfkD, YolA, YoaW, YlxY, SleB, YxiT, NucB, YpuA, YwaD, YpjP, WprA, YjcM, MotB, YdhT/GmuG, LytE, PhrF, YlxW, YobV, YocH, YodV/Phy, YoqM, YraJ, YuaB, YusW, YvgO, YvnB, YxiA/AbnB, CwlD, DltD, FliL, LytR, MreC, PbpB, PhoA, SpoIIP, SpoIIR, YrrR/PbpI, YrrS, YveB/LevB, LytH, YbdG, PbpX, YddT, YjcN, YolIBdbA, YqzC, YlxF, YndA, YkoJ, YwtD/PgdS, YdbK, YbbR, YknX, YnzA/TatAC, YpmS, YvgVBdbD, AmyE, YybN, YwfM, YhdC, YxaK/YxaL, YopL, YhjA, PhrG, YvpA/PelC and PhrA; preferably PhoA, YncM, YqgA, YjfA, YfkD, StoA, YdjM, DacF, Vpr, YbfO, AbnA, LytC, PgdS, YckD, YpuA, YuaB, YvnB, YxaL YoqH and YbdN.
  5. The method according to any of the preceding claims, wherein the bacterial secretion signal sequence consists of an amino acid sequence having a sequence identity of at least 70% compared to any of SEQ ID NO:39 to SEQ ID NO:206, preferably to any of SEQ ID NO:39 to SEQ ID NO:75, more preferably to any of the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:68, SEQ ID NO:70 and SEQ ID NO:74, yet more preferably to any of the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52 and SEQ ID NO:56.
  6. The method according to any of the preceding claims, wherein step (a) involves processing the preproenzyme into a proenzyme upon expression and secretion, such that: (i) the bacterial secretion signal sequence is cleaved off the preproenzyme during or after secretion from the Bacillus host cell; and (ii) the resulting proenzyme is located in the culture medium and comprises the prosequence and the trypsin-like protease.
  7. The method according to any of the preceding claims, wherein step (a) involves autocatalytically activating the proenzyme such that the prosequence is cleaved off from the trypsin-like protease thereby releasing the mature trypsin-like protease having trypsin-like activity.
  8. The method according to any of the preceding claims, wherein the autocatalytic activation takes places in the culture medium without rebuffering steps.
  9. The method according to any of the preceding claims, wherein the mature trypsin-like protease exhibits - an increased volumetric activity; and/or - an increased volumetric activity recoverable; compared to mature the trypsin-like protease prepared by the method according to claim 1 wherein the prosequence consists of SEQ ID NO:2 (wild-type).
  10. The method according to any of the preceding claims, wherein the host cell is selected from the group consisting of B. amyloliquefaciens, B. paralicheniformis, B. aerius, B. aerophilus B. altitudinis, B. alvei, B. anthracis, B. aquimaris, B. atrophaeus, B. badius, B. boroniphilus, B. butanolivorans, B. circulans, B. coagulans, B. coahuilensis, B. cohnii, B. firmus, B. globisporus, B. horikoshii, B. hwajinpoensis, B. idriensi, B. infantis, B. laterosporus, B. luciferensis, B. mojavensis, B. muralis, B. mycoides, B. pasteurii, B. polymyxa, B. pseudofirmus, B. pseudomycoides, B. safensis, B. selenatarsenatis, B. shackletonii, B. simplex, B. sonorensis, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis subsp. spizizenii, B. subtilis subsp. subtilis, B. tequilensis, B. thermoleovorans, B. thuringiensis, B. vallismortis, B. velezensis, B. vietnamensis, and B. vireti; preferably B. amyloliquefaciens, B. brevis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, and B. subtilis; more preferably B. amyloliquefaciens.
  11. The method according to any of the preceding claims, wherein step (a) involves growing the host cell at a temperature range of about 28°C to about 37°C, preferably at about 30°C and in a medium with a pH range of about 6.0 to about 8.5, preferably with a pH range of about 6.3 to about 7.5.
  12. A preproenzyme, preferably as defined in any of claims 1 to 9, comprising or consisting of - a trypsin-like protease having a sequence identity of at least 70% compared to SEQ ID NO:1; - preferably, a prosequence located upstream of the trypsin-like protease and having at its C-terminus a basic amino acid residue, more preferably K or R; even more preferably wherein the prosequence consists of an amino acid sequence having a sequence identity of at least 70% compared to any of SEQ ID NO:7 to SEQ ID NO:37, yet more preferably compared to any of SEQ ID NO:7 to SEQ ID NO:26; and - a bacterial secretion signal sequence located upstream of the prosequence, wherein the bacterial secretion signal sequence is capable directing secretion in a Bacillus host cell, preferably which originates from Bacillus.
  13. An expression vector comprising a DNA sequence coding for the preproenzyme according to claim 12.
  14. A host cell comprising a DNA sequence coding for the preproenzyme according claim 12.
  15. The host cell according to claim 14, wherein the host cell is a bacterium; preferably a gram-positive bacterium; more preferably a species selected from the genus Bacillus; still more preferably selected from the group consisting of B. amyloliquefaciens, B. paralicheniformis, B. aerius, B. aerophilus B. altitudinis, B. alvei, B. anthracis, B. aquimaris, B. atrophaeus, B. badius, B. boroniphilus, B. butanolivorans, B. circulans, B. coagulans, B. coahuilensis, B. cohnii, B. firmus, B. globisporus, B. horikoshii, B. hwajinpoensis, B. idriensi, B. infantis, B. laterosporus, B. luciferensis, B. mojavensis, B. muralis, B. mycoides, B. pasteurii, B. polymyxa, B. pseudofirmus, B. pseudomycoides, B. safensis, B. selenatarsenatis, B. shackletonii, B. simplex, B. sonorensis, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. subtilis subsp. spizizenii, B. subtilis subsp. subtilis, B. tequilensis, B. thermoleovorans, B. thuringiensis, B. vallismortis, B. velezensis, B. vietnamensis, and B. vireti; yet more preferably B. amyloliquefaciens, B. brevis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, and B. subtilis; even more preferably B. amyloliquefaciens.

Description

The invention relates to a method for producing a trypsin-like protease in Bacillus, the method comprising the steps of (a) expressing in a Bacillus host cell a gene coding for a preproenzyme comprising or consisting of a trypsin-like protease having a sequence identity of at least 70% compared to SEQ ID NO:1, which is the wild-type trypsin-like protease from the fungus Fusarium oxysporum, and preferably, a prosequence located upstream of the trypsin-like protease and having at its C-terminus a basic amino acid residue, more preferably K or R, and a bacterial secretion signal sequence located upstream of the trypsin-like protease and of the optionally present prosequence, wherein the bacterial secretion signal sequence is capable of directing secretion in a Bacillus host cell to a culture medium, preferably wherein the bacterial secretion signal sequence originates from Bacillus, thereby providing a culture medium comprising the mature trypsin-like protease. Optional steps may include (b) working-up the cell culture obtained in step (a) thereby providing a solid cell culture residue and a liquid cell culture supernatant, optionally (c) purifying the mature trypsin-like protease from the cell culture supernatant, optionally (d) formulating the mature trypsin-like protease, and optionally (e) packaging the purified mature trypsin-like protease. The invention further relates to a preproenzyme, preferably as defined according to any embodiment of the first aspect of the invention. The invention also relates to an expression vector comprising a DNA sequence coding for the preproenzyme according to the second aspect of the invention. Moreover, the invention relates to a host cell comprising a DNA sequence coding for the preproenzyme according to the second aspect of the invention or the third aspect of the invention. Trypsin (EC 3.4.21.4) is a serine protease with stringent cleavage specificity for the carboxyl termini of arginine and lysine and is integral to numerous biological functions, e.g., proteolysis in digestion, signal transduction and immune response. In its natural form, trypsin is synthesized as an inactive precursor, the preproenzyme, having an N-terminal secretion signal peptide, followed by a short prosequence. The secretion signal peptide is removed upon extracellular trafficking and activation of the proenzyme (trypsinogen), the second protease precursor, is achieved by proteolytic removal of the prosequence, catalyzed e.g., by a second enzyme like an enterokinase or by trypsin through autoactivation. Trypsin or trypsin-like proteases are found in various different organisms, including mammals, birds, fishes, amphibians, primitive vertebrates, insects, fungi, and bacteria. Up to date, trypsin has been utilized in many industrial applications, e.g., for food processing, leather bating, detergents, or pharmaceutical applications like the manufacturing of insulin, vaccines or viral vectors. The commercial production of trypsin usually involves animal sources from bovine or porcine origin. However, the usage of animal-derived trypsin harbors the potential risk of contamination with infectious agents like viruses or other pathogens, which cannot be neglected when used in the production of medicinal products like vaccines or viral vectors. Recombinant trypsin eliminates this risk, making it a safer alternative for use in biopharmaceutical manufacturing. Furthermore, the activity and quality of recombinant trypsin can be more consistent as it is not subject to the same biological variability as animal-derived trypsin. The use of recombinant trypsin additionally avoids ethical issues associated with the use of animal products. Further benefits of some forms of recombinant trypsin are advantages in room-temperature stability, an enhanced performance, or avoiding the need of protease inhibitors. In summary, recombinant trypsin offers a safer, more consistent, and potentially more efficient alternative to animal-derived forms, why it is gradually being adopted in various industrial processes. The trypsin of the fungus Fusarium oxysporum (FOT) is a serine protease with similar characteristics to mammalian trypsin and thus appears to be a suitable target for the recombinant production of a non-animal derived trypsin variant. However, it has a distinct prosequence, which does not enable autoactivation and is naturally hydrolyzed by a specific extracellular Fusarium aspartyl protease. Recombinant expression of FOT has been shown in different expression hosts, e.g., in Aspergillus oryzae, Pichia pastoris and Escherichia coli. However, when expressed in a non-Fusarium host, low yields and difficulties with trypsin-activation have been reported. Therefore, other expression hosts have to be considered to provide an industrially advantageous production of trypsin. An increasingly preferred host for the expression of recombinant proteins with medical, biotechnological, and industrial value is Bacillus, e.g., Bacillu