(12)UK Patent Application (1S1GB ,„>2577037 ,,3,A (43) Date of A Publication 18.03.2020 (21) Application No: 1812997.3 (51) INT CL: C12N 15/52 (2006.01) C12P 33/02 (2006.01) (22) Date of Filing: 09.08.2018 C12R 1/32 (2006.01) C12R 1/365 (2006.01) (56) Documents Cited: GB 2102429 A EP 3112472 A (71) Applicant(s): WO 2001/031050 A US 4345029 A Cambrex Karlskoga AB US 4320195 A (Incorporated in Sweden) Appl Environ Microbiol, published online 4 May 2018, S-691 85 Karlskoga, Sweden Liu et al, "Characterization and engineering of 3- ketosteroid 9a-hydroxylases in Mycobacterium Rijksuniversiteit Groningen neoarum ATCC 25795 for the development of (Incorporated in the Netherlands) androst-1,4- diene3,17-dione and 9a-hydroxy- Broerstraat 5, 9712 CP Groningen, Netherlands androst-4-ene-3,17-dione-producing strains" Appl Environ Microbiol, Vol 77 (2011), Wilbrink et al, (72) Inventor(s): "FadD19 of Rhodococcus rhodochrous DMS43269, a Jonathan Knight steroid-coenzyme A ligase essential for degradation Cecilia Kvarnstrom Branneby of C-24 branched sterol side chains", pp 4455-4464 Lubbert Dijkhuizen FEMS Microbiol Letts, Vol 205 (2001), van der Geize et Janet Maria Petrusma al, "Unmarked gene deletion mutagenesis of kstD, Laura Fernandez De Las Heras encoding 3-ketosteroid deltal-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as a counter-selectable marker", pp 197-202 (74) Agent and/or Address for Service: J Steroid Biochem Mol Biol, Vol 172 (2017), Guevara Potter Clarkson LLP et al, "Functional characterization of 3-ketosteroid 9a- The Belgrave Centre, Talbot Street, NOTTINGHAM, hydroxylases in Rhodococcus ruber strain chol-4", NG1 5GG, United Kingdom pp 176-187 J Bacteriol, Vol 194 (2012), Mohn et al, "Gene cluster encoding cholate catabolism in Rhodococcus spp." pp 6712-6719 Microb Cell Fact, Vol 17 (May 2018), Zhang et al, "Identification, function, and application of 3- ketosteroid delta 1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons", pp 77 J Steroid Biochem Mol Biol, Vol 138 (2013), Bragin et al, "Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains", pp 41-53 (58) Field of Search: INT CLC12N, C12P, C12R Other: EPODOC, WPI, Patent Fulltext, BIOSIS, MEDLINE (54) Title of the Invention: Genetically-modified bacteria and uses thereof Abstract Title: Bacteria modified in the steroid metabolism pathway GB (57) A genetically-modified bacterium, for example of the class Actinobacteria, and the use ofsuch a bacterium in the bioconversion of a steroidal substrate into a steroidal product of interest is disclosed. The bacterium is blocked in the steroid metabolism pathway prior to the degradation of the polycyclic ring system, and at the steroid side chain degradation pathway. In a preferred embodiment the genetic modification comprises inactivation of the genes 2577037 kshA1, kshA2, kshA3, kshA4 and kshA5. A The provisions of paragraph 6 and 7 of Schedule 1 to the Patents Rules 2007 have effect in respect of this application, restricting availability of samples of specified biological material to experts in accordance with those provisions. At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy. side chain, activation 4 3~oxo~9~hydroxy~4~cholenic acid 3-oxo-7,9-dihydroxy-4-choienic aci 29 01 20 GM est® ! (Pwe ί ( P io S fetad) or 7*H^t hcfeteral PIC 1 <R FJV3. 0 29 01 20 souepunqe 9Appy θ θ θ C:> CZ5 θ Ο ΟΟ CD <Ν 93uepunqv9Appy Qouepimqy θλι|Β|Θ^ eouepunqv 9λι|Β|θ^ 29 01 20 souBpunqyQAppy 10/21 ! Λ HU.Γ “ Ίϋ 11/21 29 01 20 FIG. 12 29 01 20 nw 29 01 20 eouepunqv θΛμθιθ^ 29 01 20 nw 3-oxo-7-hydroxy-4-cholenic acid co 20 01 29 CM CZ> O> OO i'-'— CO LO CO OsJ -y C^j nvni 20 81 OH 01 29 co lo o uo <o> lo cS lo o lo co cS co cS CO CO CO -^r CO CO CxJI CM ^-- 'c— ’ v™ nw 20 6l· ΌΗ 01 29 C2> LO cS UO O UO O LX'S kO cS UO O UO O co uo m co e*> c-si cn x— ! nvni 29 01 20 nw FIG. 20 CO O CO> O CO O O CO> O O CO O CZ> C21> C21") C2> ■^r c-si cz> CO CO Oxi CM nw 1 GENETICALLY-MODIFIED BACTERIA AND USES THEREOF The present invention relates to genetically-modified bacteria and the use of such bacteria in the bioconversion of steroidal substrates into steroidal compounds of interest. The 5 genetically-modified bacteria may be from the genera Rhodococcus or Mycobacterium. Steroids are a large and diverse class of organic compounds, with many essential functions in eukaryotic organisms. For example, naturally occurring steroids are involved in maintaining cell membrane fluidity, controlling functions of the male and female 10 reproductive systems and modulating inflammation. As signalling through steroid controlled pathways is important in a wide variety of processes, the ability to modulate these pathways using synthetically produced steroid drugs means they are an important class of pharmaceuticals. For example, corticosteroids 15 are used as anti-inflammatories for the treatment of conditions such as asthma and rheumatoid arthritis, synthetic steroid hormones are widely used as hormonal contraceptives and anabolic steroids can be used to increase muscle mass and athletic performance. 20 The synthesis of steroids for use as pharmaceuticals involves either semi-synthesis from natural sterol precursors or total synthesis from simpler organic molecules. Semi­ synthesis from sterol precursors such as cholesterol often involves the use of bacteria. The advantages of using bacteria to carry out these bioconversions are that the synthesis involves less steps and the reactions performed by the enzymes are 25 stereospecific, resulting in the production of the desired isomers without the need for protection and deprotection used in traditional chemical synthesis. The products of bacterial bioconversions can then be used as pharmaceuticals or as precursors for further chemical modification to produce the compound of interest. 30 Steroids naturally occur in both plant, animal and fungal species, and are produced by certain species of bacteria. Despite them only occurring naturally in only a few bacterial species, several bacterial species are able to metabolise sterol compounds as growth substrates. Examples of bacteria that can degrade sterol compounds include those from the genera Rhodococcus and Mycobacterium. 35 The bacterial sterol metabolism pathway involves progressive oxidation of the sterol side­ chain, and breakdown of the polycyclic ring system. The pathway of sterol side-chain 2 degradation in Rhodococcus has been previously investigated using mutant strains (Wilbrink et al, 2011. Applied and Environmental Microbiology, 77(13):4455-4464) and an overview of the cholesterol catabolic pathway is shown in Figure 1. It has now been found that bacterial species may be used for steroid compound production by genetic 5 modification to block the degradation pathway prior to breakdown of the polycyclic ring system and at various points in side-chain oxidation to allow accumulation of the steroidal compounds of interest in order to improve the yields obtained. In a first aspect, the invention provides a genetically-modified bacterium blocked in the 10 steroid metabolism pathway prior to degradation of the polycyclic steroid ring system, wherein the bacterium is disrupted in the steroid side-chain degradation pathway, and wherein the bacterium converts a steroidal substrate into a steroidal product of interest. By “steroid” or “steroidal” compounds we include the meaning of a class of natural or 15 synthetic organic compounds derived from the steroid core structure represented below (with lUPAC-approved ring lettering and atom numbering): 20 Steroidal compounds generally comprise four fused rings (three six-member cyclohexane rings (rings A, B and C above) and one five-member cyclopentane ring (ring D above)) but vary by the functional groups attached to that four-ring core and by the oxidation state of the rings. For example, sterols are a sub-group of steroidal compounds where one of the defining features is the presence of a hydroxy group (OH) at position 3 or the structure 25 shown above. The structure formed by the atoms labelled 20 to 27 (including positions 241 and 242) in the above diagram is referred to as the steroid side-chain. Non-limiting examples of steroids include: sterols, 3-oxo-4-cholenic acid, 3-oxo-chola-4,22-dien-24-oic acid, 3-oxo-7-hydroxy-4-cholenic acid, 3-oxo-9-hydroxy-4-cholenic acid, 3-oxo-7,9- dihydroxy-4-cholenic acid, 3-oxo-23,24-bisnor-4-cholene-22-oic acid (4-BNC), 3-oxo- 30 23,24-bisnor-1,4-choladiene-22-oic acid (1,4-BNC), 4-androstene-3,17-dione (AD), 1,4- androstadiene-3,17-dione (ADD), sex steroids (e.g. progesterone, testosterone, estradiol), 3 corticosteroids (e.g. cortisol), neurosteroids (e.g. DHEA and allopregnanolone), and secosteroids (e.g. ergocalciferol, cholecalciferol, and calcitriol). Non-limiting examples of steroidal compounds are also shown in Figure 4. 5 By “disrupted in the steroid side-chain degradation pathway” we include the meaning of a bacterium in which the normal degradation of the steroid side-chain is impaired. Normally, degradation of the steroid side-chain involves the initial cycle of side-chain activation followed by three successive cycles of β-oxidation (i.e. first, second, and third cycles of β- oxidation). In an unimpaired side-chain degradation pathway, the final product of the side­ 10 chain degradation steps is usually 4-androstene-3,17-dione (AD). Thus, a bacterium disrupted in the steroid side-chain degradation pathway will accumulate steroidal products that are upstream of the production of AD.