Hesselink, Paulus Gerhardus Maria

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   University of GroningenSterol side chain cleavage by mycobacterium.Hesselink, Paulus Gerhardus MariaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1989Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Hesselink, P. G. M. (1989). Sterol side chain cleavage by mycobacterium. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-202211SUMMARY AND CONCLUDING REMARKSMicrobial sterol side chain cleavageThe selective microbial sterol side chain cleavage is an attractive way of convertingcheap and abundantly available sterols inlo AD and ADD. These two compounds areexcellent intermediates of high added value in the synthesis of nearly all steroidhormone drugs. Sterols of choice are the zoosterol cholesterol and phytosterols such asB-sitosterol, campesterol and stigmasterol. However, the microbial side chain cleavageis characterized by some fundamental problems. Most organisms carry out sidereactions such as the sterol nucleus degradation because they use sterols as a source ofcarbon. Therefore, there is a requirement for inhibitors of undesirable side reactions orfor stable mutants lacking the genes responsible for such reactions. The majority of theside chain degrading microorganisms have low growth rates and a tendency toflocculate and float or adhere to vessel walls. Moreover, substrates and products areonly very poorly soluble in aqueous media leading to hardly accessible substrates and tomixed crystal precipiradon. Conversion in the presence of organic solvents whichincrease the reactant solubilities is often impossible as the microorganisms are notcompatible with these reaction systems. As a consequence, the microbial sterol sidechain cleavage often results in low conversion rates and poor yields.In tiis thesis, I described the investigations into the conversion of cholesterol toAD(D) by Mycobacterium sp. NRRL-B 3683 side chain cleavage. This bacterium is aUV-mutagenized organism with reduced nucleus degradation activity. The aim of theproject was the characterization and the improvement of the AD(D) production processby this strain in a broad sense. Therefore, the microbiology, biochemistry, genetics andthe reactor performance of this process were studied.Physiology and biochemistryMycobacterium sp. NRRL-B 3683 is a Gram positive rod shaped bacterium of ca. 0.7 x1.7 pm. The bacterium is prototrophic, strictly aerobic and contains a yellow pigment.Cells grow mainly as free cells in liquid media. Optimum $owth conditions in a 1 Ifermentor include pH = 7, T -- 32 "C and a stirrer speed of 200-300 rpm with a sixbladed stirrer of 5.3 cm diameter. Taxonomically, Mycobacterium sp. NRRL-B 3683resembled M. phlei and to a minor extent M. smegmatis and M. vacaae. However, noabsolute correspondence with these genera was observed. Mycobacterium sp. NRRL-B3683 showed remarkable metabolic versatility, being able to use many organicEspecially succinate was found to support rapid cell grcwth (to = 3.5-+.0 h) in themineral E2-medium. Steroids did not support cell growth, with the exception ofcholesterol which was metabolized only slowly.Mycobacterium sp. NRRL-B 3683 was able to convert a variety of steroids toAD(D) with molar efficiencies ranging from 1 Vo with progesterone to 25-30 Vo withcholesterol or 47 Vo with testosterone as a substrate. Suitable substrates included varioussterols, A5- and Aq-cholestenone, lithocholate, As-cholenic acid, BNC and testosterone.A definite preference for the 3-hydroxy-As structure and the relatively simply structuredcholesterol side chain was observed. The steroid carboxylic acids were poorly convertedwhereas the C-21 steroids pregnenolone and progresterone were hardly degraded. Theratio of AD/ADD shifted towards more ADD at prolonged incubation times whencholesterol was converted.The highest specific cholesterol side chain cleavage activity was observed during(early) exponential growth whereas the activity of the stationary phase cells was only1-5 Vo of these values. In contrast, the steroid nucleus was mainly degraded bystationary phase cells. Consequently, a nearly threefold increase in AD(D) yield couldbe realized by keeping cells in their growth phase during the entire cholesterolfermentation by using a mixture of succinate and glucose as carbon sources. Cholesterolwas needed to induce nucleus degradation; the presence of AD or ADD only did notresult in any steroid losses. AD and ADD severely inhibited Mycobacterium cell growthrates while leaving ttre specific side chain cleavage activity unaffected.Suppression of side reactionsInhibitors of one of the nucleus degrading enzymes are widely applied in order tosuppress the undesirable nucleus degradation. Attention has largely been focusscd onthe inhibition of the 9o-hydroxylase activity. This enzyme complex contains FeSclusters within an electron transport chain and is therefore susceptible to Fe2+ chelatingagents, Fe2+ replacing metal ions, sulphydryl reagents and redox dyes. In contrast, wedeveloped inhibitors of cholesterol oxidase, another enzyme involved in the steroidnucleus degradation. According to our interpretation of literature data and to some ofour experiments, this reaction does not necessarily precede the process of sterol sidechain cleavage. Several dimethylmorpholine fungicides were tested as microbialcholesterol oxidase inhibitors as we expected an analogy with the inhibition of sterol[8+7-isemsrase and C-14 reductase in higher plants and fungi by tiese compounds. Wefound that fenpropimorph, ridemorph and to a lesser extent fenpropidin were effectiveinhibitors of the isomerase activity of the cholesterol oxidases from Nocardiaery thropolis, P seudomonas testo steroni, S treptomye s sp. and S c hizo p hyllwn c ommunein vitro. These enzymes differed in reaction mechanism, steroid substrate specificity,cofactor required, optimal assay conditions and resistance to isopropanol, which was156used in enzyme assays to solubilize steroids and inhibitors. Inhibitor effectiveness withN. erythropolis cholesterol oxidase was geatly influenced by structural features. Themost important of these included a tertiary nitrogen atom able to become amorpholinium ion and the presence of the two morpholine methyl groups, preferably incis-configuration. Inhibition of cholesterol oxidase tn vivo was also observed withArthrobacter simplex, Corynebacterium sp. ATCC 31458, N. erythropolis and N.restricta, next to toxic side effects of the inhibitors on these strains. AD(D) accumulatedmaximally to 5 and 25 Vo molar conversion in experiments with N. erythropolis andMycobacteriwn sp. NRRL-B 3683, respectively, suggesting that the cholesterol sidechain cleavage itself was not inhibited by fenpropimorph or tridemorph. In addition,steroid nucleus degradation was completely inhibited. When no dimethylmorpholineswere present, only trace amounts of AD(D) were formed in cholesterol fermentations byN. erythropolts and nucleus degadarion caused severe steroid losses. In Gram negativestrains, cell toxicity was absent, implying that application in steroid transformations bybacteria like engineered Pseudomoncr strains might be feasible. A better separation ofcell toxicity and cholesterol oxidase inhibition is expected to further increase theapplicability of dimethylmorpholines in several other steroid transformations. More-over, the resemblance between microbial cholesterol oxidase inhibition and the inhibi-tion of sterol biosynthesis in higher plants and fungi was demonstrated. This mightresult in rapid and convenient fungicide screening systems for agronomic purposeswhen compared to the laborious and expensive in yivo test systems used at present.Optimization of reactor performanceSteroids such as cholesterol have low solubilities and dissolution rates in aqueousmedia, resulting in low reactant ransport rates to and from cells and in suboptimalbiocatalyst performance. As the use of organic solvents was found to be incompatiblewith cholesterol side chain cleavage by Mycobacteriurn sp. NRRL-B 3683, weinvestigated the effects of the addition of several detergents and clathrating agen8 toaqueous fermentation broths.A l.l - 3.0 fold increase of the specific side chain cleavage activity was observedwhen cyclodextrins were used in the optimized I : 2 molar ratio of sterol : cyclodextrin.For cholesterol, addition of B-cyclodextrin conhining 6 glucose residues was found tobe optimal for AD(D) production, whereas the 7 glucose units containing y-cyclodextrinwas the best clathrant. for B-sitosterol, campesterol and Aq-cholestenone. The enhance-ment of steroid solubilities and transport rates was thought to be fully responsible forthe increase in AD(D) production as cell growth was not affected by cyclodexnins.Moreover, nucleus degradation was decreased, resulting in over 85 7o molar conversionof sterols to AD(D) after 140 h of incubation, in conEast o 35 Vo molu conversion inthe absence of cyclodextrins.Ircithins were effective enhancers of sterol side chain degradation by Mycobac-teriurn. Especially egg-yolk lecithin increased the apparent solubilities of cholesteroland sitosterol and their transport rates to the biocaralyst. Thus, a rapid degradation of thecholesterol side chain and the sterol nucleus by Mycobacterium were observed. Thisresuhed in a 1.5 - 2.0 fold increase in AD(D) yields. Fatty acid analysis indicated thatthe combined presence of a saturated and a mono-unsaturated fatty acid, preferablyconsisting of 12 or more carbon atoms, in the single lecithin molecules was optimal forthis purpose.A comparison of the stimulating effects on AD(D) production of some lecithins andvarious synthetic detergents including several Tweens, Spans and Brijs indicated thatthe type of polar head group of these detergents was more important than the length ofthe fatty acyl side chains. However, a minimal length of 16 or 12 carbon atoms wasessential for AD(D) yields over 70 Vo wirh synthetic detergents and lecithins, respec-tively.GeneticsAlthough Mycobacterium sp. NRRL-B 3683 degrades the cholesterol side chain, thissrain has some undesirable characteristics such as low growth rates, sensitivity toproduct inhibition and incompatibility with organic solvents. Therefore, the total DNAwas cloned in the broad host range cosmid pLAFRI and transferred to E. coli. Thisresulted in a gene bank of over 32,000 recombinants. When the pLAFRI constructswere introduced in P. rcstosteroni,22 recombinants out of 2000 were able to grow oncholesterol as the sole source of carbon. During cholesterol fermentation, 8 of these 22P. testosteroni recombinants produced an unidentified product, most probably ahydroxylated cholestenone, and 3 strains showed rapid nucleus degradation. Many ofthe 22 rerombinant plasmids had common Mycobacterizm DNA inserts. A provisionalmap could be constructed of a 12.45 kb total common insert which presumablycontained the genes responsible for growth on cholesterol, rapid product formation andnucleus degradation.Concluding remarksThis thesis describes the results of physiological, biochemical, genetic and engineeringinvestigations in the field of microbial sterol side chain cleavage. New insights weredeveloped and progress was made in the characterization and handling of Myco-bacterium sp. NRRL-B 3683 as well as in its application as a biocaralyst in detergent orclathrant containing aqueous media. This resulted in much faster conversion rates ofcholesterol or sir.,osterol to AD(D) and in considerably higher yields of rhis product. Anovel class of inhibitors of the cholesterol oxidase activities was developed and proved158to be effective with cholesterol oxidases from a variety of sources, both in vitro and invivo. This finding has potential applications in steroid biotransformations and inagronomic fungicide research. Finally, genes encoding steroid transforming enzymes inMycobacteriurn were cloned and successfully expressed in Pseudomanas. Thisdevelopment of genetically engineered reaclor compatible strains seems to be the mostpromising option for further improvement of the microbial sterol side chain cleavageand of many other steroid biotransformations. From the industrial point of view, the useof stable mutants which catalyse no side reactions and are compatible with organicsolvents is to be preferred over the use of enzyme inhibiors or biotransformations indetergent containing aqueous media. The use of suitable organic solvents whichincrease reactant solubilities further than the addition of detergents to aqueous reactionsystems is likely to result in higher biotransformation rates. Moreover, purification ofhydrophobic products from organic solvents is generally much easier than from aqueousemulsions. Several PseudomonoJ strains including P. oleovorans are well known fortheir capacity to carry out biotransformations of hydrophobic compounds in reactionsystems containing over 99 Ea organic solvent. Information on their genetics,metabolism, regulation and handling as biocatalysts is rapidly becoming available.Therefore, application of the information described in this thesis in combination withthus genetically engineered strains should be interesting and worth while, both from ascientific and an economic point of view.

Sterol side chain cleavage by mycobacterium.

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Sterol side chain cleavage by mycobacterium.

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