Aerobic Microbial Transformations of Sterols and , a personal Approach

Bernardo Servín-Massieu

CONTENTS

Overview

The following article has the purpose to publish a malicious bias to give the impression that the development of the industry in was only the contribution from alumni from the National School of Chemistry Sciences and the Chemistry Institute, both belonging to the National Autonomous University of Mexico in Mexico City promoted by the Mexican Chemistry Society.

Not at all, alumni from the National School of Biological Sciences from the National Polytechnic Institute in Mexico City made a substantial contribution to the aerobic microbial transformation of, initially steroid molecules and afterwards in the microbial transformation of sterols.

In the 1940’s of the XX century Russell E. Marker designed the transformation of to using wild yams collected in the Metlac ravine near the town of Orizaba in the state of Veracruz about 200 miles east Mexico City. It is known as Marker’s degradation being the kick-off of a new industry in Mexico.

These yams were from Dioscorea mexicana known by the local inhabitants as “cabeza de negro” negro’s head, a vine that grows wild in moist subtropical forest in Veracruz, Puebla, Chiapas and Oaxaca states, further along Marker discovered that “Barbasco” Dioscorea compositae and Dioscorea floribunda contained higher contents of diosgenin.

Marker consulted the Mexico City yellow pages after checking at the Hotel Geneve, London Street, Pink Zone and found an entry “Laboratorios Hormona, S.A.”, he called Federico Lehmann and agreed to meet.

It can be said that the steroid industry boom in Mexico was indirectly due to Adolph Hitler’s Nazism and Francisco Franco’s Fascism that respectively caused the Jewish diaspora in Germany and “refugiados” after the socialist civil war in Spain.

Laboratorios Hormona was founded by Federico Lehmann (Germany) and Emeric Somlo (Hungary) and in there team was Jose Erdos (hungary), Francisco Giral, E. Muñoz-Mena, A. Medinaveytia, J. Viró, L. Abramson from Spain, and C. Widmer, a Swiss citizen.

During the 1930s-1940s period the only sea port of entry from Europe to Mexico was Veracruz, founded by Hernán Cortés in 1519, via Havana Cuba.

Lehmann, Marker and Somlo founded a new company that was called ( from Synthesis and Mexico), after Marker left, S. Kauffman, G. Rosenkranz and J. Pataki joined Syntex and were able to reproduce Marker’s degradation, that was the beginning of Syntex boom. The operation was set in the outskirts of Mexico City in the Molino de Bezares borough on the road to Toluca. Additionaly the yams were processed in the Ojo de Agua plant in Ixtaczoquitlán, Ver. Near Orizaba, that later was merged by Schering AG Berlin and nowadays is part of Bayer Germany, called PROQUINA (Productos Químicos Naturales). Research:

After The Upjohn Company researchers in Kalamazoo, Mich. Discovered that molds could transform the steroid nucleus by microbial hydroxylating carbon 11, among other molds, species of genus Rhizopus could commercially perform in one step this reaction in replacement of the organic chemistry synthesis reactions that could be done in many steps and very small yields, Syntex, began exploring this and other reactions with a wide range of bacteria and mold genera.

At Syntex, S.A., Mexico City during the 1950-1960 period a microbial research team formed part of the Syntex Research Division in the outskirts of Mexico City. This group was leaded by Carlos Casas-Campillo, integrated by Fernando Esparza-García, Dora Balandrano, Alfonso Galarza, Ignacio Magaña-Plaza, José Ruiz-Herrera, Miguel Bautista-Susunaga, and several interns from which Bernardo Servín-Massieu and Luis Jiménez-Zamudio formed part.

About 50 patents were granted to this group in Mexico, USA, Canada, England, Holland, Japan, Switzerland, France and Germany. Microorganisms of several genera were studied: Panaeolus, Fusarium, Psilocybe, Arthrobacter (Corunebacterium, Mycobacterium, Rhizopus, Curvularia, Streptomyces, Arthrobotrys, Rhizobium, Fusarium, Streptomyces, Mycecoccus.

The following substrates were studied: Progesterone (21-hydroxylation); Progesterone as an antimicrobial agent; 19-norprogesterone (11-alpha hydroxylation); 21-21 dimethoxy progesterone antimicrobial properties;19-nortestorenone (1-2 dehydrogenation); Reichstein compound S (11-beta and 6-beta hydroxylation); Reichstein Compound (11-beta hydroxylation); Estrane derivatives (Hydroxylation).

The reader should consult Charney & Herzog for details.

At Searle de México, S.A. after Marker left Syntex, A group of researchers and engineers formed Productos Esteroides, S.A. (PESA), this company was merged by G.D. Searle of Skokie, Ill. A research group was formed originally led by Luis E. Miramontes-Cárdenas that initially did research on the microbial transformation of dioscin in barbasco yams and the production of glucose oxidase by Aspergillus niger.

Luis Miramontes left, and I took the microbiology research team leadership supervised by Robert P. Graber, and a change in scope towards the evaluation of Mycobacteria in the transformation of sterols to AD (4- Androstene-3,17-Dione) and ADD (1,4-Androstadiene-3,17-Dione) as start-up compounds for the synthesis of Spironolactone (7-(Acetylthio)-17-hydroxy-3-oxo-pregn-4-ene-21-carboxylic acid), a major market drug by Searle as Aldactone©®.

Among others, this group was formed by Javier Hebrero-Rodríguez, Gilberto García-Gutiérrez, Alejandro Canale-Guerrero, Felipe M. Vera-Solís, Silvia Calva-Salgado. Analytical support was from Alfonso Maldonado’s chemical research group.

Several species of Mycobacteria were evaluated using sterols marketed by General Mills as Generols, that are a mixture of Campesterol (24R)-Ergot-5-en-3ß-ol) and ß-Sitosterol (3ß-Stigmast-5-en-3-ol). Also, a by- product of the Upjohn Co. from the separation of Stigmasterol ((3ß,22E)-Stigmsta -5-22-dien-3-ol), that is used by Upjohn in the synthesis of PROVERA ©® , a major market drug by Upjohn.

The major source from these sterols is a by-product of the edible oil industry called soapstock that after milling and or solvent extraction of the seeds, the free fatty acids are neutralized with potassium or sodium hydroxide forming a precipitate, down the refining process the high vacuum deodorization of the oil also provides more sterols depending of the oil seed consumed. The pioneering research on this sources was done by Percy Julian that accidentally soya bean oil was mixed with water from which he was able to isolate stigmasterol. Years on, Julian was involved in the Mexican steroid industry boom founding STEROMEX, that he later sold to Smith Kline and French.

A thorough literature search was made as to which culture media were used for Mycobacteria and different sterol concentration levels were studied up to 500 grams/liter with several carbon and nitrogen sources combinations using factorial designs as published by Box, Draper, Wilson and others. Promising results obtained in shaked flasks , and 28 and 70 liter fermenters. In particular Mycobacterium phlei NRRL 3805.

The obtained results encouraged Searle de Mexico general Manager, Dave Baird to lease a pilot plant from the Laboratori Ricerche of Società Prodotti Antibiotici S.p.A. Milano Italy. All nutrients for two runs were shipped from México to Italy except water and the strain was carried personally by me. This pilot plant consisted of 18 175 liter fermenters and the group supervised by Robert P Graber, included Javier Hebrero- Rodríguez, Gilberto García-Gutiérrez, Alfonso Maldonado and myself.

After six weeks in Milan more promising results were obtained, so G.D. Searle leased an old fermentation plant from Hercules Corp. in Harbor Beach, Mich. To scale-up to 9000 liter and 200 000 liter fermenters in early 1975 the company announced the successful start-up of the plant, which reflected favorably on the stock value at Wall Street. The Mexican group to Harbor Beach consisted of Javier Hebrero-Rodríguez, Gilberto García-Gutiérrez, Alejandro Canale-Guerrero, Felipe M. Vera-Solís and myself as group leader. In late 1974 the Harbor Beach was de-mexicanized and the WASPs took over the operation.

In the following years Searle de México shut-down its Chemical Division operations based on Dioscorea wild yams in Naucalpan, México and moved the synthesis of AD to Spironolactone to Gosport, England.

During the 1971-1974 my group was frequently bullied by the Development group leaded by Humberto Flores and his boss Héctor Flores-Gallardo.

Environmental Issues.

Last but not least, the presence of steroids in the environment have become an important issue, generally known as Endocrine disruptors, cause disturbances in fauna and to some extent in flora, fish show abnormal sexual features and

References:

Budavari, Susan (Ed.) (1989) The Merck Index, Eleventh Edition. Merck & Company, Inc. Rahway

Casas-Campillo, Carlos, visit www.colegionacional.org.mx

Charney, William. & Herzog, Hershel, L. (1967) Microbial Transformations of Steroids. Academic Press, New York

Esparza-García, Fernando, (2011) visit www.biotecnologia.cinvestav.mx

Fahrbach, Michael (2006) Anaerobic degradation of steroid hormones by novel denitrifying bacteria, a dissertation from the Rheinnisch-Westfälischen Technischen Hochschule Aachen.

Jiménez-Zamudio, Luis, (2011) visit www.sepi.encb.ipn.mx Kieslich, Klaus (1976) Microbial Transformations of Non-Steroids Cyclic Compounds. Georg Thieme Stuttgart.

Lehmann, P.A. (1992) Early History of Steroid Chemistry in Mexico: the Story of Three Remarkable Men. Steroids 57(8):403-408.

Martínez-Cruz, Jovita (1982) Catálogo de Cultivos Microbianos. Departamento de Biotecnología y Bioingeniería, CIEA, IPN, México, D.F.

Ruiz-Herrera, José, (2011) visit www.ira.cinvestav.mx

Steraloids, Inc. (1976) Steroids Catalog