Floyd Milton Huber

The formation of daptomycin by supplying decanoic acid to Streptomyces roseosporus cultures producing the antibiotic complex A21978C

Antibiotic substance A21978C is a complex of compounds having a common cyclic polypeptide nucleus and different fatty acid sidechains. Daptomycin is a semi-synthetic antimicrobial substance derived from the A21978C complex by a very elaborate chemical process. To obtain the daptomycin, the A21978C complex was first isolated from culture filtrates of Streptomyces roseosporus by several resin procedures. The resulting material was then ‘blocked’ and added to an Actinoplanes culture for deacylation. The protected A21978C nucleus was subsequently isolated by the same procedure as the parent complex and reacylated with the desired fatty acid (decanoic acid). The acylated compound was deblocked to yield daptomycin. This report describes the experimentation undertaken to establish a strategy to supply a very toxic precursor to cultures of S. roseosporus and, thereby, produce daptomycin biosynthetically.

Sporulation ofStreptomyces roseosporus in submerged culture

SummaryA soil isolate ofStreptomyces roseosporus was found to produce spores in stirred submerged culture. Both biological mass and respiratory activity increased during the sporulation process. Contrary to other reports, the differentiation process was not purposefully initiated by critical manipulation of either nutritional or environmental conditions.

High yielding culture conditions for the biosynthesis of D-amino acid oxidase byTrigonopsis variabilis

SummaryA process was developed for the production ofTrigonopsis variabilis and D-amino acid oxidase. Yields for the yeast were in excess of 220 g/l wet weight and 62 g/l dry weight. Using cephalosporin C as a substrate the enzyme concentration was 7000 units per liter.

Sterilization of bioreactor media on the basis of computer calculated thermal input designated asF0

SummaryIndustrial fermentation media are normally sterilized with steam to destroy the indigenous microbial population prior to inoculation with a specific microorganism. Because biological validation of each sterilization cycle is impractical, an ‘overkill’ approach is commonly employed on the basis that alteration of heat-sensitive nutrients is less detrimental than survival of indigenous microbes. However, the heat destruction of microbes is known to be a probability function amenable to calculation. A computer has been programmed to calculate the on-line heat input asF0 values during sterilization of media in stirred bioreactors. The accumulation ofF0 values is then announced verbally to bioreactor operators by a communications controller with voice synthesizer.

Interaction of media components during bioreactor sterilization: definition and importance of R0

SummarySterilization of bioreactor media, to destroy viability of the indigenous microbial population, is normally accomplished by autoclaving, or heating with pressurized steam. However, simultaneous chemical changes in media can also be expected to result from the high temperatures. A kinetic procedure involving on-line computer calculation of heat input, designated asF0 values, was previously developed to estimate sterility achievement. A similar kinetic procedure, based on a general purpose Arrhenius ‘pseudo’ rate equation and designated asR0 values, has now been designed to evaluate, and control the effects of temperature and heating time on chemical reactions occurring in the media. Data are presented indicating thatR0 may be a useful parameter for reducing variability in culture metabolism and ‘scale-up’ when these variations result from different nutrient concentrations produced by non-standard heating during media sterilization in stirred bioreactors.

Development of Real Time Expert System Applications for the On-line Analysis of Fermentation Respiration Data

Abstract A knowledge and model based expert system approach for the control of industrial fermentations at Eli Lilly and Company is under development. This paper reports the integration of the G2 Real-Time Expert System (Gensym Corporation Cambridge, MA) into a fermentation pilot plant. Process specific knowledge has been combined with simple modeling techniques to provide on-line expert decision making. A convenient testing utility was created to allow the rapid development of knowledge based applications. Key features of this utility are a) its ability to run historical data files “through” the application at 100 times the rate of real time data acquisition and b) its access to literally thousands of files, any one of which can serve as a test case.

Defined media strategies for the biosynthesis of Cephalosporin C

SummaryA stirred reactor process is described for the production of Cephalosporin C in high yields. Using a chemically defined medium, fermentation control strategies were developed to optimize the production of the antibiotic. The highest yields were obtained with carbon as the limiting substrate during both the rapid growth and synthetic phases of the process; the relative merits of glucose and lipid as carbon sources were examined.

The synthesis of A21978C analogs byStreptomyces roseosporus cultivated under carbon limitation and fed fatty acids

SummaryBy employing carbon limited fed batch techniques, fatty acids of length C8 to C12 were successfully employed as precursors for biosynthesis of A21978C antibiotics. The efficiency of incorporation was related to the length of the lipid precursor, with C10-C12 fatty acids being the preferred substrates.

Characterization of the process for the biosynthesis of the actaplanin complex by Actinoplanes missouriensis

Abstract The production of the antibiotic actaplanin by Actinoplanes missouriensis was characterized in a high yielding process. The data generated, indicate that the antibiotic is synthesized during a period of declining cell mass as estimated by dry weight and viability. Further study demonstrated that the producing culture binds actaplanin and is inhibited by the antibiotic in the soluble form.