Jinyou Zhang

Chemically defined media for commercial fermentations

Abstract The use of chemically defined media is gaining popularity in some commercial fermentations, particularly for the preparation of biological products. Although these media are still not frequently developed for industrial processes, they do exhibit favorable characteristics at large scale that are not observed with traditional complex media. This review focuses on the application, development, and practical considerations, especially process economics, of fermentations in chemically defined media in an industrial environment.

Chiral bio-resolution of racemic indene oxide by fungal epoxide hydrolases

Eighty fungal strains were evaluated for their production of an epoxide hydrolase capable of catalyzing the kinetic resolution of racemic indene oxide into 1(S),2(R) indene oxide. This screen identified Diplodia gossipina ATCC 16391 as the best catalyst. Process development studies of this bio-resolution demonstrated that the optical purity of the 1(S),2(R) indene oxide produced was dependent upon pH and substrate concentration. A shake-flask-scale bio-resolution process supported the production of preparative quantities of optically pure (ee=100%) 1(S),2(R) indene oxide.

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimers disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.

Development of a bioconversion process for production of cis-1S,2R-indandiol from indene by recombinant Escherichia coli constructs

Abstract Recombinant Escherichia coli cells expressing the toluene dioxygenase (TDO) genes from Pseudomonas putida convert indene to cis-1S,2R-indandiol, a potentially important intermediate for the chemical synthesis of the HIV-1 protease inhibitor, Crixivan. A bioconversion process was developed through optimization of medium composition and reaction conditions at the shake-flask and 23-l fermentor scales. A cis-1,2-indandiol productivity of approx. 1000 mg/l was achieved with construct TDO123, which represents a 50-fold increase over the initial titer. Varying the bioconversion conditions did not change the enantiomeric excess (e.e.) for the 1S,2R enantiomer from about 30%, suggesting that toluene dioxygenase intrinsically converts indene to 1S,2R- and 1R,2S-indandiols at a ratio of 2:1. Further inclusion of the Pseudomonas dehydrogenase gene in construct D160-1 led to the production of chirally pure cis-1S,2R-indandiol (e.e. > 99%) as a result of the selective degradation of the 1R,2S enantiomer, with the overall yield (650 mg/l) proportionally reduced. A single stage process was developed for D160-1 and scaled up to the 23-l fermentor, achieving a cis-1S,2R-indandiol titer of 1200 mg/l.

Bioconversion of indene to trans-2S,1S-bromoindanol and 1S,2R-indene oxide by a bromoperoxidase/dehydrogenase preparation from Curvularia protuberata MF5400

Abstract 1S,2R-Indene oxide is the precursor of cis -1S,2R-aminoindanol, a key intermediate for the Merck HIV–1 protease inhibitor, Crixivan®. As an alternative to the challenging chemical synthesis of this chiral epoxide from indene, the biotransformation route using an enzyme catalyst was examined. Approximately 3% of the 400 fungal cultures isolated from high salt environments were found to possess neutral haloperoxidase activities. Subsequent studies revealed that indene conversion by these positive cultures could only be obtained when both hydrogen peroxide and bromide ions were present. The products were generally racemic trans -bromoindanols which upon basification yielded racemic epoxides. Finally, it was found that a crude enzyme preparation from the fungal culture Curvularia protuberata MF5400 converted indene to the chiral 2S,1S-bromoindanol which can be chemically converted to the desired 1S,2R-epoxide through basification or used directly in the asymmetric synthesis of cis -1S,2R-aminoindanol. The bioconversion rate and the enantiomeric excess (ee) achieved with this cell-free system were heavily pH dependent. An initial 1.5-h reaction at pH 7.0 gave ∼10% yield of the chiral bromoindanol or epoxide from indene, and the yield was rapidly improved to >30% for trans -2S,1S-bromoindanol with an ee of 80%. Reaction mechanistic studies revealed that the stereoselectivity observed was apparently due to a specific dehydrogenase activity present in MF5400 which was also found to resolve chemically synthesized racemic trans -2-bromoindanols.

Scale-up studies on a defined medium process for pilot plant production of illicicolin by Gliocladium roseum.

Illicicolin was cultivated at the 600‐L pilot scale for purposes of material generation and process development. The initial medium containing oat flour was difficult operationally as a result of excessive foaming during sterilization, so a new defined medium process (with either glucose or sucrose as the carbon source), developed at the 23‐L scale, was scaled up and improved for pilot scale needs. Pilot scale media development efforts focused on exploring the highest concentration of media (1.0× to 3.0×) that could be cultivated at the pilot scale and not be limited by mixing or oxygen mass transfer. The process was scaled up successfully and peak titers improved 7.5‐fold, from about 200 mg/L in the initial complex medium to 1500 mg/L in the final defined medium.

Use of glucose feeding to produce concentrated yeast cells.

A defined medium and fed-batch feeding process for the production of a yeast biocatalyst, developed at the 23-L scale, was scaled up to the 600-L pilot scale. Presterilized 100-L-vol plastic bags were implemented for the pilotscale nutrient feeding. Medium of increased concentration Ôqs implemented at the pilot scale, and equivalent dry cell weights were reached with a medium 80% more concentrated than that used at the laboratory scale. The higher medium concentration was believed to be necessary at the pilot scale owing to the additional heat stresses on key components (e.g., complexing of magnesium sulfate with phosphate), increased dilution during sterilization, lower evaporation rate owing to the lower vessel volume per minute air flow rate, and increased dilution owing to nutrient feeding or shot additions. Peak cell density was found to be somewhat insensitive to variations in residual glucose levels. These results suggest that defined medium developed at the laboratory scale may need to be further optimized at the pilot scale for equivalent performance.

Physostigmine production byStreptomyces griseofuscus NRRL 5324: Process development and scale-up studies

A reliable and scalable fermentation process was developed for production of the acetylcholine esterase inhibitor physostigmine employingStreptomyces griseofuscus NRRL 5324. Initial fermentation in small-scale bioreactors reached physostigmine levels of approximately 60 mg L−1 after 139 h. Optimization of both process operating parameters and production medium composition rapidly yielded a seven-fold increase in physostigmine titer. The scaled up process routinely produced physostigmine titers of approximately 400 mg L−1 during a fermentation cycle of 180 h, and supported the rapid production of large amounts of physostigmine. A physostigmine production of 500 mg L−1 representing an eight-fold improvement over the original performance, was achieved using a glucose/ammonium fed-batch process.