Figure 2: Examples of active ingredients for which biocatalytic routes are being developed: rosuvastatin (1) and esomeprazole
(2). (ALL MOLECULES IN FIGURE ARE COURTESY OF US FOOD AND DRUG ADMINISTRATION)
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Codexis is developing intermediates and APIs for the generic equivalents of several branded pharmaceutical products, according
to the company's S-1 filing. The company plans to launch an intermediate for Merck & Co.'s "Singulair" (montelukast); esomeprazole,
the API in AstraZeneca's "Nexium" (see Figure 2); and rosuvastatin, the API in AstraZeneca's "Crestor" (see Figure 2). Other
products for which it plans to sell generic APIs are levetiracetam, the API in "Keppra" by UCB Pharmaceuticals (Brussels) and duloxetine, the API in "Cymbalta" by Eli Lilly (Indianapolis, IN), according to the company's S-1 filing.
Last year, Codexis developed biocatalyst panels to allow innovators to screen biocatalysts for activity against existing drug
compounds and pipeline candidates. Once a useful biocatalyst is discovered through the panels or in-house screening, Codexis
provides the biocatalyst for commercial manufacture or provides further screening and optimization if needed. Merck & Co.
was Codexis's first customer for the panels.
To develop its biocatalysts, Codexis uses gene shuffling to manipulate the genetic code for a biocatalyst to produce variants
of the enzyme with improved industrial characteristics. It also uses whole genome shuffling to manipulate the genome of a
microbe to produce new microbial variants. This technique entails protoplast fusion, which fuses two or more cells into one,
followed by the regeneration of normal cells, to shuffle the genome. High-throughput screening methods screen the biocatalysts
for activity. Codexis's proprietary "ProSAR" bioinformatics software quantifies the effect of specific mutations in an improved
biocatalyst variant.
Phosphite dehydrogenase-based NADP regeneration technology
Enzyme-catalyzed reactions that require stoichiometric amounts of reduced nicotinamide cofactors, nicotinamide adenine dinucleotide
(NADH) and its phosphorylated form (NADPH) have much potential in biocatalysis. A drawback, however, is these cofactors are
expensive. Preparative applications require regeneration of the cofactors in situ, usually by a second enzyme with high specificity for a sacrificial substrate (2).
Researchers have successfully addressed this problem. Huimin Zhao, professor in the Department of Chemical and Engineering,
Wilfred A. van der Donk, professor in the Department of Chemistry, and William Metcalf, professor in the Department of Microbiology
at the University of Illinois, used directed evolution and rational-design approaches to develop a novel phosphite dehydrogenase (PTDH)-based NADPH regeneration
system to improve the enzyme activity toward NADP by 1000-fold, to increase the overall activity sixfold, and to increase
the thermostability by more than 22,000-fold. PTDH is considered more efficient than technology based on formate/formate dehydrogenase
(2, 7).
Zhao and his team discovered and characterized a wild-type PTDH enzyme from Pseudomonas stutzeri that catalyzes the oxidation of phosphite to phosphate with the reduction of NADP+ to NADPH. His team engineered a PTDH variant with improved stability, activity, and cofactor specificity. The variant PTDH
also has higher specific activity, a higher thermodynamic equilibrium constant, and a broader pH-rate maximum. The phosphite
substrate is also inexpensive (2, 7).
The team used a membrane bioreactor for the synthesis of L-tert-leucine and xylitol using the variant PTDH for cofactor regeneration. The PTDH system has many applications for the industrial
synthesis of unnatural amino acids, polyols, and chiral alcohols. The technology was licensed to BASF (Ludwigshafen, Germany) and BioCatalytics, now part of Codexis (2, 7).
Patricia Van Arnum is a senior editor at Pharmaceutical Technology, 485 Route One South, Bldg F, First Floor, Iselin, NJ, 08830, tel. 732.346.3072, pvanarnum@advanstar.com
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