Pharmaceutical Review Pharm. Bioprocess. (2013) 1(2), 179–196 Recent advances in biocatalyst development in the pharmaceutical industry Biocatalysts are increasingly employed as more efficient and environmentally safer Mingzi M Zhang†1, Xiaoyun alternatives to traditional chemical catalysts in the manufacturing of fine chemicals. Su†1, Ee Lui Ang1 & This is driven by advances in recombinant DNA technology, protein engineering and Huimin Zhao*1,2 bioinformatics, all of which are critical in the discovery, tailoring and optimization of 1Metabolic Engineering Research enzymes for industrial processes. In this article we review these key technological in- Laboratory, Institute of Chemical & Engineering Sciences, Agency for novations, as well as highlight the strategic application of these tools in the develop- Science, Technology & Research, ment of biocatalysts in the production of advanced pharmaceutical intermediates. Singapore 2Departments of Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA The synthesis of pharmaceutical products covery and development for pharmaceutical *Author for correspondence: and therapeutic agents using biological sys- processes. For a comprehensive summary of Tel. : +1 217 333 2631 tems is becoming increasingly important for recent examples of biocatalyst application E-mail: zhao5@illinois. edu the healthcare industry. Integral to pharma- in the pharmaceutical industry, readers can †Authors contributed equally ceutical bioprocessing is the development refer to the article by Bornscheuer et al. [2]. of biocatalysts for synthesis of desired com- This review focuses on the recent techno- pounds. While biocatalysts are capable of ac- logical innovations for biocatalyst discovery cepting a wide range of substrates with high and engineering (Figure 1). Selected case stud- enantioselectivity and regioselectivity [1], they ies highlight how these methods were inte- are often not very active or stable under the grated and applied to develop enzyme-based process conditions frequently required for and whole-cell biocatalysts for the production commercial-scale production, making many of active pharmaceutical ingredients. Emerg- biocatalytic processes unscalable. However, ing trends that promise to shape the future of technical advances have enabled biocatalysis biocatalyst development are also presented in to become economically feasible and a green- this review. er alternative to traditional chemical synthe- sis routes, especially in the pharmaceutical Tools employed in biocatalyst sector [2]. discovery & engineering One of the key technological break- » Biocatalyst discovery tools throughs that aided the development of DNA sequencing & genomics/ many industrial biocatalytic processes is the metagenomics introduction of directed evolution strategies The biocatalysts used for pharmaceutical in the mid to late 1990s [3–5]. Since then, di- purposes can be individual or a combina- rected evolution has found many applications tion of enzymes. Alternatively, whole cells in the healthcare industry including areas can also be used as a biocatalyst, in which such as therapeutic protein development [6,7], several enzymes coordinate to bring about and active pharmaceutical ingredient synthe- chemical transformations. In any case, en- sis [2]. In recent years, state-of-the-art gene zymes are the core of biocatalysis and there discovery tools and DNA synthesis technolo- is a huge demand in the pharmaceutical in- gies have further accelerated biocatalyst dis- dustry for novel enzymes with more desirable future science group 10.4155/PBP.13.14 © 2013 Future Science Ltd ISSN 2048-9145 179 Review Zhang, Su, Ang & Zhao Key Terms characteristics in terms of substrate called third-generation DNA sequencing. The Nano- Enantioselectivity: Preference of a specificity, and regio- and enantio- pore DNA sequencing technology also belongs to the chemical reaction for one enanti- selectivities. The exponentially in- third-generation DNA sequencing, but it does not omer over the other. Enantiomers creasing genome and metagenome require fluorescent labeling and the expensive charge- are two molecules with non- sequencing datasets allow access to coupled device cameras [13] . The Helicos BioSciences superimposable chemical structures that are mirror images vast biodiversity, providing an in- HeliScope Single Molecule Sequencer can obtain over of each other. valuable resource for such novel bio- 1 Gb of usable nucleotide sequence per day [204]. Regioselectivity: Preference of a catalysts. Genome mining based on the rapidly evolving DNA chemical reaction (bond breaking For two decades, the Sanger meth- sequencing technologies mentioned above uncovers or formation) for a single isomer od has been a workhorse technology novel enzymes with alternative characteristics, such over the other products in the reaction. for DNA sequencing before the ap- as different substrate specificity, regio- and enantio- pearance of next-generation sequenc- selectivities. For example, cytochrome P450 enzymes Metagenome: Genetic material directly extracted ing (NGS), which is widely employed (P450s) are a large and highly diverse superfamily of from environmental samples. in recent genomic, metagenomic and heme-containing enzymes that can transfer an oxygen Metagenomics is the study of transcriptomics endeavors [8]. Three atom from molecular oxygen to an organic molecule. metagenomes. This approach is of the most popular methods include P450s have two prominent characteristics that make useful for identifying genes from unculturable microorganisms, the 454™ Genome Sequencer FLX them attractive candidates for pharmaceutical biocat- which represent the vast majority instrument from Roche Applied Sci- alysts. First, they catalyze insertion of oxygen in less (>99%) of microbial diversity. ence (Germany), the Solexa™ from r eactive carbon–hydrogen bonds under mild condi- Illumina (CA, USA), and SOLiD™ tions. Second, they act on diverse substrates, including from Applied Biosystems (CA, USA). The 454 DNA fatty acids, terpenes, steroids, prostaglandins, polyaro- sequencing method and the Illumina Solexa method are matic and heteroaromatic compounds [14] . Since many sequencing-by-synthesis methods. The 454 method is drugs or their intermediates are also P450 substrates, based on emulsion polymerase chain reaction (PCR) [9] there have been efforts in mining sequenced genomes to amplify the DNA followed by pyrosequencing tech- for novel P450s. For example, 18 putative P450 genes nique [10] and can acquire over 1 million reads with were identified from the genome ofStreptomyces coeli- read lengths of up to 1000 base pairs (bp) in 23 h [201] . color A3 [15], 21 from Sorangium cellulosum, 18 from For the Illumina Solexa method, the HiSeq2500/1500 Stigmatella aurantiaca, 17 from Haliangium ochraceum, system can acquire a maximum of 600 gigabase (Gb) 7 from Myxococcus xanthus [16] and a total of 12,456 of sequences with 6 billion reads with a read length putative P450 genes were predicted from all kingdoms of 2 × 100 bp in paired-end reads [202]. The SOLiD of life [17] . Prediction of the functions of P450s can be system involves sequencing by ligation instead of syn- difficult because of their diversity. The genome mining thesis and can obtain sequence data above 20 Gb per method also presents a way of prediction by researching day with a 50 bp average read length [203]. While these into the genome context of the P450s [16] . methods use clonally amplified templates, other meth- The plant and microbial genomes also encode a vast ods, such as those developed by Helicos BioSciences number of non-ribosomal peptide synthetases (NRPSs) (MA, USA) [11] and Pacific Bio sciences (CA, USA) and polyketide synthases (PKSs), two important fami- [12], utilize single-molecule templates, which are more lies of enzymes involved in producing pharmaceuti- demanding in image sensing. These technologies are cally important non-ribosomal peptide natural prod- ucts and polyketides. Many of these natural products, such as erythro- Tools for biocatalyst discovery and engineering in pharmaceutical bioprocessing mycin and vancomycin, are useful in antibiotic, anticancer and immu- Discovery tools Engineering tools nosuppressant treatments. Similar to P450s, elucidation of the exact roles of putative NRPSs or PKSs DNA sequencing Other -omics tools DNA synthesis Enzyme biocatalyst engineering tools is not easy and is often time-con- suming. Lautru et al. described the Genomics Transcriptomics Rational design prediction of substrates recognized Metagenomics Proteomics Directed evolution by the adenylation domains that are Semi-rational design commonly present in NRPS. The De novo design prediction guided the identification of a tris-hydroxamatetetrapeptide Figure 1. Tools for biocatalyst discovery and engineering in pharmaceutical bioprocessing. iron c helatorcoelichelin produced 180 Pharm. Bioprocess. (2013) 1(2) future science group Recent advances in biocatalyst development in the pharmaceutical industry Review by the Streptomyces coelicolor CchH [18]. Functional as- (R)-4-cyano-3-hydroxybutyric acid, a key intermedi- signment to a putative NRPS or PKS is also hindered ate of atorvastatin [27]. Similarly, 137 nitrilases were by silencing of the corresponding genes under standard identified from screening over 600 biotope-specific fermentation conditions. In Aspergillus nidulans, no environmental DNA libraries [28]. By mining of pub- PKS–NPRS metabolites