Pharmaceutical Biocatalysis

Drugs, Genetic Diseases, and Epigenetics

Peter Grunwald

• Publisher: Jenny Stanford Publishing
• Year: 2020
• Print ISBN: 9789814877145
• eBook ISBN: 9781000067408
• Topic: Medicine
• Subtopic: Biotechnology in Medicine

Volume 7 of the Jenny Stanford Series on Biocatalysis deals with several different aspects of pharmaceuticals, which include not only various applications of drugs and their metabolism but also natural resources for active pharmaceutical ingredients as well as the removal of pharmaceutical pollution. In detail, novel approaches for developing microbial fermentation processes to produce vitamin B6 using microorganisms are described together with novel routes for vitamin B6 biosynthesis. The other topics discussed are new approaches for producing the successful anticancer drug Taxol from naturally occurring precursors, molecular farming through plant engineering as a cost-effective means to produce therapeutic and prophylactic proteins, and successful screening of potent microorganisms producing L-asparaginase for various chemotherapeutic applications. Furthermore, microbial biotransformations in the production and degradation of fluorinated pharmaceuticals are described. The other chapters inform the reader about the biotransformation of xenobiotics/drugs in living systems, the degradation of pharmaceuticals by white-rot fungi and their ligninolytic enzymes, and the removal of pharmaceutical pollution from municipal sewage using laccase.

Chapter 1

Fermentative Production of Vitamin B6

Jonathan Rosenberg^a, Björn Richts,^a and Fabian M. Commichau^b

^aDepartment of General Microbiology, Georg-August-University Goettingen, Grisebachstr. 8, D-37077 Göttingen, Germany

^bBTU Cottbus-Senftenberg, Institut für Biotechnologie, FG Synthetische Mikrobiologie, Universitätsplatz 1, 01968 Senftenberg, Germany

[email protected]

1.1 Introduction

Vitamin B6 has been discovered almost one century ago and is an essential organic micronutrient for organisms from all kingdoms of life (György, 1956; Hellmann and Mooney, 2010; Kraemer et al., 2012; Eggersdorfer et al., 2012). Vitamin B6 collectively designates the water-soluble vitamers pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), and their respective phosphate esters pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate (PNP), and pyridoxamine 5′-phosphate (PMP) (Fig. 1.1A; Rosenberg, 2012; Rosenberg et al., 2017). PLP is the most important vitamer serving as a cofactor for a plethora of proteins and enzymes (Mehta et al., 1993; Jansonius, 1998; Mehta and Christen, 2000; Christen and Mehta, 2001; Eliot and Kirsch, 2004; Phillips, 2015). Estimations revealed that over 160 enzymes with distinct catalytic activities require vitamin B6 as a cofactor (about 4% of all described catalytic activities) (Percudani and Peracchi, 2009). Most of the PLP-dependent enzymes are involved in biosynthesis of amino acids, decarboxylation and racemization reactions, cleavage of C_α-C_β bonds, α-, β- and γ-elimination or replacement reactions (John, 1995; Mehta and Christen, 2000; Christen and Mehta, 2001; Eliot and Kirsch, 2004). Moreover, PMP serves as a cofactor for enzymes of deoxysugar biosynthetic pathways (Burns et al., 1996; Romo and Liu, 2011). Furthermore, PLP modulates the activity of DNA-binding transcription factors in eukaryotes and prokaryotes (Oka et al., 2001; Huq et al., 2007; El Qaidi et al., 2013; Belitsky, 2004a; Belitsky, 2014; Tramonti et al., 2015, 2017 2018; Suvorova and Rodionov, 2016; Tramonti et al., 2017). The finding that about 1.5% of the genes of many free-living prokaryotes code for PLP-dependent proteins underlines the importance of the B6 vitamer for the function of proteins and catalytic enzymes (Percudani and Peracchi, 2003). There is also evidence that vitamin B6 is implicated in oxidative stress responses in plants (Bilski et al., 2000; Mooney and Hellmann, 2010; Moccand et al., 2014; Vanderschuren et al., 2013). Thus, vitamin B6 fulfils a variety of vital functions in different cellular processes (Fitzpatrick et al., 2007; Mooney et al., 2009; Vanderschuren et al., 2013; Parra et al., 2018).

Animals and humans have to ingest vitamin B6 with their diet because these organisms are unable to synthesize the micronutrient (Fitzpatrick et al., 2007, 2010; Kraemer et al., 2012). Vitamin B6 limitation has been associated with neurological disorders such as epileptic encephalopathy due to inherited errors in the enzymes interconverting B6 vitamers in the socalled “salvage pathway” (Mills et al., 2005; Bagci et al., 2008; di Salvo et al., 2012). Vitamin B6 deficiency can also be caused by interactions between drugs, such as contraceptives, and enzymes of the salvage pathway (Lumeng et al., 1974; Lussana et al., 2003; di Salvo et al., 2011). Therefore, vitamin B6 is of commercial interest for improving the quality of the food and for applications in the pharmaceutical industry (Rosenberg et al., 2017; Acevedo-Rocha et al., 2019). In the food industry, the hydrochloride salt of the B6 vitamer PN is usually used in combination with other vitamins in a variety of food products (Domke et al., 2005; Eggersdorfer et al., 2012). Vitamin B6 is also added to the food that is used for intensive animal farming to improve animal health and to enhance the yield (Johnson et al., 1950; Verbeek, 1975; Eggersdorfer et al., 2012). Many studies report positive effects of vitamin B6 although a large number of commercial products contain this compound. Only a few studies revealed that vitamin B6 can be toxic. A recent case described photosensitive skin darkening, hyperemesis and diarrhea as toxic effects, which disappeared soon after intoxication stopped (Cupa et al., 2015). Moreover, long-time supplementation of PN in higher doses is known to cause sensory neuropathy (Schaumburg et al., 1983; Albin et al., 1987). This effect is also used as a model for neuropathy (Hong et al., 2009; Potter et al., 2014).

So far the B6 vitamers are fully chemically synthesized via five different routes with variations, partly using expensive and/or toxic chemicals such as hydrogen cyanide, phosphorous pentoxide, and 1,4-butenediol (Pauling and Weimann, 1996; Kleemann et al., 2008; Eggersdorfer et al., 2012; Acevedo-Rocha et al., 2019). Several extensive attempts have been made by the biotech industry and academia to engineer microorganisms for vitamin B6 production by classical mutagenesis and by genetic modification (Pflug and Lingens, 1978; Ischikawa et al., 1997; Yocum et al., 2004; Hoshino et al., 2006a,b,c; Commichau et al., 2014, 2015; Rosenberg et al., 2017; Acevedo-Rocha et al., 2019). Unfortunately, none of the attempts were promising enough to establish an effective fermentation process (Rosenberg et al., 2017; Acevedo-Rocha et al., 2019). However, there is still considerable interest on the industrial side to shift from chemical synthesis processes to environmentally sustainable fermentation technologies (Acevedo-Rocha et al., 2019).

This chapter summarizes recent findings regarding biosynthesis and homeostasis of vitamin B6 metabolism with a focus on prokaryotic microorganisms (bacteria). We also describe the approaches for developing microbial fermentation processes to produce vitamin B6 using microorganisms. Furthermore, we describe novel routes for vitamin B6 biosynthesis that have been discovered recently and discuss their potential for overproducing the commercially valuable substance. Finally, we highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies that might help to circumvent these limitations to improve vitamin B6 production.

1.2 De novo Synthesis of Vitamin B6

Two non-homologous pathways for de novo vitamin B6 biosynthesis are known (Fig. 1.1B; Mittenhuber, 2001; Tanaka et al., 2005; Fitzpatrick et al., 2007, 2010; Rosenberg et al., 2017; Parras et al., 2018). The longer vitamin B6 biosynthetic pathway that was first discovered in the Gram-negative model bacterium Escherichia coli depends on the sugar deoxyxylulose 5-phosphate (DXP). The DXP-dependent pathway consists of two branches and seven enzymatic steps. In the longer branch of this pathway, the erythrose 4-phosphate (E4P) dehydrogenase (Epd), the 4-phosphoerythronate (4PE) dehydrogenase (PdxB and PdxR in E. coli and in the Gram-negative bacterium Sinorhizobium meliloti, respectively), and the 3-phosphoserine aminotransferase (SerC) convert E4P, which is derived from the pentose phosphate pathway, to 4-phosphohydroxy-L-threonine (4HTP) (Fig. 1.1B; Zhao et al., 1995; Drewke et al., 1996; Yang et al., 1998; Tazoe et al., 2006; Rudolph et al., 2010). 4HTP is then oxidized by the 4HTP dehydrogenase (PdxA) to 2-amino-3-oxo-4-phosphohydroxy)butyric acid that spontaneously decarboxylates to 3-phosphohydroxy-1-aminoacetone (PHA) (Cane et al., 1998; Laber et al., 1999; Banks and Cane, 2004). The PNP synthase PdxJ produces the B6 vitamer PNP from PHA and DXP, of which the latter substrate is generated by the DXP synthase Dxs from glyceraldehyde 3-phosphate (G3P) and pyruvate in the short branch of the DXP-dependent vitamin B6 pathway (Cane et al., 1999; Laber et al., 1999). The final step is catalyzed by the PNP oxidase PdxH and yields in the biologically active B6 vitamer PLP (Fig. 1.1B; di Salvo et al., 1998, 2002, 2003). The DXP-dependent vitamin B6 pathway has been intensively studied in E. coli. However, an in silico analysis revealed that it is only present in α- and γ-proteobacteria, who acquired it with the function of PdxB after the DXP-independent vitamin B6 pathway had been lost in their ancestral lineage (Mittenhuber, 2001; Tanaka et al., 2005; Rosenberg et al., 2017). Additionally, the pathway was constituted by members of the α-proteobacteria who acquired pdxR, which is not homologous to pdxB, but fulfils the same enzymatic function (Tazoe et al., 2006). It is also interesting to note that two enzymes of the DXP-dependent vitamin B6 route are involved in other metabolic pathways. SerC is essential for de novo biosynthesis of serine and the Dxs provides DXP as a precursor for thiamine and isoprenoids to the cell (Dempsey and Itoh, 1970; Sprenger et al., 1997).

Figure 1.1 (A) The B6 vitamers pyridoxal (PL), pyridoxal 5′-phosphate (PLP), pyridoxine (PN), pyridoxine 5′-phosphate (PNP), pyridoxamine (PM), and pyridoxamine 5′-phosphate (PMP). PLP is the physiologically most...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• 1. Fermentative Production of Vitamin B6
• 2. Exploring Alternative Taxol Sources: Biocatalysis of 7-β-Xylosyl-10- Deacetyltaxol and Application for Taxol Production
• 3. Molecular Farming through Plant Engineering: A Cost-Effective Approach for Producing Therapeutic and Prophylactic Proteins
• 4. Microbial Biotransformations in the Production and Degradation of Fluorinated Pharmaceuticals
• 5. Successful Screening of Potent Microorganisms Producing L-Asparaginase
• 6. Biotransformation of Xenobiotics in Living Systems—Metabolism of Drugs: Partnership of Liver and Gut Microflora
• 7. Degradation of Pharmaceutically Active Compounds by White-Rot Fungi and Their Ligninolytic Enzymes
• 8. Removal of Pharmaceutical Pollutants from Municipal Sewage Mediated by Laccases
• 9. Mechanism of Drug Resistance in Staphylococcus aureus and Future Drug Discovery
• 10. Genome Editing and Gene Therapies: Complex and Expensive Drugs
• 11. Epigenetic and Metabolic Alterations in Cancer Cells: Mechanisms and Therapeutic Approaches
• Index