Developing novel systems for expression of intracellular membrane-bound human cytochrome P450 enzymes in baker’s yeast

Human cytochrome P450 (CYP) enzymes belong to a family of monooxygenase enzymes that are responsible for the metabolism, in a stereo-specific and stereo-selective manner, of diverse chemicals. These chemicals are often referred to as xenobiotics which are usually harmful and toxic to the human organism. CYP enzymes play a key role in the metabolism of pharmaceutical drugs, making them more soluble for excretion from the human body. Human CYPs are also naturally involved in the biosynthesis of vitamins, steroids, fatty acids, lipids and cholesterol. A CYP enzyme requires a CYP reductase (CPR) for it to be active. Both CYPs and CPR are bound to the intracellular endoplasmic reticular (ER) membranes. The presence of a CPR is absolutely essential for membrane-bound CYP activity. It seems another ER-bound protein, cytochrome b5, is required for the activity of some CYPs. However, cytochrome b5’s role in CYP activity is not clearly understood. Drug metabolism research in pharmaceutical industry involves accurately (a) determining the potential of a drug and its metabolites to inhibit CYP enzymes and (b) identifying its metabolites which are formed through CYP-mediated biotransformation reactions. The degree of success in drug metabolism research is a crucial measure for approval/rejection of a drug by regulatory authorities. However, drug metabolism studies take place only in the pre-clinical – clinical interface which is quite late in the drug discovery process for a failure to occur, which unfortunately happens quite often. Drug metabolism studies are not performed any earlier primarily because CYP enzymes are extremely costly and are difficult to use because of their inherent instability at room temperature. The experiments performed in this thesis attempt to address these issues. The main aim of the work was to create a new set of tools that would facilitate the drug discovery process in general but, more specifically, allow these reagents to be used by chemists/biologists in early pre-clinical phase, where they are most needed, by trying to overcome the major hurdles of CYP’s costs and stability. There are five experimental Chapters (Chapters 3 to 7) of this thesis. Chapter 3 describes the expression of 17 human CYP enzymes in baker’s yeast, using human CYP genes chemically synthesized with yeast-biased codons. There are 61 codons which code for the 20 amino acids that are essential for protein syntheses. It is known that the highly expressed genes, in a particular organism, use specific codons which would indicate that there is codon bias. Hence, codons used by highly expressed genes in baker’s yeast were used to create genetic sequences for human CYP enzymes. These codon-optimised human CYP genes, in theory, should provide high expression in yeast. Therefore, expression of synthetic genes, from episomal (i.e. ‘extra-chromosomal’) plasmids, was compared with the expression of native genes, isolated from a human liver cDNA library to assess the assumption that yeast-biased codons would provide better expression of human CYPs. It was clearly shown that, per constant number of yeast cells, the synthetic genes expressed far more CYP activity than the native genes. It was also shown that some CYPs which have been claimed to require cytochrome b5 for their activity may not require its presence. Chapter 4 describes the expression of one copy of synthetic genes (chemically synthesized with yeast-biased codons), from a single yeast ‘chromosomal’ locus to find out which locus gives the best expression of human CYP proteins. Expression of genes from chromosomal loci allows growth of yeast cells in cheap, ‘non-selective’ growth media, continuously over 5 days, or longer, in shake-flasks or fermentors. In contrast, expression from extra chromosomal, episomal plasmids demand growth of yeast cells in ‘selective’ growth media, which are expensive, where cell numbers are relatively low, and cell growth is restricted to 24 h. It has been speculated that, heterologous (i.e. foreign) gene expression from yeast depends on the yeast proteins that reside in the neighbourhood of the human protein that is being expressed from a particular yeast chromosome. Hence, CYP gene expression cassettes (consisting of a promoter, the gene of interest and a transcription terminator) were integrated into different chromosomal loci, using homologous recombination, a technology which also facilitates gene therapy in human cells. The results obtained clearly show that there is differential expression of a human CYP enzyme when expressed from the neighbourhood of the yeast ADE2, HIS3 and URA3 genes. Indeed, the best human CYP expression occurs from a particular locus of chromosome XV, where the yeast HIS3 gene resides. Chapter 5 is a continuation of Chapter 4 where two copies of chromosomally integrated human CYP genes (chemically synthesized with yeast-biased codons and integrated at two different chromosomal loci, via homologous recombination), have been used to express human CYPs from baker’s yeast. The goal was to obtain high yields and activities of human CYP enzymes. In order to achieve this, a new process was developed for isolation of microsomal [i.e. endoplasmic reticular (ER) membrane bound] enzymes. CYPs are totally inactive when shorn off these ER membranes. The activities of the baker’s yeast produced human CYP enzymes have been compared, head-to-head, with the three commercially available enzymes which are produced either from insect cells or bacterial cells and which are sold worldwide. Global market size of recombinant CYPs is considered to be at least $250 million. The results obtained show that the human CYP enzymes, produced from baker’s yeast, are much more active than the commercially available enzymes. The results also suggest that, without embarking on production of these enzymes in a cheaper environment, the human CYP enzymes could easily be produced in the UK and sold at half the price quoted by the current manufacturers. Yet these cheap, highly active enzymes, if marketed, could still provide a profitable margin. Because of their cheapness, they could be made widely available for early pre-clinical research. Chapter 6 explores the use of three CYP-producing recombinant cells for biotransformation reactions. It has been reported that, until now, whole cell mediated biotransformation reactions generally yield, at best, no more than 10-15% of the product. Whole recombinant yeast cells, expressing (a) CYP1A1, (b) variants of human CYP2D6 and (b) human CYP3A4 have been used to transform (i) chrysin, a natural product, (ii) codeine, a CYP2D6 substrate, and (ii) the CYP3A4 substrate, AZD-2014, an Astra-Zeneca drug in multicentre Phase II/Phase III clinical trials. The results from LC/MS, HPLC and TLCs clearly show that there is >80% product formation. They also indicate that, in future, organic chemists in pre clinical drug discovery could use this type of robust whole yeast cells, harbouring human CYPs, for bioorganic reactions. Chapter 7 describes the successful simultaneous co-production of two or three different human CYPs within the same cells. It is likely that two specific CYPs function, in tandem, on a substrate. There is also a necessity of determining inhibition of a particular human CYP, in the presence of another CYP or other CYPs. It is believed that the inhibition of a CYP is likely to be altered in the presence of another CYP through a biochemical process known as the ‘crowding effect’. These hypotheses can, in future, be tested using the recombinant cells reported in this Chapter. The cells, co-expressing two or three human CYPs, could also be used for both biotransformation reactions and isolation of microsomal enzymes. The concept of co-expression of CYPs could also be used, in the future, as a stepping stone for creation of liver-like yeast cells which have the ability to express multiple CYPs of choice. In summary, this thesis describes various yeast systems that have been created for efficient Drug Metabolism studies and Biotransformation reactions.