Studies on yeast host strains for heterologous P450 expression

The Saccharomyces cerevisiae strain AH22 was capable of human P4501A1 expression without detectable background of yeast P450, unlike ATCC44773. Repeated backcrossing to AH22 produced a strain allowing transformation by vectors carrying various common selectable markers. Background yeast xenobiotic metabolism was observed only with growth on complex medium.     

Protein synthesis inhibitors and ethanol selectively enhance heterologous expression of P450s and related proteins in Escherichia coli.

The antibiotics chloramphenicol (Cm), tetracycline, and erythromycin, which inhibit bacterial protein synthesis and are known to induce the cold shock response, unexpectedly enhance the heterologous expression of P450s and related proteins in Escherichia coli. In contrast, antibiotics that mimic heat shock in E. coli such as puromycin, streptomycin, and kanamycin decrease the expression of the same proteins. A sublethal dose of Cm (1 microgram/ml) effectively enhances the expression of both membrane-bound proteins (microsomal and mitochondrial P450s) and a soluble mitochondrial protein (adrenodoxin) over the range of two- to eightfold. The expression level of N-terminal truncated P450c17 (1600 nmol/liter culture without Cm), for instance, reached 3500 nmol/liter culture by the addition of Cm, approximately 8.4% of the total cellular protein. Cm also enabled expression at useful levels of active P450s previously difficult to express in E. coli. In contrast, the expression of P450scc, a mitochondrial protein, is decreased by Cm but enhanced by ethanol, a powerful elicitor of heat shock response in E. coli. These results show that both the cold shock response induced by some antibiotics and the heat shock response induced by ethanol may lead to enhanced expression of certain heterologous proteins in E. coli. This study also indicates that protein synthesis inhibitors associated with the cold shock response may act as protein synthesis enhancers under certain conditions.     

Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor

A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/ Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.     

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.     

High-level heterologous expression of fungal cytochrome P450s in Escherichia coli

A thorough understanding of the sequence–structure–function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5′ end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.     

In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins

The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-l-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.     

Combined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase

Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.     

Phosphorylation of ribosomal proteins from eukaryotes in homologous and heterologous cell-free systems

The phosphorylation of ribosomal proteins from eukaryotes in homologous and heterologous cell-free systems has been studied. The ribosomes and protein kinases from yeast (Saccharomyces cerevisiae, strain Bu), wheat (Triticum vulgare) and rabbit (Orystolagus cuniculus) have been used.It has been found that five ribosomal proteins incorporate γ-³²P from ATP during the incubation of wheat ribosomes with wheat protein kinase. When the phosphorylation of isolated wheat ribosomal proteins was examined more phosphoproteins were detected. These data confirm the suggestion that the ribosomal structure affects the phosphorylation. Probably some ribosomal proteins remain hidden for the action of protein kinase.The results from the crossed experiments show that there is no barrier for phosphorylation of yeast ribosomes with liver protein kinase, of wheat ribosomes with yeast and liver protein kinases and of liver ribosomes with yeast and plant protein kinases. The wheat protein kinase does not phosphorylate the yeast ribosomes under these experimental conditions. Some differences in the set of phosphoproteins obtained with various protein kinases have been detected. These data suggest that the ribosomal protein phosphorylation is not highly species specific although it is not universal.     

Sequence analysis and heterologous expression of a new cytochrome P450 monooxygenase from Rhodococcus sp. for asymmetric sulfoxidation

In this study, a 3.7-kb DNA fragment was cloned from Rhodococcus sp. ECU0066, and the sequence was analyzed. It was revealed that the largest one (2,361 bp) of this gene fragment encodes a protein consisting of 787 amino acids, with 73% identity to P450RhF (accession number AF45924) from Rhodococcus sp. NCIMB 9784. The gene of this new P450 monooxygenase (named as P450SMO) was successfully expressed in Escherichia coli BL21 (DE3), and the enzyme was also purified and characterized. In the presence of reduced nicotinamide adenine dinucleotide phosphate, the enzyme showed significant sulfoxidation activity towards several sulfides, with (S)-sulfoxides as the predominant product. The p-chlorothioanisole, p-fluorothioanisole, p-tolyl methyl sulfide, and p-methoxythioanisole showed relatively higher activities than the other sulfides, but the stereoselectivity for p-methoxythioanisole was much lower. The optimal activity of the purified enzyme toward p-chlorothioanisole occurred at pH 7.0 and 30°C. The current study is the first to report a recombinant cytochrome P450 enzyme of Rhodococcus sp. which is responsible for the asymmetric oxidation of sulfides. The new enzymatic activity of P450SMO on the above compounds makes it an attractive biocatalyst for asymmetric synthesis of enantiopure sulfoxides.     

The use of synthetic genes for the expression of ciliate proteins in heterologous systems

The common fish parasite, Ichthyophthirius multifiliis, expresses abundant glycosylated phosphatidylinositol (GPI)-anchored membrane proteins known as immobilization antigens, or i-antigens. These proteins are targets of the host immune response, and have been identified as potential candidates for recombinant subunit vaccine development. Nevertheless, because Ichthyophthirius utilizes a non-standard genetic code, expression of the corresponding gene products, either as subunit antigens in conventional protein expression systems, or as vector-encoded antigens in the case of DNA vaccines, is far from straightforward. To overcome this problem, we utilized 'assembly polymerase chain reaction' to manufacture synthetic versions of two genes (designated IAG52A[G5/CC] and IAG52B[G5/CC]) encoding approximately 52/55 kDa i-antigens from parasite strain G5. This approach made it possible to eliminate unwanted stop codons and substitute the preferred codon usage of channel catfish for the native sequences of the genes. To determine whether the synthetic alleles could be expressed in cells that use the standard genetic code, we introduced IAG52A[G5/CC] into a variety of heterologous cell types and tested for expression either by immunofluorescence light microscopy or Western blotting. When cloned downstream of appropriate promoters, IAG52A[G5/CC] was expressed in Escherichia coli, mammalian COS-7 cells, and channel catfish where it elicited antigen-specific immune responses. Interestingly, the localization pattern of the corresponding gene product in COS-7 cells indicated that while the protein was correctly folded, it was not present on the cell membrane, suggesting that the signal peptides required for GPI-anchor addition differ in ciliate and mammalian systems. Construction of synthetic alleles should have practical utility in the development of vaccines against Ichthyophthirius, and at the same time, provide a general method for the expression of ciliate genes in heterologous systems.     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Function of heterologous and truncated RNase P proteins in Bacillus subtilis

Bacterial RNase P is composed of an RNA subunit and a single protein (encoded by the rnpB and rnpA genes respectively). The Bacillus subtilis rnpA knockdown strain d7 was used to screen for functional conservation among bacterial RNase P proteins from a representative spectrum of bacterial subphyla. We demonstrate conserved function of bacterial RNase P (RnpA) proteins despite low sequence conservation. Even rnpA genes from psychrophilic and thermophilic bacteria rescued growth of B. subtilis d7 bacteria; likewise, terminal extensions and insertions between beta strands 2 and 3, in the so-called metal binding loop, were compatible with RnpA function in B. subtilis. A deletion analysis of B. subtilis RnpA defined the structural elements essential for bacterial RNase P function in vivo. We further extended our complementation analysis in B. subtilis strain d7 to the four individual RNase P protein subunits from three different Archaea, as well as to human Rpp21 and Rpp29 as representatives of eukaryal RNase P. None of these non-bacterial RNase P proteins showed any evidence of being able to replace the B. subtilis RNase P protein in vivo, supporting the notion that archaeal/eukaryal RNase P proteins are evolutionary unrelated to the bacterial RnpA protein.     

Evidence for requirement of NADPH-cytochrome P450 oxidoreductase in the microsomal NADPH-sterol Delta7-reductase system

Rabbit antibodies raised against the hydrophilic part of microsomal NADPH-cytochrome P450 oxidoreductase (denoted fpT) demonstrated a marked ability to inhibit NADPH-sterol Delta7-reductase activity. In addition, trypsin and proteinase K treatment of microsomes removed almost all microsomal electron transfer constituents from the microsomes, but the Delta7-reductase activity could be reconstituted by adding detergent-solubilized NADPH-cytochrome P450 oxidoreductase (denoted OR). Furthermore, after solubilization from microsomes, the Delta7-reductase activity could be reconstituted with OR in a DEAE-cellulose column chromatography eluate fraction, which contained little OR activity. In the microsomal system, carbon monoxide, ketoconazole, and miconazole, specific inhibitors of cytochrome P450, had no effect on Delta7-reductase activity. These results provide the first evidence of an essential requirement of OR, which is distinct from cytochrome P450, in the NADPH-sterol Delta7-reductase system. EDTA, o-phenanthroline and KCN markedly lowered Delta7-reductase activity in a dose-dependent manner. Among metal ions tested, only ferric ion restored the reductase activity in the EDTA-treated microsomes. These results sugguest that NADPH-sterol Delta7-reductase is membrane-bound iron-dependent protein embedded in the microsomal lipid bilayer.     

Directed evolution of proteins for heterologous expression and stability

Recent developments have been made in the application of directed evolution to achieve the efficient heterologous expression of proteins in Escherichia coli and yeast by increasing the stability and solubility of the protein in the host environment. One interesting conclusion that emerges is that the evolutionary process often improves the stability and solubility of an intermediate (apoprotein, proprotein or folding intermediate) that otherwise constitutes a bottleneck to functional expression, rather than altering the protein's final state.     

Cynomolgus monkey cytochrome P450 2C43: cDNA cloning, heterologous expression, purification and characterization

The cDNA of cytochrome P450 (CYP) 2C43 was cloned from cynomolgus monkey liver by RT-PCR. The deduced amino acid sequence showed 93% and 91% identity to human CYP2C9 and CYP2C19, respectively. The cDNA was expressed in Escherichia coli and purified by a series of chromatography steps, yielding a specific content of 11.5 nmol P450/mg protein. The substrate specificity of the purified CYP2C43 was examined in a reconstitution system comprising NADPH-P450 reductase, lipid, cytochrome b(5) and CYP2C marker substrates. The purified CYP2C43 showed high activity for testosterone 17-oxidation and progesterone 21-hydroxylation, which were also observed for CYP2C19 but not CYP2C9. In addition, CYP2C43 showed activity for (S)-mephenytoin 4'-hydroxylation, a marker reaction for CYP2C19. With CYP2C9 marker substrates, CYP2C43 exhibited low activity for diclofenac 4'-hydroxylation and no activity for tolbutamide p-methylhydroxylation. Therefore, in terms of substrate specificity, our results indicate that CYP2C43 is similar to CYP2C19, rather than CYP2C9.     

Molecular cloning and heterologous expression in E. coli of cytochrome P45017alpha. Comparison of structural and functional properties of substrate-specific cytochromes P450 from different species

To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017 from species of the Bovidae family (sheep, goat, and bison), which catalyze 17-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C₁₉-steroids. Recombinant cytochromes P45017 were expressed in E. coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography. Highly purified cytochromes P45017 were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC. It is shown that each form of cytochrome P45017 is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b5 in the reaction mixture. The analysis of the activity of the known forms of cytochrome P45017 in view of the data obtained in the present work allows the division of known cytochromes P45017 into three main group: group A (pig, hamster, rat), cytochromes P45017 catalyze the reaction of 17-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17-hydroxyprogesterone and 17-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017, which have no or have insignificant 17,20-lyase activity in relation to 17-hydroxyprogesterone; group C (guinea pig), cytochrome P45017 which either has no or has insignificant 17,20-lyase activity on transformation 17-hydroxypregnenolone to dehydroepiandrosterone.     

Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast

Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.     

Yeast systems biotechnology for the production of heterologous proteins

Systems biotechnology has been established as a highly potent tool for bioprocess development in recent years. The applicability to complex metabolic processes such as protein synthesis and secretion, however, is still in its infancy. While yeasts are frequently applied for heterologous protein production, more progress in this field has been achieved for bacterial and mammalian cell culture systems than for yeasts. A critical comparison between different protein production systems, as provided in this review, can aid in assessing the potentials and pitfalls of applying systems biotechnology concepts to heterologous protein producing yeasts. Apart from modelling, the methodological basis of systems biology strongly relies on postgenomic methods. However, this methodology is rapidly moving so that more global data with much higher sensitivity will be achieved in near future. The development of next generation sequencing technology enables an unexpected revival of genomic approaches, providing new potential for evolutionary engineering and inverse metabolic engineering.