growth on methanol, enzymes required for catabolism of this substrate are expressed at very high levels with alcohol oxidase, the first enzyme in the methanol utilization pathway, encoded by the gene AOX1, representing as much as 30% of the cellular protein. Transcription of AOX1 is tightly regulated; in the absence of methanol, the AOX1 gene is completely turned off but responds rapidly to the addition of methanol to the medium. Therefore, the AOX1 promoter is an excellent candidate for producing large amounts of recombinant protein under controlled conditions. Moreover, the induction of the cloned gene can be timed to maximize recombinant protein production during large-scale fermentations. In contrast to S. cerevisiae, P. pastoris does not synthesize ethanol; therefore, very high cell densities of P. pastoris are attained, with the concomitant secretion of large quantities of protein. In addition, P. pastoris normally secretes very few proteins, thus simplifying the purification of secreted recombinant proteins.
Many P. pastoris expression vectors have been devised, each one having more or less the same format. The basic features include a gene of interest under the control of promoter and transcription termination sequences from the P. pastoris AOX1 gene, an E. coli origin of replication and selectable marker gene, and a yeast selectable marker gene (Fig. 3.31). The addition of a signal sequence from either the P. pastoris phosphatase PHO1 gene or another yeast gene facilitates the secretion of a recombinant protein. To avoid the problems of plasmid instability during long-term growth, most P. pastoris vectors are designed to be integrated into the host genome, often within the AOX1 gene or the HIS4 gene for histidine biosynthesis. Both the engineered gene of interest and a yeast selectable marker gene are inserted together into a specific chromosome site by either a single (Fig. 3.32A) or a double (Fig. 3.32B) recombination event. The P. pastoris expression system has been used to produce more than 100 different biologically active proteins from bacteria, fungi, invertebrates, plants, and mammals, including humans. Many of these proteins, such as the hepatitis B virus surface antigen, human serum albumin, and bovine lysozyme, are identical to the native proteins and thus authentic.
Figure 3.31 P. pastoris integrating expression vector. The gene of interest (GOI) is cloned between the promoter (AOX1p) and transcription termination-polyadenylation sequence (AOX1t) of the P. pastoris alcohol oxidase 1 gene. The HIS4 gene encodes a functional histidinol dehydrogenase of the histidine biosynthesis pathway. The ampicillin resistance (Ampr) gene and an origin of replication (oriE) function in E. coli. The segment marked 3′ AOX1 is a piece of DNA from the 3′ end of the alcohol oxidase 1 gene of P. pastoris. A double recombination event between the AOX1p and 3′ AOX1 regions of the vector and the homologous segments of chromosome DNA results in the insertion of the DNA carrying the gene of interest and the HIS4 gene.
Figure 3.32 Integration of DNA into a specific P. pastoris chromosome site by single (A) or double (B) recombination. (A) A single recombination (dashed line) between the HIS4 gene of an intact circular plasmid and a chromosome his4 mutant gene results in the integration of the entire vector, including the gene of interest (GOI) with the AOX1 promoter in the 5′ AOX1 DNA segment and the transcription termination–polyadenylation sequence from the AOX1 gene (TT), into the chromosome. The inserted DNA is flanked by recombined mutant his4 and functional HIS4 genes. The dot in the his4 gene represents the mutation. (B) A double recombination (dashed lines) between the cloned 5′ AOX1 and 3′ AOX1 DNA segments of a restriction endonuclease (RE) linearized DNA fragment from the vector and the corresponding chromosome regions results in the integration of the gene of interest (GOI) with the AOX1 promoter in the 5′ AOX1 segment, the transcription termination–polyadenylation sequence from the AOX1 gene (TT), and a functional HIS4 gene. The chromosome AOX1 coding region is lost as a result of the recombination event.
Authentic heterologous proteins for industrial and pharmaceutical uses have also been generated in other yeasts. For example, the α- and β-globin chains of human hemoglobin A were produced from cDNAs in the methylotrophic yeast Hansenula polymorpha. The thermotolerant dimorphic yeasts Arxula adeninivorans and Yarrowia lipolytica have demonstrated promising potential as hosts for high levels of heterologous-protein expression. These yeasts can grow at temperatures up to 48°C and can survive at higher temperatures (55°C) for several hours. At higher temperatures, the fungi grow in a mycelial form and revert to budding cells below 42°C. Some secreted proteins, such as glucoamylase and invertase, are produced at higher levels in mycelia. Cell morphology also influences posttranslational modification, with O-linked glycosylation predominating in budding cells while N-glycosylation occurs in both mycelial and budding cells. An additional advantage of A. adeninivorans is the ability to grow on a wide range of inexpensive carbon and nitrogen sources.
It is often necessary to try several host types in order to find the one that produces the highest levels of a biologically active recombinant protein. Differences in the processing and productivity of a particular protein can occur among different yeast strains. For example, both S. cerevisiae and H. polymorpha produced a truncated version of the human protein interleukin-6 (IL-6), whereas A. adeninivorans produced a full-length version of the protein. The construction of a wide-range yeast vector for expression in several fungal species has facilitated this trial-and-error process (Fig. 3.33). The basic vector contains features for propagation and selection in E. coli and a multiple cloning site for insertion of interchangeable modules that are chosen for a particular yeast host, including a sequence for vector integration into the fungal genome, a suitable origin of replication, a promoter to drive expression of the heterologous gene, and selectable markers to complement a range of nutritional auxotrophies or to confer resistance to antifungal compounds, such as hygromycin B (Table 3.12). In other words, by selecting from a range of available modules, customized vectors can be rapidly and easily constructed for expression of the same gene in several different yeast cells to determine which host is optimal for heterologous-protein production.
Figure 3.33 A wide-range yeast vector system for expression of heterologous genes in several different yeast hosts. The basic vector contains a multiple cloning site (MCS) for insertion of selected modules containing appropriate sequences for chromosomal integration (rDNA module), replication (ARS module), selection (Selection module), and expression (Expression module) of a target gene in a variety of yeast host cells (Table 3.12 shows examples of interchangeable modules). Sequences for maintenance (oriE) and selection (Ampr) of the vector in E. coli are also included.
Table 3.12 Examples of modules available for wide-range yeast vector systems
Expression vectors with appropriate transcription and translation control elements for the expression of recombinant proteins in filamentous fungi are also commercially available. Distinct from unicellular yeasts, filamentous fungi are multicellular, microscopic fungi that produce long, branching strands of cells called hyphae. This group of fungi includes the common mold genera Penicillium, Rhizopus, Trichoderma, and Aspergillus. Many species of these genera of filamentous fungi are a rich natural resource for commercially important metabolites and enzymes and have also been used as cell factories for the production of recombinant proteins for the food, beverage, pulp and paper, and pharmaceutical industries (Table 3.13).
