P), which is considered as a standard promoter for methanol-free expression systems [35][36]. This promoter is regulated by the metabolism of carbon sources, and its transcription level is the highest in the presence of glucose and is the lowest when cells are fed with methanol [37].
P
GAP is generally weaker than P
AOX1 and attempts have hence been made to improve the strength of P
GAP. Ata et al. [42] analyzed the transcription factor binding sites of P. Ata et al. [38] analyzed the transcription factor binding sites of P GAP. Via targeted deletion or overexpression of these sites, a promoter library was constructed with different strength in initiating gene expression.
3.3.3. Other Promoters
Although the use of methanol as the sole source of carbon and energy is a major advantage of the P. pastoris cell factories, the toxicity and safety issues of methanol impose many limitations on practical industrial applications [44]. To solve this problem, methanol-free induction systems have been developed based on P cell factories, the toxicity and safety issues of methanol impose many limitations on practical industrial applications [39]. To solve this problem, methanol-free induction systems have been developed based on P AOX1. Chang et al. synthesized a positive feedback circuit in which P
AOX2-driven Mxr1 promotes the transcription of P
AOX1AOX1 is induced under glycerol starvation or in the absence of carbon sources [45]. Kinases have been proposed as potential targets for regulating the repression of P is induced under glycerol starvation or in the absence of carbon sources [40]. Kinases have been proposed as potential targets for regulating the repression of P AOX1 in the presence of a common carbon source such as glycerol [46]. By targeting the genes encoding glycerol kinase ( in the presence of a common carbon source such as glycerol [41]. By targeting the genes encoding glycerol kinase ( gut1) and dihydroxyacetone kinase (
dak), non-methanol-inducible P
AOX1 expression systems were constructed using glycerol and dihydroxyacetone as carbon sources to induce P
AOX1 expression, respectively, although the induction was not as efficient as that induced by methanol [47].
expression, respectively, although the induction was not as efficient as that induced by methanol [42].
3.3.4. Synthetic Core Promoter Engineering
The core promoter region plays a pivotal role in the regulation of gene expression, and its genetic modification is an important content of promoter engineering. The core promoter is the region necessary for RNA polymerase to recognize and initiate transcription, and it consists of the RNA polymerase binding site, the TATA box, and the transcription start site. In
P. pastoris, fully synthetic core promoters and the 5′-untranslated region have been designed and applied to P
AOX1, resulting in a series of promoter libraries with different expression levels
[59][43].
3.4. Signal Peptides
Secretory expression of proteins is generally dependent on cleavable signal peptides (usually 15–50 amino acids) that direct the transmembrane transfer of the newly synthesized peptides and proteins. These signal peptides, mostly located at the N-termini of the secreted proteins, usually contain three domains, i.e., the positively charged basic N-terminus (1–5 amino acids), the hydrophobic center that forms a helical structure (7–15 amino acids), and the highly polar C-terminus (3–7 amino acids) that serves as the cleavage site
[61][44]. With a great impact on the extent of protein folding and the rate of protein secretion, signal peptides play a crucial role in high-level expression and secretion of functional proteins
[62][45].
The signal peptides commonly used for protein secretion in
P. pastoris include the signal sequence of α-factor and invertase-2 (SUC2) of
S. cerevisiae, and
P. pastoris acid phosphatase signal peptide (PHO1). Among these, α-factor is used most frequently and is mainly suitable for the secretory expression of peptides and small proteins. To further improve the efficiency of secretion, this signal peptide has been engineered through codon optimization, modification of the hydrophobic region, addition of spacer sequences, and site-directed mutagenesis
[63,64,65][46][47][48].
3.5. CRISPR/Cas9 Genome Editing in Pichia pastoris
Since
P. pastoris is an important workhorse for the synthesis of various bio-products, it is crucial to establish efficient and concise gene editing technologies for the genetic modifications of this microbe. Traditionally, gene insertion/deletion/replacement of
P. pastoris relies on homologous recombination, which is inefficient with a low rate of success even when long homologous arms (sometimes more than 1 kb) are used. This is due to the domination of NHEJ over homologous recombination in this yeast
[11]. Deletion of
KU70 impairs NHEJ and significantly facilitates homologous recombination at the expense of a lower transformation efficiency and slower cell growth.
CRISPR/Cas9 introduces breaks in DNA sequences complementary to the sgRNA, which are then repaired by host cells. Thereby, genetic modifications can be introduced programmably at desired locations by using sgRNA with particularly designed sequences. Compared with traditional homologous recombination-guided genomic modification, CRISPR/Cas9 is highly flexible in the sense that only sgRNA needs to be re-designed for each independent genomic modification process. As one of the most potent and convenient gene editing technologies, CRISPR/Cas9 has been explored extensively in the engineering of
P. pastoris [8,69,70][8][49][50].
CRISPR/Cas9-mediated genomic editing in
P. pastoris relies on the correct expression of Cas9 and sgRNA in the nucleus, which can be affected by a series of factors. To improve the expression, researchers used RNA Pol II promoter for sgRNA expression, added ribozyme sequences both upstream and downstream of sgRNA, and optimized the codon of Cas9. After such optimization, near 100% efficiency could be achieved in
P. pastoris for gene deletion, and multiplex gene deletion and targeted gene insertion was achieved efficiently with the aid of NHEJ
[71][51]. This system was further introduced into a
KU70-knockout strain, so that DNA breaks could be repaired through homologous recombination. Despite lower cell viability, near 100% efficiency of gene integration was achieved
[72][52].
The biosynthetic pathways of value-added compounds (natural products and bulk chemicals) are often complex and involve multiple pathway genes. Therefore, a competent genetic tool that can manipulate multi-gene pathways has important implications for the application of
P. pastoris as a cell factory. Based on the
KU70 knockout strain, a CRISPR/Cas9-mediated marker-less multi-site gene integration method has been developed, for which various sgRNA targets are designed within 100 bp upstream of the promoter or downstream of the terminator. Using this method, the integration efficiency of double-locus could reach 57.7%–70%
[73][53]. The same method was used to establish a standardized CRISPR-based synthetic biology toolkit, in which the integration efficiency of double-locus could reach ~93%
[74][54]. This toolkit allowed for one-step assembly of the biosynthetic pathways of 2,3-butanediol,
β-carotene, zeaxanthin, and astaxanthin
[74][54].
4. Adaptive Laboratory Evolution of Pichia pastoris
Adaptive laboratory evolution (ALE) is a method to artificially simulate the mutation and selection process in natural evolution under laboratory conditions such that the directed evolution of microorganisms can be achieved within a short period of time and mutated microbes with desired traits can be screened
[76][55]. Compared with metabolic engineering, ALE only focuses on the generation of appropriate interference factors without detailed information on the intricate and intersecting metabolic networks, thus demonstrating broad applicability and strong practicability. ALE is one of the most effective methods of strain construction toward high-level synthesis of bio-products
[77,78][56][57]. Although widely used in
S. cerevisiae and
E. coli, ALE only has limited applications in
P. pastoris, and there is still a large space for development.
Efficient use of carbon sources and substrates is key to the high-level microbial production of bio-products, and ALE has been adopted in promoting the metabolic performances of
P. pastoris on various nutrients or substrates. Moser et al. investigated the effect of growth media on cell growth and recombinant protein production in
P. pastoris X-33 using methanol as a carbon source for continuous subculture in eutrophic medium YPM and low-nutrient medium BMM. After approximately 250 generations, evolved strains showed higher growth rates. Whole genome sequencing identified mutations in the
AOX1 gene involving the methanol binding region and its vicinity, leading to, surprisingly, a decline in AOX activity, possibly due to less intracellular accumulation of the toxic compound formaldehyde. Such methanol adaptation led to significantly higher titers of recombinant human serum albumin and fused lobes hexosaminidases
[79][58].
5. Practical Applications of Pichia pastoris as a Cell Factory
Since Philips Petroleum Company released the
P. pastoris expression system to academic research laboratories in 1993, the expression system has developed rapidly
[83][59].
P. pastoris has gradually replaced
S. cerevisiae as the eukaryotic expression system because it secretes very few endogenous proteins and has glycosylation similar to that in mammalian cells.
P. pastoris has thus been gradually developed into a common host for the expression of medical and industrial enzymes, and thousands of recombinant proteins have been successfully produced
[84][60]. In addition, natural products with diverse structures have also been synthesized in this host (
Figure 4), which overcomes the disadvantages associated with chemical synthesis or extraction from plants that are traditionally used for their production. These accomplishments have promoted the engineering and development of
P. pastoris as a potent and potential microbial platform
[8].
Figure 4. The portfolio of typical compounds produced by engineered
P. pastoris.
5.1. Recombinant Proteins
5.1.1. Nanobodies
Nanobodies, the natural antibodies first found in the serum of camels and sharks, are the smallest units known to bind antigens [100,101]. Compared with traditional antibodies, nanobodies have unique properties such as strong antigen binding, low immunogenicity, high solubility and stability, and low molecular weight, which offer potential advantages in disease diagnosis and treatment [102]. For example, nanobody neutralization therapy has been employed in the treatment of the coronavirus COVID-19 [103]. Nanobodies can be stably expressed in .
5.1. Recombinant Proteins
5.1.1. Nanobodies
Nanobodies, the natural antibodies first found in the serum of camels and sharks, are the smallest units known to bind antigens [61][62]. Compared with traditional antibodies, nanobodies have unique properties such as strong antigen binding, low immunogenicity, high solubility and stability, and low molecular weight, which offer potential advantages in disease diagnosis and treatment [63]. For example, nanobody neutralization therapy has been employed in the treatment of the coronavirus COVID-19 [64]. Nanobodies can be stably expressed in P. pastoris besides prokaryotic hosts [104].
5.1.2. Human Proteins
Human serum albumin (HSA) is the major protein in human plasma and is widely used for drug delivery. Its recombinant expression in besides prokaryotic hosts [65].
5.1.2. Human Proteins
Human serum albumin (HSA) is the major protein in human plasma and is widely used for drug delivery. Its recombinant expression in P. pastoris GS115 has been reported, and a high yield of 8.86 g/L was obtained after process optimization [109]. Human coagulation factor XII plays an important role in thrombosis, and its abundant supply is necessary for inhibitor screening in the development of antithrombotic drugs. The recombinant serine protease domain of human coagulation factor XII has been expressed in GS115 has been reported, and a high yield of 8.86 g/L was obtained after process optimization [66]. Human coagulation factor XII plays an important role in thrombosis, and its abundant supply is necessary for inhibitor screening in the development of antithrombotic drugs. The recombinant serine protease domain of human coagulation factor XII has been expressed in P. pastoris X-33 with a yield of 20 mg/L and a clotting activity similar to that of its natural counterpart [110]. Compared with these cellular proteins which can be expressed easily in the soluble form, high-level microbial expression of human membrane proteins can be a real challenge. Nonetheless, a human multichannel membrane protein named sterol ∆8-∆7 isomerase has been successfully expressed in the form of a GFP fusion in X-33 with a yield of 20 mg/L and a clotting activity similar to that of its natural counterpart [67]. Compared with these cellular proteins which can be expressed easily in the soluble form, high-level microbial expression of human membrane proteins can be a real challenge. Nonetheless, a human multichannel membrane protein named sterol ∆8-∆7 isomerase has been successfully expressed in the form of a GFP fusion in P. pastorisE. coliS. cerevisiae, with the best expression achieved in
P. pastoris at 200 mg/L in shake flasks and 1000 mg/L in condensed culture [111].
5.2. Value-Added Compounds
at 200 mg/L in shake flasks and 1000 mg/L in condensed culture [68].
5.2. Value-Added Compounds
5.2.1. Terpenoids
Terpenoids are secondary metabolites with isoprene as the basic structural unit, and they mainly include monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids, and polyterpenoids
[115][69]. Most of these compounds have anti-tumor, anti-inflammatory, and immunomodulatory effects, and are widely used in food processing and pharmaceutical manufacturing industries
[116][70]. Yeast cells generally produce more precursors than bacteria for terpenoid biosynthesis, including DMAPP (dimethylallyl diphosphate) and IPP (isopentenyl diphosphate)
[117][71]. Compared with other yeast chasses,
P. pastoris can reach a higher fermentation density, produce fewer metabolic byproducts, and present strong tolerance to complex environments. These traits make
P. pastoris suitable for terpenoid biosynthesis.
5.2.2. Polysaccharides
Polysaccharides widely exist in nature and participate in important cellular processes as an energy source and organizational structure. Polysaccharides can be used as efficient drug carriers due to their biodegradability, biocompatibility, and generally low costs
[120][72]. Polysaccharide biosynthesis using engineered microorganisms has been attracting increasing attention, with some of the focus laid on
P. pastoris due to its abundant supply of sugar precursors.
5.2.3. Polyketides
Polyketides are a class of widely distributed natural products produced during the secondary metabolism of plants or microorganisms, with rich chemical properties and unique physiological activities. Polyketide biosynthesis in
P. pastoris was first reported in 2013 for the fungal polyketide compound 6-methylsalicylic acid (6-MSA)
[92][73]. The genes encoding
Aspergillus nidulans phosphopantetheinyl transferase (
npgA) and
Aspergillus terrus 6-MSA synthase (
atX) were integrated into the genome of
P. pastoris via homologous recombination, thus producing 2.2 g/L of 6-MSA in a 5 L bioreactor upon 20 h of methanol induction. On this basis, a more structurally complex polyketide citrinin was also synthesized in
P. pastoris by assembling
npgA,
Monascus purpureus citrinin polyketide synthase gene
pksCT, and several genes in the citrinin gene cluster
[93][74].
6. Conclusions
Pichia pastoris has been developed to produce proteins for four decades and has emerged as a new chassis to produce diverse chemicals and natural products in recent years. With the unique property of utilizing methanol as a sole carbon source, metabolic engineering of
P. pastoris is attracting increasing attention amid the great concern on global energy security, given the potential of methanol as a supply of energy and carbon source for biomanufacturing.
P. pastoris has shown great success in the biosynthesis of some natural products with the titers reaching >1 g/L, indicating very promising applications for industrialization and commercialization.