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Gene of interest number of copies in transformed K. pastoris

Gene of interest number of copies in transformed K. pastoris


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When transforming a K. pastoris strain, is there a way to predict how many copies of the gene will be integrated following the homologous recombination? More specifically is it something that a molecular biologist is able to control in a simple way, or, is it usually left to the chance (and determined afterward using gene sequencing)?

Alternatively : if I want to transform an electrocompetent wild type strain such as Pichia X33 using a plasmid vector, is the multiple integration an iterative process I control or is it random?)

Edit: Thank you all for the answers. Finally if I understand well the process is pretty random but you can control it to some extent. @gaspanic: I keep the idea of using fluorescent reporters and FACS for that ;)


To predict the number integrations is probably not possible, but you can favor multiple integrations, if that's what you are after. At least in Saccharomyces cerevisiae integration of a first plasmid appears to stimulate the insertion of additional plasmids at the same site (1). It is therefore possible to favor multiple integrations (2) by increasing the DNA-to-cell ratio in the transformation, thereby increasing the chance that any competent cell receives more than one copy of the plasmid, which could then integrate in tandem. The easiest way is to simply use more DNA, but depending on your cell competence you might need to compensate this increase by using less cells for your transformation (or simply plate less cells on each plate) as you will probably also increase the overall colony yield.

One way to actively select for multiple integrations is to use a competitive inhibitor for the selection marker. Again, in Saccharomyces cerevisiae transformations with HIS3 selection can be combined with 3-AT (3). This allows you to inhibit HIS3 activity just enough to only permit the growth of cells that have integrated a desired number of copies of the plasmid. Of course, this method is far from fool-proof, as you might also select for mutants that have increased HIS3 expression for other reasons. Edit: I actually found one paper doing this in K. pastoris (4).

Finally, if your implementation allows it you can facilitate the screening for multiple integrations by including a fluorescent protein-expressing cassette in your plasmid. That way you can easily screen for different numbers of integrations by fluorescence microscopy or FACS.

Sources:

  1. Orr-Weaver et al. (1983): https://mcb.asm.org/content/3/4/747
  2. Plessis and Dujon (1993): https://doi.org/10.1016/0378-1119(93)90172-Y
  3. Wikipedia (retrieved 15 Aug 2020): https://en.wikipedia.org/wiki/3-Amino-1,2,4-triazole
  4. Menéndez et al. (2018): https://doi.org/10.2144/00295rr05

Pichia pastoris as an expression host for membrane protein structural biology

Pichia pastoris is a well established recombinant expression system.

A number of integral membrane proteins have been produced in this system.

The protein has been used to solve several high resolution structures.

Key features of the P. pastoris expression system are described.

A summary of the tools available for membrane protein expression is included.

The methylotrophic yeast Pichia pastoris is a widely used recombinant expression host. P. pastoris combines the advantages of ease of use, relatively rapid expression times and low cost with eukaryotic co-translational and post-translational processing systems and lipid composition. The suitability of P. pastoris for high density controlled culture in bioreactors means large amounts of protein can be obtained from small culture volumes. This review details the key features of P. pastoris, which have made it a particularly useful system for the production of membrane proteins, including receptors, channels and transporters, for structural studies. In addition, this review provides an overview of all the constructs and cell strains used to produce membrane proteins, which have yielded high resolution structures.


Guide to Protein Purification, 2nd Edition

James M. Cregg , . Thomas Chappell , in Methods in Enzymology , 2009

Abstract

The yeast Pichia pastoris has become the premier example of yeast species used for the production of recombinant proteins. Advantages of this yeast for expression include tightly regulated and efficient promoters and a strong tendency for respiratory growth as opposed to fermentative growth. This chapter assumes the reader is proficient in molecular biology and details the more yeast specific procedures involved in utilizing the P. pastoris system for gene expression. Procedures to be found here include: strain construction by classical yeast genetics, the logic in selection of a vector and strain, preparation of electrocompetent yeast cells and transformation by electroporation, and the yeast colony western blot or Yeastern blot method for visualizing secreted proteins around yeast colonies.


Guide to Protein Purification, 2nd Edition

James M. Cregg , . Thomas Chappell , in Methods in Enzymology , 2009

Abstract

The yeast Pichia pastoris has become the premier example of yeast species used for the production of recombinant proteins. Advantages of this yeast for expression include tightly regulated and efficient promoters and a strong tendency for respiratory growth as opposed to fermentative growth. This chapter assumes the reader is proficient in molecular biology and details the more yeast specific procedures involved in utilizing the P. pastoris system for gene expression. Procedures to be found here include: strain construction by classical yeast genetics, the logic in selection of a vector and strain, preparation of electrocompetent yeast cells and transformation by electroporation, and the yeast colony western blot or Yeastern blot method for visualizing secreted proteins around yeast colonies.


Romanos, M.A., Scorer, C.A. and Clare, J.J. 1992. Foreign gene expression in yeast: a review. Yeast 8: 423–488.

Cregg, J.M., Tschopp, J.F., Stillman, C., Siegel, R., Akong, M., Craig, W.S., Buckholz, R.G., Madden, K.R., Kellaris, P.A., Davies, G.R., Smiley, B.L., Cruze, J., Torregrossa, R., Velicelebi, G. and Thill, G.P. 1987. High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Bio/Technology 5: 479–485.

Sreekrishna, K., Nelles, L., Potenz, R., Cruze, J., Mazzaferro, P., Fish, W., Motohiro, F., Holden, K., Phelps, D., Wood, P. and Parker, K. 1989 High-level expression, purification, and characterisation of recombinant human tumour necrosis factor synthesised in the methylotrophic yeast Pichia pastoris. Biochemistry 28: 4117–4125.


Introduction

The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production, due to a highly efficient secretion mechanism and the possibility of reaching high product titres for simpler enzymes such as phytase and complex proteins containing multiple post-translational modifications, e.g. monoclonal antibodies 1,2,3,4,5 . Research by Kurtzman et al. 6,7 resulted in the reclassification of the P. pastoris genus as Komagatella, including the subspecies K. phaffii and K. pastoris. However, they are still commonly referred to as P. pastoris. In recent years, the genetic toolbox for P. pastoris has been markedly expanded with several newly discovered native promoters, synthetic promoters and other regulatory elements 8,9,10,11 . The construction of optimized strains and vectors enabled new applications, e.g. the production of metabolites or expression of proteins lacking yeast specific hypermannosylation patterns 12,13 . Additionally, in a very recent publication Weninger et al. 14 reported the first CRISPR/Cas9 system for P. pastoris opening up new possibilities for genetic engineering approaches.

Nevertheless, the most frequently used approach for introducing the target gene in P. pastoris is still the integration of an expression cassette into the genome via homologous recombination. The most popular target for integration is the AOX1 (alcohol oxidase 1) locus that represents the stronger expressed of the two alcohol oxidases in P. pastoris. This approach usually involves the utilization of the AOX1 promoter (pAOX1) as homologous sequence and as promoter of the target gene, because it offers very high expression levels and tight regulation 15 . After a successful integration, the gene expression can be induced with methanol. However, a clone with an intact AOX1 can metabolize methanol at a higher rate, designated as “methanol utilization plus” (Mut + ), complicating the maintenance of a constant induction 16,17 . Therefore, the application of expression cassettes with two homologous sequences targeted for mediating the replacement of AOX1 is one possible technique. A knock-out mutation of AOX1 leads to the phenotype “methanol utilization slow” (Mut S ) easing process control and allowing to select for correct integration based on the phenotype.

Despite using comparatively long homologous sequences (ca. 1000 bp), a high variance in targeting efficiency is observed, indicating the prevalence of the non-homologous end joining (NHEJ) pathway in P. pastoris 18,19,20 . As a result, a high clonal variability is found after transformation, necessitating a time and labour-intensive screening process for the clone with the desired characteristics 21,22 . Concerning productivity characteristics, the different expression levels of clones typically originate from varying gene copy numbers 23,24 . The clonal variability is regarded as an inherent property of P. pastoris that is a by-product of the available and established transformation techniques in combination with the strong NHEJ pathway in this yeast species. Many other yeasts, filamentous fungi and higher eukaryotes that are used in biotechnological applications display similar or more pronounced clonal variabilities due to a predominant NHEJ pathway 25,26,27 , while in the model yeast Saccharomyces cerevisiae homologous recombination is dominant over the NHEJ pathway 28 . Different techniques to reduce the clonal variability in P. pastoris have been proposed 14,18,20,29 . However, disadvantages like high complexity, lower strain fitness or not yet fully realized methods for donor cassette integration have kept these techniques from replacing the established ones for the efficient construction of producer strains. Therefore, successive genetic manipulation steps, e.g. for the construction of biosynthetic pathways, have been comparatively challenging in P. pastoris and only a few applications have been reported so far 30,31,32,33,34 . To date, the humanisation of the N-glycosylation pathway in P. pastoris via multiple consecutive cloning steps has been the most sophisticated and successful genetic engineering endeavour 13,35 . Similar observations have been made for other non-conventional yeast like Hansenula polymorpha, Kluyveromyces lactis and Yarrowia lipolytica 25,36,37 .

In our previous study 24 , we concentrated on analysing the clonal variability found in 845 P. pastoris strains transformed with a GFP expression cassette targeted for AOX1 replacement. By screening all clones for their Mut-phenotype, GFP productivity and gene copy number 31 clones with interesting features were selected for genome sequencing. The combination of experimental and genome data allowed the discovery of novel insights into the correlation between productivity and integration event. In the majority of cases variations in the productivity could be traced back to gene copy number related effects. No off-target integrations were found in clones with a high productivity, suggesting that the impact of such events on productivity was low.

During the Mut-phenotype assay, strains were discovered that displayed an abnormal colony morphology on plate. Some of these clones were also selected for genome sequencing. Here, we discuss the results for these strains, elucidating a variety of as yet unreported off-target integration events. Besides the disruption of random genes, the co-integration of DNA elements from the plasmid host E. coli KRX as well as the relocation of the AOX1 locus to another chromosome were discovered. The detrimental effect of these events on the genetic integrity and strain morphology presents an additional burden for the screening process. These observations are especially relevant for metabolic engineering or knock-out experiments in P. pastoris, while the integration events detailed in our previous study 24 are more pertinent to experiments targeting the creation of high producer strains.

In recent years, metabolic engineering of non-conventional yeast in general and P. pastoris in particular has gained more interest 11,38 . Therefore, the discussed integration events and the proposed theories explaining their origin will enable scientists working in this field to modify existing transformation and mutagenesis protocols in order to avoid these events. Based on this strategy, clonal diversity can be reduced and genetic engineering processes of higher complexity can be realized.


5. OTHER BIOMOLECULES PRODUCED BY P. pastoris

The P. pastoris has also been established as a versatile cell factory for the production of thousands of biomolecules both on a laboratory and industrial scale. Some of the new recombinant biological molecules that have been expressed in the P. pastoris system are listed in Table  6 .

Table 6

Pichia pastoris as a suitable host for the production of recombinant biological molecules

ProductUsed strainUsed vectorUsageReference
Lycopene and β�roteneX�pGAPZAFeed supplementsAraya‐Garay, Feijoo‐Siota, Rosa𠄍os‐Santos, Veiga𠄌respo, and Villa (2012)
PlectasinX�pPICZ㬚Antibacterial peptideJ. Zhang et al. (2011)
Bovine lactoferrinKM71HpJ902Transferrin and antibacterial proteinIglesias𠄏igueroa et al. (2016)
Bovine IFN‐αGS115pPIC9KPrevention and therapy of viral diseasesTu et al. (2016)
ApidaecinSMD1168pPIC9KAntibacterial peptideX. Chen et al. (2017)
hPAB‐βGS115pPIC9KAntibacterial peptideZ. Chen et al. (2011)
Tachyplesin IGS115pGAPZ㬛Antibacterial peptideH. Li et al. (2019)
Snakin𠄁GS11pPIC9Antimicrobial peptideKuddus et al. (2016)
PAF102X�pGAPZAAntifungal peptidePopa, Shi, Ruiz, Ferrer, and Coca (2019)
Pisum sativum defensin 1GS115pPIC9KAntifungal peptideCabral, Almeida, Valente, Almeida, and Kurtenbach (2003)
Class I chitinaseKM71HpPICZ㬚Antifungal peptideLandim et al. (2017)
Ch‐penaeidinKM71HpPIC9KAntimicrobial peptideL. Li et al. (2005)
HispidalinGS115pPICZ㬚Antimicrobial peptideMeng et al. (2019)
Fowlicidin𠄂X�pPICZ㬚Antimicrobial peptideXing et al. (2016)
Parasin IX�pPICZ㬚Antimicrobial peptideH. Zhao et al. (2015)
CecropinA‐thanatinX�pPICZ㬚Antimicrobial peptideZ. Liu et al. (2018)
Type I collagen Connective tissueNokelainen et al. (2001)
Human serum albuminGS115pPIC9KMaintaining osmolarity and carrier in bloodW. Zhu et al. (2018)
LegumainX�pPICZ㬚Lysosomal proteaseT. Zhao et al. (2018)
Goat chymosinX�pPICZ㬚Hydrolysis of κ�seinTyagi et al. (2016)
Carrot antifreeze proteinGS115pPIC9KInhibition of gluten deteriorationM. Liu et al. (2018)
ProinsulinSuperMan5pPICZ㬚Treatment of diabetes mellitusBaeshen et al. (2016)
hIFN‐γX�, GS115, KM71H, CBS7435pPICZα, pPIC9, pPpT4aSCritical cytokine for innate and adaptive immunityRazaghi et al. (2017)
IL𠄁βGS115, SMD1168, X�pPICZ𠄊Proinflammatory cytokineLi et al. (2016)
IL𠄃X�pPICZ㬚Multipotent hematopoietic cytokineDagar and Khasa (2018)
IL�GS115pPINKαHCThrombopoietic growth factorYu et al. (2018)
IL�X�pPICZ㬚Differentiation and proliferation of T, B, and NK cellsW. Sun et al. (2016)
Cyanate hydrataseGS115pPICZ㬚Detoxification of cyanate and cyanideRanjan, Pillai, Permaul, and Singh (2017)
Human antiplatelet scFv antibodyX�pPICZ㬚Treatment of atherosclerosisVallet𠄌ourbin et al. (2017)
α𠄊mylaseX�pPICZ㬚Starch saccharificationParashar and Satyanarayana (2017)
Human epidermal growth factorGS115pPIC9KGeneration of new epithelial and endothelial cellsEissazadeh et al. (2017)
BromelainKM71HpPICZ㬚Oedematous swellingsLuniak, Meiser, Burkart, and Müller (2017)
Keratinocyte growth factorX�pPICZ㬚Epithelialization‐phase of wound healingKalhor (2016)
DM64X�pPICZ㬚Anti‐myotoxicVieira, da Rocha, da Costa Neves�rreira, Almeida, and Perales (2017)
TrypsinGS115pPIC9KHydrolysis of proteins in the digestive systemY. Zhang et al. (2018)
Human sialyltransferaseKM71HpPICZ㬛Pharmacological usesLuley‐Goedl et al. (2016)
TransglutaminaseGS115pPIC9KRestructured meat productsX. Yang and Zhang (2019)
StreptokinaseX�pPICZ㬚Thrombolytic medicationDagar, Devi, and Khasa (2016)
StaphylokinaseGS115, KM71HpPICZ㬚Thrombolytic medicationFaraji et al. (2017)
TFPR1X�pPICZ㬚AdjuvantNing et al. (2016)

Abbreviations: hIFN‐γ, human interferon γ IL, interleukin NK, natural killer.


Steps in Gene Cloning

The basic 7 steps involved in gene cloning are:

  1. Isolation of DNA [gene of interest] fragments to be cloned.
  2. Insertion of isolated DNA into a suitable vector to form recombinant DNA.
  3. Introduction of recombinant DNA into a suitable organism known as host.
  4. Selection of transformed host cells and identification of the clone containing the gene of interest.
  5. Multiplication/Expression of the introduced Gene in the host.
  6. Isolation of multiple gene copies/Protein expressed by the gene.
  7. Purification of the isolated gene copy/protein

A. Isolation of the DNA fragment or gene

  • The target DNA or gene to be cloned must be first isolated. A gene of interest is a fragment of gene whose prod­uct (a protein, enzyme or a hormone) interests us. For example, gene encoding for the hormone insulin.
  • The desired gene may be isolated by using restriction endonuclease (RE) enzyme, which cut DNA at specific recognition nucleotide se­quences known as restriction sites towards the inner region (hence endonuclease) producing blunt or sticky ends.
  • Sometimes, reverse transcriptase enzyme may also be used which synthesizes complementary DNA strand of the desired gene using its mRNA.

B. Selection of suitable cloning vector

  • The vector is a carrier molecule which can carry the gene of interest (GI) into a host, replicate there along with the GI making its multiple copies.
  • The cloning vectors are limited to the size of insert that they can carry. Depending on the size and the application of the insert the suitable vector is selected.
  • The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes (MACs).
  • However, the most commonly used cloning vectors include plasmids and bacteriophages (phage λ) beside all the other available vectors.

C. Essential Characteristics of Cloning Vectors

All cloning vectors are carrier DNA molecules. These carrier molecules should have few common features in general such as:

  • It must be self-replicating inside host cell.
  • It must possess a unique restriction site for RE enzymes.
  • Introduction of donor DNA fragment must not interfere with replication property of the vector.
  • It must possess some marker gene such that it can be used for later identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell).
  • They should be easily isolated from host cell.

D. Formation of Recombinant DNA

  • The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA fragment.
  • The mixture of donor DNA fragment and plasmid vector are mixed together.
  • In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid vector occurs.
  • The result­ing DNA molecule is a hybrid of two DNA molecules – the GI and the vector. In the ter­minology of genetics this intermixing of dif­ferent DNA strands is called recombination.
  • Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is referred to as the recom­binant DNA technology.

E. Transformation of recombinant vector into suitable host

  • The recombinant vector is transformed into suitable host cell mostly, a bacterial cell.
  • This is done either for one or both of the following reasons:
    • To replicate the recombinant DNA mol­ecule in order to get the multiple copies of the GI.
    • To allow the expression of the GI such that it produces its needed protein product.
    • Some bacteria are naturally transformable they take up the recombinant vector automatically.

    For example: Bacillus, Haemophillus, Helicobacter pylori, which are naturally competent.


    Expression of Recombinant Proteins in Pichia Pastoris

    Pichia pastoris has been used extensively and successfully to express recombinant proteins. In this review, we summarize the elements required for expressing heterologous proteins, and discuss various factors in applying this system for protein expression. These elements include vectors, host strains, heterologous gene integration into the genome, secretion factors, and the glycosylation profile. In particular, we discuss and evaluate the recent progress in optimizing the fermentation process to improve the yield and stability of expressed proteins. Optimization can be achieved by controlling the medium composition, pH, temperature, and dissolved oxygen, as well as by methanol induction and feed mode.

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    Results and discussion

    GoldenPiCS, our Golden Gate-derived P. pastoris cloning system, consists of three hierarchical backbone (BB) levels for flexible generation of overexpression plasmids containing multiple transcription units (up to eight per plasmid), four different selection markers and five loci either for targeted genome integration or episomal plasmid maintenance (Fig. 1). We mainly designed the system to enable advanced cell engineering or the expression of whole metabolic pathways. In the lowest cloning level, individual parts such as promoters, coding sequences (e.g. reporters or GOIs) and transcription terminators are incorporated into backbone 1 (BB1) plasmids, which are subsequently assembled to one transcription unit in BB2. This is followed by the assembly of multiple expression units into one BB3, which is designed for subsequent genome integration in P. pastoris (four different selection markers and four loci for targeted genome integration are available). Contrary to other cloning techniques, Golden Gate Assembly avoids the need for excessive sequencing, because BB2 and BB3 constructs are assembled by ligation instead of overlap-extension PCR (only BB1 inserts require sequencing after assembly). Correct ligation is assured by defined fusion sites (see Fig. 1): Fusion sites Fs1 to Fs4 are linked to individual parts by PCR and required for their assembly into a transcription unit (BB2) e.g. promoters with coding sequences (‘CATG’, fusion site Fs2). Fusion sites FsA to FsI are used for the assembly of multiple expression units in BB3 e.g. for fusing the first transcription unit to the second (‘CCGG’, fusion site FsB). The different BB3 vectors with fusion sites FsA-FsC, FsA-FsD, to FsA-FsI are designed for the assembly of two, three, … up to eight transcription units in one single plasmid. Internal BsaI and BpiI restriction sites must be removed in all modules by introducing point mutations, with consideration of the codon usage of P. pastoris.

    Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and GoldenPiCS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a BsaI Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a BpiI GGA reaction. The transcription units in BB2 are further used for BsaI assembly into multigene BB3 constructs. Single transcription units can be obtained by direct BpiI assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. GoldenPiCS additionally includes module-containing BB1s specific for P. pastoris: 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2)

    Previously, strain engineering approaches with P. pastoris often relied on pGAPz, pPIC6 (Invitrogen) or related expression vectors, which harbor only one transcription unit (Fig. 2). Cloning of concatemers containing more than two transcription units proved to be highly time consuming and also led to unpredictable integration events when using repetitive promoter and terminator sequences [23, 24]. Therefore, conventional overexpression of n genes requires n cycles of preparing competent cells and transforming them, and the use of n different selection markers. For overexpression of three factors plus screenings using consecutive transformations, the procedure would take at least 31 days (four days for transformation and re-streak, five days for screening and two days to prepare competent cells Fig. 2, upper panel). Furthermore, selection markers can be removed and recycled, with the cost of an additional cycle of competent-making and transformation (Fig. 2, middle panel). In addition to that, the use of different integration loci must be included to prevent ‘loop-out’ incidents of integrated DNA. Alternatively, co-transformation of multiple vectors can be considered, but this requires the use of several independent selection markers, otherwise in our experience transformation efficiency is low and it is very unpredictable if all vectors get integrated into the genome [25]. Our backbone BB3 Golden Gate plasmids can carry multiple transcription units and hence significantly simplify and shorten the procedure to one single transformation step. Integration of multiple transcription units plus screening lasts only nine days (Fig. 2, lower panel). The thereby generated strains benefit from decreased generation numbers and milder selection procedures.

    Comparison of conventional and Golden Gate based strain engineering strategies for P. pastoris. Overexpression of multiple genes (GOIs) in P. pastoris using conventional cloning plasmids requires several rounds of competent making (2 days), transformation (4 days including second streak-out) and clone screening (5 days) and takes at least 31 days for three genes with three selection markers. Alternatively, multiple vectors can be co-transformed at once, but resulting transformation efficiencies are usually very low and clonal variation increases. Appropriate flanking sites for the selection marker (loxP or FRT sites) enable marker recycling by recombinases (Cre or Flp, respectively), but require one more round of competent making and transformation which takes at least eight additional days. Golden Gate plasmids carry several transcription units at once and thereby enable transformation and screening in only nine days

    Recombination events by repetitive sequences disturb full vector integration in P. pastoris

    Initially, we started with Golden Gate vectors carrying up to four transcription units with the same promoter and transcription terminator (P GAP and ScCYC1tt). High clonal variation prompted us to analyze gene copy numbers (GCN) of integrated genes and we found that individual transcription units were lost. Similar results have been obtained with P GAP or P AOX1 based multicopy vectors [24], thus showing that incomplete vector integration is not due to the Golden Gate backbone. We positively confirmed post-transformational integration stability for three of the transformants in three consecutive shake flask- batch cultivations without selection pressure (gene copy numbers were stable for more than 15 generations Additional file 1: Table S1). Therefore, we conclude that repetitive homologous sequences within the expression vector (P GAP and ScCYC1tt sequences) resulted in recombination events (internal ‘loop out’) during transformation.

    The high occurrence of incomplete vector integration prompted us to establish a collection of 20 different promoters and 10 transcription terminators (Table 1), in order to avoid repetitive sequences when creating constructs carrying multiple expression units. Promoters and terminators were selected based on the transcriptional regulation and expression strength of their natively controlled genes in published microarray experiments [26]. All of them were screened in several production-relevant conditions (Additional file 1: Table S2). In addition to established promoters [3, 12], we selected novel yet uncharacterized promoter sequences based on their expression behavior in transcriptomics data from P. pastoris cells cultivated on different carbon sources [26]. By applying this collection we aimed to gain the ability to fine-tune the expression of integrated genes. Ideally, promoters for cell engineering purposes should cover a wide range of expression strengths and allow constitutive as well as tunable expression. Transcription terminators were selected from constitutively highly expressed genes [26], many of them being derived from ribosomal protein genes. Also ribosomal genes were reported to be regulated at the level of mRNA stability [27], which is one of the main functions of the 3’UTR contained in the transcription terminator fragments.

    Validation of Kozak sequence mutations in the P GAP sequence of P. pastoris

    In the GoldenMOCS setup, fusion site Fs2 that links the promoter to the GOI contains the start codon ATG and part of the Kozak sequence, which is important for translational initiation in eukaryotes. As this fusion site is a fixed variable in the Golden Gate system, we evaluated the effect of the ‘-1’ position of the P GAP promoter (position in front of the start codon, Fig. 3d) on reporter gene expression in P. pastoris. Due to the ‘CATG’ fusion site, the native ‘A’ in position ‘-1’ of the P GAP promoter (‘-8’ to ‘-1’: AAAACACA) is changed to ‘C’ (AAAACACC). While P GAP variants with ‘A’, ‘T’ and ‘C’ at position ‘-1’ performed similarly, the variant with ‘G’ resulted in a lower eGFP level (P GAP_GATG about 40% lower compared to the other variants). The Kozak consensus sequence of P. pastoris was analyzed and found to be similar to that of S. cerevisiae, which is rich in ‘A’ and poor in ‘G’ bases (Fig. 4). Based on these results, eight bases of the A-rich Kozak consensus sequence were tested in an additional P GAP variant (P GAP_A8ATG position ‘-4’ and ‘-2’ replaced by ‘A’: AAAAAAAA) and a slightly increased expression of eGFP was found (Fig. 3d). Nevertheless, we chose the ‘CATG’ fusion site for our GoldenPiCS system and kept the ‘GCTT’ fusion site for the GOI-terminator assembly.

    Relative eGFP levels obtained with various elements of the GoldenPiCS toolbox. Expression strength of different promoters in comparison to P GAP tested on different carbon sources (a, b), expression levels for P GAP-controlled expression in combination with different transcriptional terminators (c), and comparison of P GAP variants with alternative ‘-1’ nucleotides (d). At least 10 P. pastoris clones were screened to test promoter and terminator function in up to four different conditions: glycerol and glucose excess as present in batch cultivation (“G”, “D”), limiting glucose (“X”) and methanol feed (“M”), both representing fed batch. P GAP to P SHB17 were tested in ‘G’, ‘D’, ‘X’ and ‘M’ (A). P TEF2 to P PFK300 were validated in ‘D’, while P GUT1, P THI11 and MUT-related promoters were tested in putative repressed and induced conditions (‘D’/‘G’, ‘D’+/−100 μM thiamine and ‘D’/M, respectively) (B). P GAP variants with alternative ‘-1’ bases were analyzed in glucose excess (‘D’). Relative eGFP levels are related to P GAP- controlled expression (A, B), terminator ScCYC1tt (B), or presented as relative value (C)


    Watch the video: LECT C8: RECOMBINANT DNA TECHNOLOGY Steps in Gene Cloning (May 2022).