Production of protein-based polymers in Pichia pastoris

Werten, Marc W. T.; Eggink, Gerrit; Stuart, Martien A. Cohen; Wolf, Frits A. de

doi:10.1016/j.biotechadv.2019.03.012

Cited by 92 publications

(58 citation statements)

References 352 publications

(490 reference statements)

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“…Furthermore, yeast are a crucial testbed for genome-scale design 14,15 , and accurate WGS will be necessary for validating written eukaryotic genomes. Finally, engineered yeast have significant economic value as promising cell factories for the manufacture of medicines 16,17 , fuels 18,19 , materials 20,21 , and chemicals 22,23 . Given the economic importance and increasing use of engineered yeast cell factories, it is crucial that WGS methods are developed that can efficiently validate the presence of intended engineering and confirm the absence of unintended variation.…”

Section: Introductionmentioning

confidence: 99%

“…Second, the high sequence identity in many engineered constructs, such as common plasmid elements or parts derived from the host genome, can cause identical sequences to be omitted 88, 89 . In particular, OLC assemblers struggle to reproduce the expected representation and resolution of repeats 20,22,90 . Third, genome assembly software constructs either linear or circular sequences, not both.…”

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confidence: 99%

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Verification of genetic engineering in yeasts with nanopore whole genome sequencing

Collins

Keating

Jones

et al. 2020

Preprint

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Yeast genomes can be assembled from sequencing data, but genome integrations and episomal plasmids often fail to be resolved with accuracy, completeness, and contiguity. Resolution of these features is critical for many synthetic biology applications, including strain quality control and identifying engineering in unknown samples. Here, we report an integrated workflow, named Prymetime, that uses sequencing reads from inexpensive NGS platforms, assembly and error correction software, and a list of synthetic biology parts to achieve accurate whole genome sequences of yeasts with engineering annotated. To build the workflow, we first determined which sequencing methods and software packages returned an accurate, complete, and contiguous genome of an engineered S. cerevisiae strain with two similar plasmids and an integrated pathway. We then developed a sequence feature annotation step that labels synthetic biology parts from a standard list of yeast engineering sequences or from a custom sequence list. We validated the workflow by sequencing a collection of 15 engineered yeasts built from different parent S. cerevisiae and nonconventional yeast strains. We show that each integrated pathway and episomal plasmid can be correctly assembled and annotated, even in strains that have part repeats and multiple similar plasmids. Interestingly, Prymetime was able to identify deletions and unintended integrations that were subsequently confirmed by other methods. Furthermore, the whole genomes are accurate, complete, and contiguous. To illustrate this clearly, we used a publicly available S. cerevisiae CEN.PK113 reference genome and the accompanying reads to show that a Prymetime genome assembly is equivalent to the reference using several standard metrics. Finally, we used Prymetime to resequence the nonconventional yeasts Y. lipolytica Po1f and K. phaffii CBS 7435, producing an improved genome assembly for each strain. Thus, our workflow can achieve accurate, complete, and contiguous whole genome sequences of yeast strains before and after engineering. Therefore, Prymetime enables NGS-based strain quality control through assembly and identification of engineering features.Yet, applying WGS is a challenge because of the diversity of genetic backgrounds, the variety of engineering features, and the current scale of yeast strain engineering. Myriad laboratory strains of the baker's yeast Saccharomyces cerevisiae 9, 24, 25 and nonconventional yeasts like Yarrowia lipolytica 26-28 and Komagataella phaffii (formerly Pichia pastoris) 29, 30 are used to create yeast cell factories, so there are many potential genetic backgrounds. Methods of yeast engineering leave myriad sequence features behind, including standard plasmid sets with standard expression parts 31-34 , high efficiency transformation 35-37 , homologous recombination 10, 38-40 , gene knockouts using the Cre recombinase system 41 , and genome editing using RNAguided endonucleases 7,11,42,[44][45][46] . Furthermore, the scale of yeast engineering is increasing both in the...

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

Verification of genetic engineering in yeasts with nanopore whole genome sequencing

Collins

Keating

Jones

et al. 2020

Preprint

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“…Additionally, a variety of heterologous expression hosts have also been attempted to produce recombinant spidroins, e.g. bacteria, yeast, plants, mammalian cells, and transgenic animals, each with its own pros and cons in terms of cost, manipulation, expression levels and contaminations [22][23][24]. The most widely used expression system is Escherichia coli (E. coli) owing to the most e cient, simple manipulation and cost-e cient production suitable for large-scale production [20,22,25].…”

Section: Introductionmentioning

confidence: 99%

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Cheng¹,

Chen

Yu³

et al. 2020

Preprint

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Abstract Background: Spider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process. Results: Here, we present a mild solubilization strategy—one-step heating method to solubilize spidroins from IBs, with combining spidroins’ high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70–90°C for 20 min, pH 7.0–10, urea concentration 2–4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology.Conclusions: The one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

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“…Thus, bacteria are preferred to produce the antibody fragments such as dAb (single domain antibody) and 5 scFv (single chain fragment variable) [6]. Yeasts have a better proficiency to secrete and to process the disulfide bond of recombinant proteins, and thus they have been used for producing various recombinant proteins for food and industrial application [7,8]. Yeast species such as Saccharomyces cerevisiae, Pichia pastoris and Ogataea minuta were reported for recombinant production not only of antibody fragments [9] but also of full-length antibodies [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Huynh¹,

Morita²,

Sakamoto³

et al. 2020

Preprint

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Background: Monoclonal antibodies (mAbs) as one of the biopharmaceuticals take a pivotal role in the current therapeutic application. Generally, mammalian cell lines such as Chinese hamster ovary (CHO) cell lines are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, the filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy, human anti-TNFα antibody adalimumab, one of the world’s best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to attempt for producing the full length of mAbs by A. oryzae. Generally, N-glycosylation in antibody affects the immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to firstly delete the Aooch1 encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa.Results: The adalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of the commercial product – Humira. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure under the Aooch1 deletion, which suggests only a little additional activity of immune effector functions.Conclusion: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient’s welfare.

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Production of protein-based polymers in Pichia pastoris

Cited by 92 publications

References 352 publications

Verification of genetic engineering in yeasts with nanopore whole genome sequencing

Verification of genetic engineering in yeasts with nanopore whole genome sequencing

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

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