A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

Wang, Shu; Li, Yang; Jin, Zhehao; Liu, Xiangjian; Gao, Xiang; Guo, Shuyuan; Yu, Tao

doi:10.1016/j.synbio.2023.06.003

Cited by 4 publications

(4 citation statements)

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“…Considerable developments in molecular tools, including synthetic promoters for the fine-tuning of expression [ 67 ] and CRISPR/Cas9 technology [ 68 , 69 ], have led to the creation of highly secreting strains with the participation of K. phaffii as a host [ 70 , 71 ].…”

Section: Specific Features Of K Phaffii As a Produ...mentioning

confidence: 99%

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Khlebodarova,

Bogacheva,

Zadorozhny

et al. 2024

Microorganisms

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In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker’s yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species’ several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

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Section: Specific Features Of K Phaffii As a Produ...mentioning

confidence: 99%

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Khlebodarova,

Bogacheva,

Zadorozhny

et al. 2024

Microorganisms

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“…CRISPR-Cas9 induced gene targeting using linear DNA fragments K. phaffii, like many other non-conventional yeasts and higher eukaryotes, prefers to insert DNA received by transformation into the genome via the NHEJ pathway. To increase the gene-targeting efficiency, it may therefore be an advantage to eliminate the NHEJ pathway; and for K. phaffii, it has been demonstrated that gene targeting efficiencies are dramatically improved in strains containing a disrupted KU70 homolog (Näätsaari et al, 2012;Wang et al, 2023). In addition, NHEJ inactivation enables efficient use of the TAPE tool to assess whether specific sgRNA species are able to facilitate Cas9 cleavage at the target sites (Nødvig et al, 2018;Vanegas et al, 2017).…”

Section: Crispr-cas9 Tool Design and Validationmentioning

confidence: 99%

“…Gene editing in the K. phaffii is hampered by the dominance of the non-homologous end joining (NHEJ) DNA repair pathway, which favors random integration over homologous recombination mediated gene targeting (Näätsaari et al, 2012). The emergence of CRISPR-Cas9 technology has revolutionized gene engineering of many non-model yeasts (Cai et al, 2019; Stovicek et al, 2017) and has also been successfully adapted to K. phaffii (Weninger et al, 2016) where the repertoire of CRISPR-Cas9 tools steadily increases (Dalvie et al, 2020; Gu et al, 2019; Liao et al, 2021; Liu et al, 2019; Wang et al, 2023). DNA double strand breaks induced by a CRISPR nuclease allows easy gene inactivation via erroneous NHEJ based repair, as well as efficient template guided gene editing mediated by HR.…”

Section: Introductionmentioning

confidence: 99%

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering ofKomagataella phaffii

Strucko,

Gadar-Lopez,

Frøhling

et al. 2023

Preprint

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Komagataella phaffii(Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bio-production of valuable compounds is not yet fully exploited. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of non-conventional species includingK. phaffii. Yet, improvements in gene-editing methods are desirable to further accelerate engineering of industrially and scientifically relevantK. phaffiistrains. In this study, we have developed a versatile one vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences. Importantly, we show that by using our setup it is possible to catalyze single-stranded oligonucleotide-mediated mutagenesis and marker-free gene integrations. Notably, we performed site-specific point mutations and full gene deletions using single stranded 90-mers at very high efficiencies. Lastly, we present a strategy for transient inactivation of non-homologous end-joining (NHEJ) pathway, whereKU70gene is disrupted by a visual marker (uidAgene). The latter system enables precise CRISPR-Cas9 based editing (including multiplexing) and accelerates selection of the mutants that have simultaneously undergone a desired genetic modification(s) and restored NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering ofK. phaffiistrains and could potentially be coupled with high-throughput automated workflows.

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“…Notably, overexpression of RAD52 and RAD59 resulted in significant improvement in HR efficiency. 7,8 Furthermore, the overexpression of RAD52, RAD59, MRE11, and SAE2 enabled efficient genome integration of heterologous genes using short homology arms (40 bp). 9 However, the growth rate of strain with RAD52, RAD59, MRE11, and SAE2 was about 20% lower than that of control, which was not conducive to the production of heterologous proteins.…”

Section: Introductionmentioning

confidence: 99%

Heterologous Single-Strand DNA-Annealing and Binding Protein Enhance CRISPR-Based Genome Editing Efficiency in Komagataella phaffii

Deng,

Wu,

et al. 2023

ACS Synth. Biol.

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The industrial yeast Komagataella phaffii is a highly effective platform for heterologous protein production, owing to its high protein expression and secretion capacity. Heterologous genes and proteins are involved in multiple processes, including transcription, translation, protein folding, modification, transportation, and degradation; however, engineering these proteins and genes is challenging due to inefficient genome editing techniques. We employed Pseudomonas aeruginosa phage singlestranded DNA-annealing protein (SSAP) PapRecT and P. aeruginosa single-stranded DNA-binding protein (SSB) PaSSB to introduce SSAP-SSB-based homology recombination, which facilitated K. phaffii CRISPR-based genome engineering. Specifically, a host-independent method was developed by expressing sgRNA with PapRecT-PaSSB in a single plasmid, with which only a 50 bp short homologous arm (HA) reached a 100% positive rate for CRISPR-based gene insertion, reaching 18 colony-forming units (CFU) per μg of donor DNA. Single deletion using 1000 bp HA attained 100%, reaching 68 CFUs per μg of donor DNA. Using this efficient CRISPR-based genome editing tool, we integrated three genes (INO4, GAL4-like, and PAB1) at three different loci for overexpression to realize the collaborative regulation of human-lactalbumin (α-LA) production. Specifically, we strengthened phospholipid biosynthesis to facilitate endoplasmic reticulum membrane formation and enhanced recombinant protein transcription and translation by overexpressing transcription and translation factors. The final production of α-LA in the 3 L fermentation reached 113.4 mg L −1 , two times higher than that of the strain without multiple site gene editing, which is the highest reported titer in K. phaffii. The CRISPR-based genome editing method developed in this study is suitable for the synergistic multiple-site engineering of protein and biochemical biosynthesis pathways to improve the biomanufacturing efficiency.

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A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

Cited by 4 publications

References 46 publications

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering ofKomagataella phaffii

Heterologous Single-Strand DNA-Annealing and Binding Protein Enhance CRISPR-Based Genome Editing Efficiency in Komagataella phaffii

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