Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris

Cheng, Li; Lin, Ying; Zheng, Xueyun; Yuan, Qingyan; Pang, Nuo; Liao, Xihao; Huang, Yuanyuan; Zhang, Xinying; Liang, Shuli

doi:10.1038/s41598-017-11494-5

Cited by 23 publications

(21 citation statements)

References 57 publications

(92 reference statements)

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“…The efficiency results are shown in Table 1. It can be seen that, after 36 h culturing in galactose medium, the ratio of plasmid-free cells was observed to be nearly 100% (Table 1), which was higher than that obtained using Cre/loxP in yeast Pichia (65%) [29]. Thus, the engineered yeast strain could be applied to a new round of genome editing using the gRNA vector (based on P426-CL) targeting another gene of interest (knock-in or knock-out).…”

Section: Resultsmentioning

confidence: 99%

High production of valencene in Saccharomyces cerevisiae through metabolic engineering

Chen

Zhu

et al. 2019

Microb Cell Fact

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Background The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive. Results Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield. Conclusions This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.

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Section: Resultsmentioning

confidence: 99%

High production of valencene in Saccharomyces cerevisiae through metabolic engineering

Chen

Zhu

et al. 2019

Microb Cell Fact

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“…Therefore, the assembly of multistep biosynthetic pathways for the expression of desired compounds in P. pastoris is associated with low integration efficiency, in addition to being a time-consuming process [19]. Moreover, availability of selection markers is limited in P. pastoris , which allows co-integration of genes into the genome by construction of mega plasmids bearing multiple expression cassettes [20, 21], or involves labor-intensive methods of introducing recycling makers [22, 23]. Therefore, it is crucial to develop an efficient, rapid, and marker-free gene integration approach for biosynthetic pathway assembly in P. pastoris .…”

Section: Introductionmentioning

confidence: 99%

CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

et al. 2019

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Background Pichia pastoris (syn. Komagataella phaffii ) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris . Results A non-homologous-end-joining defective strain (Δ ku70 ) was first constructed using the CRISPR–Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (P AOX1 UP-g2, P TEF1 UP-g1, and P FLD1 UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR–Cas9-mediated gene co-integration method in P. pastoris . Conclusions A CRISPR–Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris . Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

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“…The transformation method used was the electroporation method described by Cregg [52] and the following parameters were used: 1.5–2.0 kV, 25 μF and 200 Ω. The vector pPICZA- FHL1 was transferred to the phytase strain GS116/P-6C (6phy) [53] and the pectinase strain GS115/Pel-4C, which were used to generate the 6 phy/AF and 4 pel/AF strains. The transformed cells were selected on YPDZ or YPDB plates.…”

Section: Methodsmentioning

confidence: 99%

Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins

Zheng

Zhang

et al. 2019

Microb Cell Fact

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BackgroundThe methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins.ResultsThe function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities.ConclusionThis study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

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Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris

Cited by 23 publications

References 57 publications

High production of valencene in Saccharomyces cerevisiae through metabolic engineering

High production of valencene in Saccharomyces cerevisiae through metabolic engineering

CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins

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