RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

Bever, Danielle; Wheeldon, Ian; Silva, Nancy Da

doi:10.1016/j.mec.2022.e00208

Cited by 5 publications

(14 citation statements)

References 70 publications

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“…To further optimize this system and eliminate plasmid stability issues that may arise from carrying and propagating two plasmids, we integrated the Cas6-NanoBiT cassette into the CBS712ΔUΔLΔK genome. 55 This keeps RNA scaffold levels high relative to Cas6-fused protein levels (as was found to be important in S. cerevisiae). With this system, we observed a 1.5-and a 1.7-fold increase in specific luminescence relative to the two controls (Figure 5B: IP + HR).…”

Section: ■ Results and Discussionmentioning

confidence: 95%

“…60 Donor fragments amplified from pXP318-cse3-VioE and pXP318-csy4-VioC were integrated into strain BYVioAB at sites X-2 and XII-4 37 to create strain BYVioAB3E4C. K. marxianus strains CBS712ΔUΔL and CBS712ΔUΔLΔK were created from strains CBS712ΔU 51 and CBS712ΔUΔK, 55 respectively, by Cas9 targeting and disruption of the native LEU2 locus using a 200 bp internally truncated LEU2 gene as the donor. 55 Strain CBS712ΔULK-Cas6NanoBiT was created by integrating the GAL1p-Cse3-SmBit-T2A-Csy4-LgBiT-CYC1t cassette at the Chr.IV-2 (IV-2) noncoding site.…”

Section: ■ Methodsmentioning

confidence: 99%

“…K. marxianus strains CBS712ΔUΔL and CBS712ΔUΔLΔK were created from strains CBS712ΔU 51 and CBS712ΔUΔK, 55 respectively, by Cas9 targeting and disruption of the native LEU2 locus using a 200 bp internally truncated LEU2 gene as the donor. 55 Strain CBS712ΔULK-Cas6NanoBiT was created by integrating the GAL1p-Cse3-SmBit-T2A-Csy4-LgBiT-CYC1t cassette at the Chr.IV-2 (IV-2) noncoding site. 61 Plasmids pDBtgr-Cas9-LEU2 and pDBtgr-Cas9-I4, both single-plasmid Cas9 systems harboring a cassette for Cas9 expression and an RNA RNAP II cassette for gRNA expression, 55 were used for targeting the LEU2 locus and IV-2 site, respectively.…”

Section: ■ Methodsmentioning

confidence: 99%

“…55 Strain CBS712ΔULK-Cas6NanoBiT was created by integrating the GAL1p-Cse3-SmBit-T2A-Csy4-LgBiT-CYC1t cassette at the Chr.IV-2 (IV-2) noncoding site. 61 Plasmids pDBtgr-Cas9-LEU2 and pDBtgr-Cas9-I4, both single-plasmid Cas9 systems harboring a cassette for Cas9 expression and an RNA RNAP II cassette for gRNA expression, 55 were used for targeting the LEU2 locus and IV-2 site, respectively. The full GAL1p-Cse3-SmBit-T2A-Csy4-LgBiT-CYC1t cassette was PCR-amplified from pXP822-Cas6-NanoBiT and acted as the donor sequence.…”

Section: ■ Methodsmentioning

confidence: 99%

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Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding

et al. 2023

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Cells often localize pathway enzymes in close proximity to reduce substrate loss via diffusion and to ensure that carbon flux is directed toward the desired product. To emulate this strategy for the biosynthesis of heterologous products in yeast, we have taken advantage of the highly specific Cas6-RNA interaction and the predictability of RNA hybridizations to demonstrate Cas6mediated RNA-guided protein assembly within the yeast cytosol. The feasibility of this synthetic scaffolding technique for protein localization was first demonstrated using a split luciferase reporter system with each part fused to a different Cas6 protein. In Saccharomyces cerevisiae, the luminescence signal increased 3.6-to 20-fold when the functional RNA scaffold was also expressed. Expression of a trigger RNA, designed to prevent the formation of a functional scaffold by strand displacement, decreased the luminescence signal by nearly 2.3-fold. Temporal control was also possible, with induction of scaffold expression resulting in an up to 11.6-fold increase in luminescence after 23 h. Cas6-mediated assembly was applied to create a two-enzyme metabolon to redirect a branch of the violacein biosynthesis pathway. Localizing VioC and VioE together increased the amount of deoxyviolacein (desired) relative to prodeoxyviolacein (undesired) by 2-fold. To assess the generality of this colocalization method in other yeast systems, the split luciferase reporter system was evaluated in Kluyveromyces marxianus; RNA scaffold expression resulted in an increase in the luminescence signal of up to 1.9-fold. The simplicity and flexibility of the design suggest that this strategy can be used to create metabolons in a wide range of recombinant hosts of interest.

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Section: ■ Results and Discussionmentioning

confidence: 95%

Section: ■ Methodsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

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Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding

et al. 2023

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“…In 2022 Bever et al developed a highly efficient CRISPR-Cas9 system in K. marxianus that allows editing of multiple genes which can be used in both NHEJ-functional and -deficient strains showing nearly 100% efficiency of gene disruption in those two strains, whereas 100% efficiency of donor integration was observed only in NHEJdeficient strains. In addition, this system achieved a dual integration efficiency of 25.5% in an NHEJ-deficient strain [85].…”

Section: Kluyveromyces Marxianusmentioning

confidence: 97%

Upgrading Non-Conventional Yeasts into Valuable Biofactories

Castillo-Mendieta¹,

Arias²,

Gonzales-Zubiate³

2023

Biomedical Engineering

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The use of synthetic biology on yeasts has enhanced the production of commercially relevant chemicals, from biofuels to recombinant therapeutic proteins, to name just a few. Despite most of these advances had already been studied and described in Saccharomyces cerevisiae, during the last years the attention has turned to the use of alternative expression systems with a higher yield and quality such as non-conventional yeasts. Recently, there has been an increase in studies about non-conventional yeasts due to advantages based on their natural capacity to tolerate harsh conditions or the wide range of carbon sources they need during the generation of specific products. This chapter, therefore, aims to describe the current status of the most used non-conventional yeasts in metabolite production as well as the engineering behind them in order to optimize or regulate protein expression: Pichia pastoris, Kluyveromyces marxianus, Kluyveromyces lactis and Yarrowia lipolytica.

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CRISPR‐Cas9 mediated genome editing of Kluyveromyces marxianus for iterative, multiplexed gene disruption and pathway integration

Wang,

Tan

et al. 2024

Biotech & Bioengineering

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Kluyveromyces marxianus, a thermotolerant, fast‐growing, Crabtree‐negative yeast, is a promising chassis for the manufacture of various bioproducts. Although several genome editing tools are available for this yeast, these tools still require refinement to enable more convenient and efficient genetic modification. In this study, we engineered the K. marxianus NBRC 104275 strain by impairing the nonhomologous end joining and enhancing the homologous recombination machinery, which resulted in improved homology‐directed repair effective on homology arms of up to 40 bp in length. Additionally, we simplified the CRISPR‐Cas9 editing system by constructing a strain for integrative expression of Cas9 nuclease and plasmids bearing different selection markers for gRNA expression, thereby facilitating iterative genome editing without the need for plasmid curing. We demonstrated that tRNA was more effective than the hammerhead ribozyme for processing gRNA primary transcripts, and readily assembled tRNA‐gRNA arrays were used for multiplexed editing of at least four targets. This editing tool was further employed for simultaneous scarless in vivo assembly of a 12‐kb cassette from three fragments and marker‐free integration for expressing a fusion variant of fatty acid synthase, as well as the integration of genes for starch hydrolysis. Together, the genome editing tool developed in this study makes K. marxianus more amenable to genetic modification and will facilitate more extensive engineering of this nonconventional yeast for chemical production.

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RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

Cited by 5 publications

References 70 publications

Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding

Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding

Upgrading Non-Conventional Yeasts into Valuable Biofactories

CRISPR‐Cas9 mediated genome editing of Kluyveromyces marxianus for iterative, multiplexed gene disruption and pathway integration

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