Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Vogl, Thomas; Kickenweiz, Thomas; Pitzer, Julia; Sturmberger, Lukas; Weninger, Astrid; Biggs, Bradley W.; Köhler, Eva-Maria; Baumschlager, Armin; Fischer, Jasmin Elgin; Hyden, Patrick; Wagner, Marlies; Baumann, Martina; Borth, Nicole; Geier, Martina; Ajikumar, Parayil Kumaran; Glieder, Anton

doi:10.1038/s41467-018-05915-w

Cited by 91 publications

(117 citation statements)

References 70 publications

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“…We investigated promoters from R. toruloides that were posited to have medium or strong transcriptional activity in both directions. Bidirectional promoters can reduce design and assembly complexity and also help keep DNA construct sizes within reasonable limits, a useful feature when engineering multigene pathways [20][21][22]. Of the 14 promoters that were predicted to be bidirectional according to RNA-sequencing data (P1, P2, P8, P9, P11, P12, P13, P19, P21, P22, P24, P25, P28 and P29) fluorescence of both reporters was detected for 8 of them, indicating bidirectional transcription (Figure 3).…”

Section: Discussionmentioning

confidence: 99%

A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

Nora

Wehrs

Kim

et al. 2019

Preprint

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Background: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. Results:Our data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. We measured promoter expression strength by flow cytometry using a dual fluorescent reporter system. From these analyses, we found a total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) of potential value for genetic engineering of R. toruloides. Conclusions:We present a list of robust and constitutive, native promoters to facilitate genetic engineering of R. toruloides. This set of thoroughly characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

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Section: Discussionmentioning

confidence: 99%

A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

Nora

Wehrs

Kim

et al. 2019

Preprint

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“…To avoid clonal variation due to different copy numbers of integrated expression cassettes as well as different integration sites and genomic alterations that can bias expression strength comparisons in P. pastoris (Schwarzhans et al 2016a, b;Vogl et al 2018a), transformants from this study underwent the following screening and rescreening procedures [the section is adapted from the open access publication (Vogl et al 2018b)]. P. pastoris cells were transformed with molar equivalents to 1 µg of the empty pPpT4_S vector SwaI linearized plasmids as 1 µg of the empty pPpT4_S vector was found to yield predominantly single copy integration (Vogl et al 2014(Vogl et al , 2018a.…”

Section: Screening Rescreening Procedures and Culture Conditionsmentioning

confidence: 99%

“…(Hartner et al 2008;Vogl et al 2018c)]. P PpAOX1 variants (Hartner et al 2008), alternative promoters (Prielhofer et al 2013), novel MUT promoters , synthetic bidirectional promoters (Vogl et al 2018b) and altering the molecular regulation of P PpAOX1 (Shen et al 2016a, b;Wang et al 2017;Vogl et al 2018c) showed derepression to varying extents in P. pastoris.…”

Section: Introductionmentioning

confidence: 99%

Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris

et al. 2020

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Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression. Despite highly similar open reading frames in the MUT pathways of the four yeasts, the regulation of the respective promoters varies strongly between species. While most endogenous Pp MUT promoters remain tightly repressed after depletion of a repressing carbon, Hp, Cb and Pm MUT promoters are derepressed to up to 70% of methanol induced levels, enabling methanol free production processes in their respective host background. Here, we have tested a series of orthologous promoters from Hp, Cb and Pm in Pp. Unexpectedly, when induced with methanol, the promoter of the HpMOX gene reached very similar expression levels as the strong methanol, inducible, and most frequently used promoter of the Pp alcohol oxidase 1 gene (P PpAOX1 ). The HpFMD promoter even surpassed P PpAOX1 up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced P PpAOX1 . These results demonstrate that orthologous promoters from related yeast species can give access to otherwise unobtainable regulatory profiles and may even considerably surpass endogenous promoters in P. pastoris.

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“…To engineer the second module, SMO genes ( styAB ), SOI gene ( styC ) and PAR gene had been engineered together with SMO_SOI to give SMO_SOI_PAR. To obtain balanced enzyme expression in the E. coli strains, many strategies could be employed, such as engineering the promoters or the ribosome‐binding sites of the genes. Herein, we utilized four plasmids with different copy numbers to optimize the co‐expression: each of module was constructed in pACYC, pCDF, pET, and pRSF, respectively.…”

Section: Resultsmentioning

confidence: 99%

One‐Pot Production of Natural 2‐Phenylethanol from L‐Phenylalanine via Cascade Biotransformations

Lukito

Saw

et al. 2019

ChemCatChem

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2‐Phenylethanol (2‐PE) is an aroma compound with a rose‐like odors widely used as food ingredient, and natural 2‐PE is much preferred for the application with high selling price but limited availability. Bioproduction of 2‐PE from bioresources could provide a green, sustainable, and economic process to manufacture natural 2‐PE. Here we report a novel artificial cascade biotransformation to convert bioderived L‐phenylalanine (L‐Phe) to natural 2‐PE via deamination, decarboxylation, epoxidation, isomerization, and reduction in one pot. Recombinant E. coli strains co‐expressing the 5 necessary enzymes were engineered by using double plasmids, modular approach, and random combination as highly active, easily available, and recyclable biocatalysts. Two‐phase biotransformation with E. coli (RE) cells using biodiesel, a green and easily available solvent for in situ product extraction to minimize the inhibition, significantly improved product concentration and yield, converting 80 mM of L‐Phe to 71 mM (8.8 g/L, 89 % yield) of 2‐PE. The inhibition of 2‐PE was also reduced through in‐situ product adsorption using XAD4 resins. Using XAD4‐biodiesel‐aqueous buffer 3‐phase system could further enhance the concentration of 2‐PE to 85 mM (10.4 g/L, 85 % yield). The developed artificial cascade biotransformation of L‐Phe to 2‐PE is much better than other reported bioproductions of 2‐PE and provides high‐yielding, sustainable, and potentially practical synthesis of high‐value natural 2‐PE.

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Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Cited by 91 publications

References 70 publications

A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris

One‐Pot Production of Natural 2‐Phenylethanol from L‐Phenylalanine via Cascade Biotransformations

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