<i>Pichia pastoris Alcohol Oxidase 1</i> (<i>AOX1</i>) Core Promoter Engineering by High Resolution Systematic Mutagenesis

Portela, Rui M C; Vogl, Thomas; Ebner, Katharina; Oliveira, Rui; Glieder, Anton

doi:10.1002/biot.201700340

Cited by 39 publications

(20 citation statements)

References 39 publications

(89 reference statements)

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“…Using the TATA boxes as a hallmark for determining the core promoter length, we observed exceptionally short core promoters in all histone BDPs (55–81 bp, compared to 160 bp in case of the well-studied P AOX1 39 ). Core promoters are the basic region needed for transcription initiation and bound by general transcription factors (TFs) and RNA polymerase II (RNAPII).…”

Section: Resultsmentioning

confidence: 95%

“…It is worth nothing that histone core promoter sequences contain the 5′ untranslated region (5′ UTRs) of the natural histone mRNAs, as these cannot easily be functionally separated from the core promoter 40,41 . Regardless of this complication, the short core promoters/5′ UTRs identified here are desirable tools for promoter engineering as they can be simply provided on PCR primers 39–41 . Concurringly, these short histone core promoters turned out to be an excellent repository of parts for promoter bidirectionalization and the creation of synthetic hybrid promoters.…”

Section: Resultsmentioning

confidence: 99%

“…This vector contains integration sequences near the ARG4 locus and was linearized with Swa I to target insertion near the ARG4 locus, as had been well established for promoter characterizations in P. pastoris 30,39,41 . Also the following vectors described below were based on this vector backbone.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature’s use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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“…Promoter engineering includes approaches such as chimeric promoter design (Blazeck, Garg, Reed, & Alper, 2012;Blazeck, Liu, Redden, & Alper, 2011), random mutagenesis (Alper, Fischer, Nevoigt, & Stephanopoulos, 2005;Portela, Vogl, Ebner, Oliveira, & Glieder, 2018;Qin et al, 2011), modification of transcription factor binding sites (Ata et al, 2017;Hartner et al, 2008;Prielhofer et al, 2018), and synthetic promoter design (Curran et al, 2014;Portela et al, 2017;Redden & Alper, 2015;Vogl, Ruth, Pitzer, Kickenweiz, & Glieder, 2014). Synthetic biology extends on promoter engineering aiming to increase the transcription rate through designing regulatory circuit(s) by engineering binding of activating TF(s).…”

Section: Introductionmentioning

confidence: 99%

Engineering of alcohol dehydrogenase 2 hybrid‐promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol

Ergün

Gasser

Mattanovich

et al. 2019

Biotech & Bioengineering

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The aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel-engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (P ADH2 ) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single-handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Cat8, and Aca1 binding, and synthetic TATA-box integration; and, (b) nucleosome optimization. P ADH2-Cat8-L2 and P ADH2-Cat8-L1 designed by the integration of synthetic Cat8 binding sites were superior, and then P ADH2-NucOpt .Compared to that with P ADH2 at t = 20 hr of the fermentations, P ADH2-Cat8-L2 allowed the highest increase in enhanced green fluorescent protein expression as 4.8-fold on ethanol and 3.8-fold on methanol; and, P ADH2-NucOpt upregulated the expression 1.5-fold on ethanol and enhanced 3.2-fold on methanol. Using the superior two tools, Cat8-L2 and NucOpt, we designed P ADH2-NucOpt-Cat8-L2 . With P ADH2-NucOpt-Cat8-L2 , the expression in the fermentation of ethanol was upregulated 3.7-fold that is distinctly higher than that with P ADH2-NucOpt but lower than that with P ADH2-Cat8-L2 ; while on methanol compared to that with P ADH2 , the expression was enhanced 8.8-fold. Extracellular recombinant human serum albumin production was also studied with P ADH2-Cat8-L2 and P ADH2-NucOpt , and average recombinant human serum albumin yield (Y P/X ) on ethanol was 1.13 and 0.38 mg/g WCW, respectively; whereas with P ADH2 , Y P/X was 0.26 mg/g WCW . We conclude that as upregulation of transcription and enhanced expression correlate with the sequence of synthetic motifs and their location in the hybrid-promoter architectures of NEPVs in coordination with trans-acting factors, which are the design parameters in the engineering of binding sites; the NEPVs generated promising recombinant protein production platforms with a high impact on industrial scale production processes, as well as would open up new avenues for research in P. pastoris. K E Y W O R D S alcohol dehydrogenase 2 promoter, Cat8, hybrid-promoter architecture, novel-engineered promoter variant, nucleosome optimization, Pichia pastoris (Komagataella phaffii), synthetic binding site

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“…To generate synthetic promoters with specific properties, many promoter engineering strategies and screening method have been developed, especially within the commonly used hosts Escherichia coli and Saccharomyces cerevisiae . Promoter libraries construction using random sequence modifications has been shown to be a useful strategy for developing new promoters, particularly by random saturation mutagenesis of the flanking regions of the promoters core elements …”

Section: Introductionmentioning

confidence: 99%

Construction of Synthetic Promoters by Assembling the Sigma Factor Binding −35 and −10 Boxes

Liu

Weng

Shi

et al. 2018

Biotechnology Journal

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Promoter is one of the key elements in regulating gene expression. Many natural or synthetic promoters have been modulated by their cis‐ or tans‐regulatory elements to confer instant gene expression change in responding to designated stimuli. In addition, bacterial cells also engage different sigma factors to control the gene expression network at different growth phases or in response to the changing environment and external stresses. In this study, a set of promoters that assimilate the endogenous regulation of different sigma factors σ70, σ38, σ32, and σ24 are synthesized. Promoters are designed to contain two or more kinds of interlocking sigma factor binding sites. The most competitive sigma factors will be automatically selected by the cell to take over the synthetic promoters during the cell growth course. Some of the synthetic promoters exhibit very strong strengths under different conditions, including stationary phase, low temperature, acidic pH, and high osmotic pressure. Comparing to the T7 promoter, synthetic promoter P21285 achieved higher yields of L‐asparaginase and acid urease in Escherichia coli. The research not only expands the synthetic biology toolbox but also provide another strategy to design and construct synthetic promoters in prokaryotes.

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Pichia pastoris Alcohol Oxidase 1 (AOX1) Core Promoter Engineering by High Resolution Systematic Mutagenesis

Cited by 39 publications

References 39 publications

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

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

Engineering of alcohol dehydrogenase 2 hybrid‐promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol

Construction of Synthetic Promoters by Assembling the Sigma Factor Binding −35 and −10 Boxes

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