The maize α-zein promoter can be utilized as a strong inducer of cellulase enzyme expression in maize kernels

Requesens, Deborah Vicuna; Romero, Maria Elena Gonzalez; Devaiah, Shivakumar P.; Chang, Yeun-Kyung; Flory, Ashley; Streatfield, Stephen J.; Ring, Rebecca; Phillips, Cassie; Hood, Nathan C.; Marbaniang, Cyrus Dean; Howard, John A.; Hood, Elizabeth E.

doi:10.1007/s11248-019-00162-1

Cited by 4 publications

(3 citation statements)

References 33 publications

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“…Endoglucanases can be secreted to the apoplast (Xiao et al 2018), but targeting to the ER (Klose et al 2015) or plastids (Schmidt et al 2019b;Fumagalli et al 2019) helps to prevent the enzymes degrading essential cellulose structures before plants are harvested and homogenized for product extraction. Alternatively, enzyme expression can be restricted to specific organs, such as maize kernels (Vicuna Requesens et al 2019), or can be induced at a particular time, such as just before harvest, thus reducing the fiber content and rigidity, which can facilitate primary product extraction. Methods to analyze the spatiotemporal activity of regulatory elements in plants are well-established, allowing the most appropriate promoters to be selected (Xiong et al 2016).…”

Section: Self-catalyzed Processing Of Residual Biomassmentioning

confidence: 99%

Targeted genome editing of plants and plant cells for biomanufacturing

2021

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Plants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as “plant molecular farming” (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose “chassis” for PMF.

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Section: Self-catalyzed Processing Of Residual Biomassmentioning

confidence: 99%

Targeted genome editing of plants and plant cells for biomanufacturing

2021

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“…A few seed-specific promoters from maize have also been isolated recently. One of them is the globulin-1 promoter where the specific expression of foreign genes in the maize embryos was reported (Requesens et al 2019). However, the amount of protein produced is very low despite the strong activity of this promoter because the embryo only accounts for 10% of the total seed weight.…”

Section: Introductionmentioning

confidence: 99%

Analysis of the Promoter of Emb5 from Zea mays Identifies a Region of 523 bp Responsible for Its Embryo-Specific Activity

Yang

Wang

et al. 2020

Plant Mol Biol Rep

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The maize Emb5 is an abscisic acid–responsive gene which is specifically expressed in the late embryo during seed maturity. To further dissect and identify the elements specific for its embryo expression pattern, we investigated the activity of the − 1653 bp upstream of the “full-length” promoter region of this gene in transgenic Arabidopsis plants. We first confirmed that the “full-length” promoter could indeed drive the expression of β-glucuronidase reporter gene (GUS) in the transgenic Arabidopsis seed embryo. Subsequently, DNA fragments of ~ 500 bp in length were generated after a series of progressive deletions from positions − 1653 bp to − 1 bp relative to the transcriptional initiation site. These fragments were fused with GUS and introduced into Arabidopsis. Measurement of the GUS activity in the immature seeds isolated from the transgenic plants revealed that the region between positions − 523 bp and − 1 bp, namely ProEm-D, is absolutely required and sufficient for the temporal and embryo-specific expression of GUS with an activity comparable with the full-length Emb5 promoter in Arabidopsis. Therefore, our results clearly demonstrated that the 523 bp ProEm-D can replace the − 1653 bp Emb5 promoter to drive embryo-specific expression in Arabidopsis seed. Because of its small size and strong embryo-specific activity, it could become the promoter of choice in metabolic pathway engineering to transfer multiple genes for the production of valuable pharmaceutical products in seeds, such as polyunsaturated fatty acids found in fish oils, or pro-vitamin A where at least three transgenes are required to assemble the entire metabolic pathways.

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“…The predominant component in residual biomass is cellulose (Sheen, 1983), and its degradation can be performed by exoglucanases and endoglucanases, which convert cellulose into oligo-and monosaccharides (Bornscheuer et al, 2014). Restricting enzyme expression to specific organs or inducing it at a precise moment, such as pre-harvest, can reduce the rigidity of the fibres, facilitating the extraction of the product (Vicuna Requesens et al, 2019). Genome editing techniques can be employed to alter the promoters of endogenous cellulase, enabling precise control over spatiotemporal expression of these genes (Buyel et al, 2021).…”

Section: Self-catalysing Residual Biomassmentioning

confidence: 99%

Enhancing Nicotiana benthamiana as chassis for Molecular Farming: targeting flowering time for increased biomass and recombinant protein production

Paola

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Plant Molecular farming (PMF) is the production of industrially relevant and commercially valuable proteins in plants. Plants exhibit many advantages as bioreactors such as scalability, cost-effectiveness, and the ability to produce complex proteins. Species of the Nicotiana genus, especially tobacco and Nicotiana benthamiana, have become increasingly important as production platforms for PMF due to their advantages such as high biomass yield, ease of transformation, robust protein expression, and nonfood crop status. Nevertheless, these species are not yet ideal production platforms -breeding goals such as flowering delay or abolition to enhance plant biomass could improve N. benthamiana as a prime chassis for molecular farming. In this chapter, our focus was the knockout of key genes for flowering, such as members of the FLOWERING LOCUS T (FT) proteins family. The flowering inducers NbFT4 and the homeologous pair NbFT5_1a/NbFT5_1b together with NbSPL13_1a, member of the SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors family, were targets of CRISPR/Cas9 editing. The lines that exhibited biallelic mutations for these genes, alone and in combination, showed delayed flowering and a remarkable increase in biomass, height and branching. These characteristics could be the foundation for the improvement of N. benthamiana as a molecular farming production platform.My contribution to this chapter was essential. I searched for the FT genes in N. benthamiana, I generated the edited lines presented in this chapter, genotyped, phenotyped them, and assayed their expression potential for eGFP. I wrote and corrected the text of the chapter and generated its figures.

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The maize α-zein promoter can be utilized as a strong inducer of cellulase enzyme expression in maize kernels

Cited by 4 publications

References 33 publications

Targeted genome editing of plants and plant cells for biomanufacturing

Targeted genome editing of plants and plant cells for biomanufacturing

Analysis of the Promoter of Emb5 from Zea mays Identifies a Region of 523 bp Responsible for Its Embryo-Specific Activity

Enhancing Nicotiana benthamiana as chassis for Molecular Farming: targeting flowering time for increased biomass and recombinant protein production

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