Continuous translation of circularized mRNA improves recombinant protein titer

Costello, Alan; Lao, Nga T.; Barron, Niall; Clynes, Martin

doi:10.1016/j.ymben.2019.01.002

Cited by 32 publications

(26 citation statements)

References 27 publications

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“…The reasoning behind the choice of these components for the production of proteins was straight‐forward, based on the premise that more messenger RNA (mRNA) is beneficial for protein overexpression. To date, protein expression systems have been optimized widely via multiple approaches, including host reconstruction, expression vector redesign, and optimization of fermentation conditions (Costello, Lao, Barron, & Clynes, 2019; S. Li et al, 2016; Rosano, Morales, & Ceccarelli, 2019).…”

Section: Introductionmentioning

confidence: 99%

Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis

Sun

Zhang

Wang

et al. 2020

Biotech & Bioengineering

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Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membrane structure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high-and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

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Section: Introductionmentioning

confidence: 99%

Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis

Sun

Zhang

Wang

et al. 2020

Biotech & Bioengineering

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“…Then we inserted a P2A self-cleavage peptide sequence between two split GFP fragments, which enables ribosome skipping and cleavage of the nascent polypeptide without translation termination (Fig. S1A, S1C) (18, 27) . Functional GFP protein translated from circ-GFP was detected by both fluorescence microscopy and anti-GFP western blot (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It has been reported that circRNAs can serve as templates for cap-independent translation via virus or cellular internal ribosome entry site (IRES) [5][6][7], m6A modification [8], short IRES-like sequence [9], and other elusive mechanisms [10]. While the translation of circRNAs and their cellular impact cannot reach consensus [11][12][13][14][15][16], tentative and massive efforts have been made to identify endogenous translatable circRNAs [7], investigate the function of endogenous circRNA-derived peptides [17,18], and explore the bioengineering application for protein production [19][20][21][22]. The circular structure renders circRNA not only tolerant to RNA exonuclease resulting in high stability [11,23], but also the possibility to enable rolling circle translation from infinite open reading frames (ORFs), ORFs without stop codons [5,9,10,19].…”

Section: Introductionmentioning

confidence: 99%

Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts

Jiang

Chen

Zhang

2021

Preprint

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In RNA field, the demarcation between coding and non-coding has been negotiated by the recent discovery of occasionally translated circular RNAs (circRNAs). Although absent of 5’ cap structure, circRNAs can be translated cap-independently. Complementary intron-mediated overexpression is one of the most utilized methodologies for circRNA research but not without bearing echoing skepticism for its poorly defined mechanism and latent coexistent side products. In this study, leveraging such circRNA overexpression system, we have interrogated the protein-coding potential of 30 human circRNAs containing infinite open reading frames in HEK293T cells. Surprisingly, pervasive translation signals are detected by immunoblotting. However, intensive mutagenesis reveals that numerous translation signals are generated independently of circRNA synthesis. We have developed a dual tag strategy to isolate translation noise and directly demonstrate that the fallacious translation signals originate from cryptically spliced linear transcripts. The concomitant linear RNA byproducts, presumably concatemers, can be translated to allow pseudo rolling circle translation signals, and can involve backsplicing junction (BSJ) to disqualify the BSJ-based evidence for circRNA translation. We also find non-AUG start codons may engage in the translation initiation of circRNAs. Taken together, our systematic evaluation sheds light on heterogeneous translational outputs from circRNA overexpression vector and comes with a caveat that ectopic overexpression technique necessitates extremely rigorous control setup in circRNA translation and functional investigation.

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“…In future applications, the choice of IRES and promoter may be adapted according to the targeted tissue and it will be of particular interest to test cellular IRESs rather than only viral IRESs in such vectors, as these IRESs are often tissue-specific in vivo (95,96). A recent study has developed a cell factory for recombinant protein production in Chinese hamster ovary (CHO) cells, based on rolling circle translation (97). Cells were transfected with a plasmid containing the sequence of human erythropoietin (EPO) ORF flanked by adequate splicing sites to obtain a circRNA.…”

Section: Future Perspectives For Biotechnological and Therapeutic Appmentioning

confidence: 99%

Circular RNA, the Key for Translation

Prats

David

Diallo

et al. 2020

Preprint

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It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m6A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5’end-independent translation initiation. Today a new family of so-called “non-coding” circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5’end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3’-5’ interaction have been reported, including m6A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and non-covalently circular mRNA translation landscape shows that RNA circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.

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Continuous translation of circularized mRNA improves recombinant protein titer

Cited by 32 publications

References 27 publications

Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis

Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis

Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts

Circular RNA, the Key for Translation

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