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In synthetic biology, precise control over protein expression is required in order to construct functional biological systems. A core principle of the synthetic biology approach is a model-guided design and based on the biological understanding of the process, models of prokaryotic protein production have been described. Translation initiation rate...
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... allow translation strengths to be estimated from the mRNA sequence so genetic designs can be better informed. Three calculators have been created with two (RBS Calculator and UTR Designer) using a thermody- namic model and run from online servers, and a third (RBS Designer) using a steady-state kinetic model with a download- able application ( Table 1). All of the models seek to sim- plify the complex natural phenomenon of translation and will continue to be improved and refined to increase predictive accuracy. ...
Similar publications
Different codon optimization algorithms are available that aim at improving protein production by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5′‐UTR structure...
Citations
... The initiation of protein translation is the rate-determining step and has a crucial impact on the efficiency of cell-free protein synthesis [40]. Modification of the cap-dependent initiation allows precise control over protein expression levels. ...
Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed.
... The mRNA secondary structures formed in this region can be modified by environmental conditions like temperature or interaction with trans-acting regulators, small RNA, or ribosomes in order to regulate protein expression (59). The SD can also modulate the formation of the initiation complex by its complementarity with the anti-SD sequence of the 16S rRNA and its position in the sequence (60)(61)(62)(63). ...
Protein synthesis efficiency is highly dependent on mRNA coding sequence. Furthermore, there is extensive evidence of a correlation between mRNA stability and protein expression level, though the mechanistic determinants remain unclear. Using yellow fluorescent protein (YFP) as a reporter gene, we herein demonstrate that adenosine (A) abundance in the first six codons is a critical determinant for achieving high protein synthesis in E. coli . Increasing A and/or decreasing guanosine (G) content in this region results in substantial increases in protein expression level both in vivo and in vitro that are correlated with steady-state mRNA concentration in vivo , and this effect is attributable to changes in the stability of the mRNA that are directly coupled to its translation efficiency. Increasing A content promotes mRNA incorporation into the functional 70S ribosomal initiation complex without altering its affinity for the 30S ribosomal subunit. These results support a model in which base composition in the first six codons modulates local mRNA folding energy to control the balance between productive translation initiation versus degradation of mRNAs bound to the 30S ribosomal subunit. Based on these findings, we developed a short N-terminal coding sequence that optimizes translation initiation efficiency for protein production in E. coli .
... In unicellular organisms, control system design has been aided by the development of standardized libraries consisting of characterized parts, typically mined from wellstudied molecular mechanisms (Chen et al. 2013;Seghezzi et al. 2011), that span both transcriptional and post-transcriptional regulation (Barbier et al. 2020;Baumschlager and Khammash 2021;Merrick et al. 2018;Reeve et al. 2014;Villa et al. 2018; Zhang and Shi 2021). As described above, each control system is developed from basic biological building blocks, such as promoters and DNA binding domains (DBDs), which can be pulled from parts libraries allowing scalable construction of synthetic pathways (McLaughlin et al. 2018;Moore et al. 2016;Redden et al. 2015). ...
Key message
Synthetic control systems have led to significant advancement in the study and engineering of unicellular organisms, but it has been challenging to apply these tools to multicellular organisms like plants. The ability to predictably engineer plants will enable the development of novel traits capable of alleviating global problems, such as climate change and food insecurity.
Abstract
Engineering predictable multicellular phenotypes will require the development of synthetic control systems that can precisely regulate how the information encoded in genomes is translated into phenotypes. Many efficient control systems have been developed for unicellular organisms. However, it remains challenging to use such tools to study or engineer multicellular organisms. Plants are a good chassis within which to develop strategies to overcome these challenges, thanks to their capacity to withstand large-scale reprogramming without lethality. Additionally, engineered plants have great potential for solving major societal problems. Here we briefly review the progress of control system development in unicellular organisms, and how that information can be leveraged to characterize control systems in plants. Further, we discuss strategies for developing control systems designed to regulate the expression of transgenes or endogenous loci and generate dosage-dependent or discrete traits. Finally, we discuss the utility that mathematical models of biological processes have for control system deployment.
... The development and standardization of biological parts (DNA or RNA fragments) to reliably achieve a predictable output in living organisms has been an often-emphasized aspect of synthetic biology [1]. Remarkable progress has been achieved in characterization of bacterial promoters [2], transcriptional terminators [3], insulators [4], translation optimization [5] and promoter inducibility [6], however advances on a higher level of systems' complexity have not been as forthcoming [7][8][9] and major synthetic biology applications (e.g. [10][11][12][13][14]) were achieved by a host of sophisticated and custom-developed genetic engineering [15,16]. ...
Synthetic biology enables the creative combination of engineering and molecular biology for exploration of fundamental aspects of biological phenomena. However, there are limited resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. We developed unigems, a set of ten plasmids that enable classroom-based investigation of gene-expression control and biological logic gates to facilitate teaching synthetic biology and genetic engineering. It is built on a high-copy plasmid backbone and is easily extensible thanks to a common primer set that facilitates Gibson assembly of PCR-generated or synthesized DNA parts into the target vector. It includes two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The set can readily be employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.
... Remarkably, in some cases SD-like motifs are not required for translation, an observation hinting at the existence of other mechanisms besides 'canonical' translation initiation (23)(24)(25)(26)(27). Further, the influence of mRNA secondary structures was studied under the hypothesis that the required unfolding of such structures during translation initiation might decrease expression (15,21,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). For example, stable secondary structures around the start codon were found to hinder translation, while structures further up-or downstream had less pronounced effects (36). ...
... To this end, we assessed mean effects of individual bases and positions in 5 -UTR and CDS along with various higher-order sequence parameters of the mRNA. We found that 25% of variance in our data can be explained by individual nucleotides and that GC-content, hybridisation with the 16S-rRNA and mRNA folding are the most significant determinants of translation initiation in line with previous studies (9)(10)(11)(12)(13)21,(28)(29)(30)(31)(33)(34)(35)(36)(37)(38)40,41,98). ...
Translation is a key determinant of gene expression and an important biotechnological engineering target. In bacteria, 5'-untranslated region (5'-UTR) and coding sequence (CDS) are well-known mRNA parts controlling translation and thus cellular protein levels. However, the complex interaction of 5'-UTR and CDS has so far only been studied for few sequences leading to non-generalisable and partly contradictory conclusions. Herein, we systematically assess the dynamic translation from over 1.2 million 5'-UTR-CDS pairs in Escherichia coli to investigate their collective effect using a new method for ultradeep sequence-function mapping. This allows us to disentangle and precisely quantify effects of various sequence determinants of translation. We find that 5'-UTR and CDS individually account for 53% and 20% of variance in translation, respectively, and show conclusively that, contrary to a common hypothesis, tRNA abundance does not explain expression changes between CDSs with different synonymous codons. Moreover, the obtained large-scale data provide clear experimental evidence for a base-pairing interaction between initiator tRNA and mRNA beyond the anticodon-codon interaction, an effect that is often masked for individual sequences and therefore inaccessible to low-throughput approaches. Our study highlights the indispensability of ultradeep sequence-function mapping to accurately determine the contribution of parts and phenomena involved in gene regulation.
... DAU101 by almost 1.5 folds. Over the last decade, several computational tools such as RBS Calculator, RBS Designer, and UTR Designer have been developed to calculate and model the translation efficiency (Reeve et al. 2014). It has been reported that the expression of proteins is strongly influenced by the codons present in the transcript. ...
Chitinases are a group of enzymes that catalyze chitin hydrolysis and are present in all domains of life. Chitinases belong to different glycosyl hydrolase families with great diversity in their sequences. Microorganisms such as bacteria and fungi produce chitinases for nutrition, and energy, and to parasitize the chitinous hosts. But chitinases from bacteria are of special interest due to their ubiquitous nature and ability to perform under extreme conditions. Chitinases produced by bacteria have been explored for their use in agriculture and industry. In agriculture, their main role is to control chitin-containing insect pests, fungal pathogens, and nematodes. In the seafood industry, they found their role in the management of processing wastes which are mainly chitinous substances. Chitinases are also used to synthesize low molecular weight chitooligomers which are proven bioactive compounds with activities such as anti-tumour, antimicrobial, and immunity modulation. Considering their importance in ecology and biotechnological applications, several bacterial chitinases have been studied in the last two decades. Despite their potential, bacterial chitinases have a few limitations such as low production and lack of secretion systems which make the wild-type enzymes unfit for their applications in industries and other allied sectors. This review is an attempt to collate significant works in bacterial chitinases and their application in various industries and the employment of various tools and techniques for improvement to meet industrial requirements.
... 39,40 We hypothesized that it would be beneficial to tune the translation of tetA so that expression levels are high enough to confer NiCl 2 sensitivity but low enough to avoid a significant burden to the cell. To this end, we created a DNA sequence library of the tetA TIR. 12 The TIR spans from the Shine− Dalgarno sequence to the fifth codon of the gene of interest 41 and has a major impact on the overall production level of a protein. 12,42−46 Randomization of the TIR has previously been successfully used to optimize the expression of different genes. ...
Engineering of bacterial genomes is a fundamental craft in contemporary biotechnology. The ability to precisely edit chromosomes allows for the development of cells with specific phenotypes for metabolic engineering and for the creation of minimized genomes. Genetic tools are needed to select for cells that underwent editing, and dual-selection markers that enable both positive and negative selection are highly useful. Here, we present an optimized and easy-to-use version of the tetA dual-selection marker and demonstrate how this tetAOPT can be used efficiently to engineer at different stages of the central dogma of molecular biology. On the DNA level, tetAOPT can be used to create scarless knockouts across the Escherichia coli genome with efficiency above 90%, whereas recombinant gene integrations can be achieved with approximately 50% efficiency. On the RNA and protein level, we show that tetAOPT enables advanced genome engineering of both gene translation and transcription by introducing sequence variation in the translation initiation region or by exchanging promoters. Finally, we demonstrate the use of tetAOPT for genome engineering in the industrially relevant probiotic strain E. coli Nissle.
... 80 Additionally, synthetic RBS sequences with diverse translation initiation levels can be designed for the control of gene expression using computational tools such as RBS calculator, UTR designer, and RBS designer. 81 For the strains used in industrial applications, the chromosomal expression of biosynthetic genes is often favored over plasmid-based expression because of the plasmid maintenance and instability problems. Recombinase systems such as the Lambda Red or RecET from E. coli have widely been employed to delete or introduce target genes in various microbial hosts. ...
The sustainable production of chemicals from renewable, nonedible biomass has emerged as an essential alternative to address pressing environmental issues arising from our heavy dependence on fossil resources. Microbial cell factories are engineered microorganisms harboring biosynthetic pathways streamlined to produce chemicals of interests from renewable carbon sources. The biosynthetic pathways for the production of chemicals can be defined into three categories with reference to the microbial host selected for engineering: native-existing pathways, nonnative-existing pathways, and nonnative-created pathways. Recent trends in leveraging native-existing pathways, discovering nonnative-existing pathways, and designing de novo pathways (as nonnative-created pathways) are discussed in this Perspective. We highlight key approaches and successful case studies that exemplify these concepts. Once these pathways are designed and constructed in the microbial cell factory, systems metabolic engineering strategies can be used to improve the performance of the strain to meet industrial production standards. In the second part of the Perspective, current trends in design tools and strategies for systems metabolic engineering are discussed with an eye toward the future. Finally, we survey current and future challenges that need to be addressed to advance microbial cell factories for the sustainable production of chemicals.
... Through a series of elegant experiments, their results revealed that although mRNA features other than the SD sequence are able to render complexes initiation-competent, there is a linear correlation between SD strength and translation efficiency (Saito et al., 2020). These findings are in good agreement with previous work that investigated the sequence and structural properties of mRNA in the vicinity of the start codon (O'Donnell and Janssen, 2001;Li et al., 2012;Reeve et al., 2014;Duval et al., 2015;Hockenberry et al., 2017). ...
A recent addition to the -omics toolkit, ribosome profiling, enables researchers to gain insight into the process and regulation of translation by mapping fragments of mRNA protected from nuclease digestion by ribosome binding. In this review, we discuss how ribosome profiling applied to mycobacteria has led to discoveries about translational regulation. Using case studies, we show that the traditional view of “canonical” translation mechanisms needs expanding to encompass features of mycobacterial translation that are more widespread than previously recognized. We also discuss the limitations of the method and potential future developments that could yield further insight into the fundamental biology of this important human pathogen.
... One aspect of this analysis that has become apparent to us was that the predicted iTSS TIR varied vastly among host genera. RBS calculator (20) determines TIRs in relative units, and the determination is based on the free energies of key molecular interactions involved in translation initiation (22). Fig. 2C shows the box plots of the distribution of the TIRs by host genera, where it is possible to see that some genus have putative iTSSs with higher predicted TIRs than others (compare, e.g., Streptococcus with Tsukamurella) and that it did not correlate with how frequently iTSS have been identified in that genus (compare Fig. 2A with 2C). ...
Bacteriophages use endolysins to cleave the host bacteria cell wall, a crucial event underlying cell lysis for virion progeny release. These bacteriolytic enzymes are generally thought to work as single, monomeric polypeptides, but a few examples have been described in which a single gene produces two endolysin isoforms.
