Adjustment of Codon Usage Frequencies by Codon Harmonization Improves Protein Expression and Folding

Angov, Evelina; Legler, Patricia M.; Mease, Ryan M.

doi:10.1007/978-1-61737-967-3_1

Cited by 49 publications

(42 citation statements)

References 28 publications

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“…This means the gene sequence still This article is protected by copyright. All rights reserved 14 retains its preferred and rare codons, these are just altered to the equivalent preferred and rare synonymous codons used by the host organism (Angov et al 2008;Angov et al 2011;Angov 2011). The codon harmonization algorithm was applied to genes encoding three Plasmodium falciparum proteins to be expressed in E. coli and gene expression was subsequently determined to increase from 4 to 1,000 fold that of non-optimized genes (Angov et al 2008).…”

Section: Development Of Algorithms For Synthetic Genesmentioning

confidence: 99%

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Webster

Teh

K.‐C.

2016

Biotech & Bioengineering

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Degeneracy in the genetic code allows multiple codon sequences to encode the same protein. Codon usage bias in genes is the term given to the preferred use of particular synonymous codons. Synonymous codon substitutions had been regarded as "silent" as the primary structure of the protein was not affected; however, it is now accepted that synonymous substitutions can have a significant effect on heterologous protein expression. Codon optimization, the process of altering codons within the gene sequence to improve recombinant protein expression, has become widely practised.Multiple inter-linked factors affecting protein expression need to be taken into consideration when optimizing a gene sequence. Over the years, various computer programmes have been developed to aid in the gene sequence optimization process. However, as the rulebook for altering codon usage to affect protein expression is still not completely understood, it is difficult to predict which strategy, if any, will design the 'optimal' gene sequence.In this review, codon usage bias and factors affecting codon selection will be discussed and the evidence for codon optimization impact will be reviewed for recombinant protein expression using plants as a case study. These developments will be relevant to all recombinant expression systems, however, molecular pharming in plants is an area which has consistently encountered difficulties with low levels of recombinant protein expression, and should benefit from an evidence based rational approach to synthetic gene design. This article is protected by copyright. All rights reserved

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Section: Development Of Algorithms For Synthetic Genesmentioning

confidence: 99%

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Webster

Teh

K.‐C.

2016

Biotech & Bioengineering

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“…Codon harmonization (Angov et al, 2008;Angov, 2011;Angov et al, 2011) is thought to assist protein folding in heterologous host systems by mimicking the translation rates of the native host in a heterologous strain. The translation rate is determined by codon usage frequencies, where the presence of infrequently used codons forces a reduction of the translation rate, allowing evolving protein to fold in phases (Angov et al, 2008;Angov, 2011;Angov et al, 2011). Both the frequency and positioning of infrequently used codons is critical for protein folding.…”

Section: Native Pdc (Goxpdc Wt ) Expression In G Thermoglucosidasiusmentioning

confidence: 99%

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Zyl

Taylor

Eley

et al. 2013

Appl Microbiol Biotechnol

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This study reports the expression, purification and kinetic characterization of a PDC from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120µM at pH 5), high catalytic efficiency (4.75 x 10 5 M -1 s -1 at pH 5), a pH opt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C (the highest yet reported for a b a c t e r i a l P D C ) . D u e t o g o o d in vitro thermostablity (approximately 40% enzyme activity retained after 30 minutes at 65°C) this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius. Initial studies using a variety of methods failed to detect activity at any growth temperature. However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45°C (as determined by enzyme activity, Western blot, mRNA detection and ethanol productivity). Here we describe the successful expression of PDC in a true thermophile. Yields as high as 0.35 g/g ±0.04 ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights that the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription / translation coupling.

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“…[285][286][287][288]). These include mRNA stability [289,290], codon usage [291][292][293][294], riboswitches [12,295], ribosome binding site potency [296,297] and even the RNA polymerase itself [298] (including photoswitchable variants [299]). …”

Section: Control Factor Expression Engineeringmentioning

confidence: 99%

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Kell¹

2018

Preprint

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The yield and ease of purification of biotechnological products are typically greatly enhanced if they can be secreted into the external medium. Although not universally appreciated, however, small molecule biotechnological products do not ‘float across’ the phospholipid bilayer portion of biological membranes, and thus they need transporters to assist their passage into the extramembrane and extracellular spaces. Some of these transporters may be reversible, equilibrative transporters that might more normally be used for uptake, while others may have an efflux directionality imposed on them via suitable energy coupling mechanisms. Despite the energetic costs of small molecule synthesis, natural evolution does in fact provide a number of mechanisms by which the secretion of such products can actually enhance fitness. Where available these provide useful starting points, and assaying for such activities is crucial. In particular, in a systems biology approach, we first need to identify such/suitable activities before we can seek to increase them. They may then be improved through promoter engineering, via manipulation of control elements, or by directed evolution of the transporter proteins themselves. Modern methods of synthetic biology provide enormous opportunities for all kinds of efflux transporter engineering; they are just beginning to be realised.

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Adjustment of Codon Usage Frequencies by Codon Harmonization Improves Protein Expression and Folding

Cited by 49 publications

References 28 publications

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Control of metabolite efflux in microbial cell factories: current advances and future prospects

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