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

Abstract

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

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots

“…Previous research has also demonstrated that introns can enhance transgene expression levels in plants, for example addition of introns in the 5 0 untranslated region of transgenes (Jeong et al, 2006;Karthikeyan et al, 2009;Mitsuhara et al, 1996) or within the coding sequence (Bartlett et al, 2009;Rose, 2004). Codon optimization (Webster et al, 2017) and the use of different terminator sequences (Ingelbrecht et al, 1989;Nagaya et al, 2010) have also been shown to influence transgene expression. The relative importance of these different factors for governing transgene expression has not been fully explored in a systematic approach, and it is therefore difficult to know which of these are most useful or appropriate.…”

Recoding of synonymous genes to expand evolutionary landscapes requires control of secondary structure affecting translation

“…This work underscores the impact that mRNA secondary structures can have on DNA sequences designed in silico for synthetic biology approaches. Although the influence of secondary structures on translation rates has been known for a long time, these are normally found in the 5′ UTR of a gene and they are still regarded as a minor factor compared to CAI (Webster, Teh, & Ma, ). In our approach, we generated two genes synonymous to intI1 that showed lower levels of activity.…”

New Breeding Techniques for Greenhouse Gas (GHG) Mitigation: Plants May Express Nitrous Oxide Reductase

“…However, producing a foreign protein in a plant cell is often a serious challenge. For example, different codon usage [133] and cellular properties between eukaryotic and prokaryotic cells are considered as unknown aspects of this strategy. At least two key questions need to be addressed in future studies to probe the probability for success of this green gene de-toxic tactic for accelerating the destruction of nitrous oxide via canopy catalysis: (1) Which candidate is the best source-organism to donate nosZ sequence for plant transformation?…”