Increasing Expression of P450 and P450-Reductase Proteins from Monocots in Heterologous Systems

Batard, Yannick; Hehn, Alain; Nedelkina, Svetlana; Schalk, Michel; Pallett, Ken; Schaller, Hubert; Werck‐Reichhart, Danièle

doi:10.1006/abbi.2000.1867

Cited by 83 publications

(56 citation statements)

References 44 publications

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“…Cytochrome P450s form one of the largest families of genes in plants (Batard et al 2000). P450s have been indicated to provide resistance to herbicides for monocotyledonous plants.…”

Section: Codon Optimization In Plant Biotechnologymentioning

confidence: 99%

“…The wheat coding sequence has a high level of GC content and a strong codon usage bias, which is very different to tobacco. Batard et al adjusted the 5'-terminus of the P450 gene sequence to resemble the codon usage of tobacco, which was sufficient to increase the levels of protein expression, from low or undetectable to detectable by Western blot and an average 5-fold increase in C4H activity (Batard et al 2000). Virus.…”

Section: Codon Optimization In Plant Biotechnologymentioning

confidence: 99%

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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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“…Cytochrome P450s form one of the largest families of genes in plants (Batard et al 2000). P450s have been indicated to provide resistance to herbicides for monocotyledonous plants.…”

Section: Codon Optimization In Plant Biotechnologymentioning

confidence: 99%

Section: Codon Optimization In Plant Biotechnologymentioning

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 optimization of the 5# region of PsCYP720B4 (Supplemental Fig. S2) recoded 561 bp (Batard et al, 2000). A transcription initiation enhancer element (Cavener and Ray, 1991) was introduced 5# of the initiation codon.…”

Section: Expression Of Diterpene Synthases and Pscyp720b4 In Yeastmentioning

confidence: 99%

Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects

et al. 2011

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Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of ent-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ent-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism.

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“…Synthetic genes, codon optimized for their target species, have been used for the in planta expression of glycosyl hydrolases to achieve high expression levels [29,49]. Although there is no direct comparison in literature between in planta expression of codon-optimized versus non codonoptimized hydrolytic enzymes, evidence from previous studies support the use of codon optimized genes for improved expression level of hydrolytic enzymes in planta [48,50].…”

Section: Strategies To Increase Expression Level Of Glycolytic Enzymementioning

confidence: 99%

Expression of Glycosyl Hydrolases in Lignocellulosic Feedstock: An Alternative for Affordable Cellulosic Ethanol Production

2016

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Ethanol produced from lignocellulosic feedstock is a promising alternative to fossil fuels and corn-sourced ethanol. However, it creates unique challenges in terms of requirements for breakdown to fermentable sugars, including the need for pretreatment and enzymatic hydrolysis of structural carbohydrates. Hydrolases from microorganisms are currently utilized for biomass hydrolysis in the production of lignocellulosic ethanol; however, expressing these hydrolases in lignocellulosic feedstock is a favorable alternative, due to the large availability of biomass and the potential for the feedstock to play a dual role as both biomass substrate and enzyme provider. This review summarizes recent achievements in hydrolytic enzyme expression in a variety of model plants and potential feedstocks, including strategies to improve enzyme yield and to prevent deleterious effects on plants hosts. We propose possible scenarios for utilizing enzyme-expressing transgenic feedstock and illustrate the potential benefits of using these crops for ethanol production. Furthermore, challenges are highlighted and potential solutions proposed to move the field forward to cost-comparable lignocellulosic ethanol.

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Increasing Expression of P450 and P450-Reductase Proteins from Monocots in Heterologous Systems

Cited by 83 publications

References 44 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

Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects

Expression of Glycosyl Hydrolases in Lignocellulosic Feedstock: An Alternative for Affordable Cellulosic Ethanol Production

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