Rare codon clusters at 5′-end influence heterologous expression of archaeal gene in Escherichia coli

Kim, Seonghun; Lee, Sun Bok

doi:10.1016/j.pep.2006.07.014

Cited by 55 publications

(38 citation statements)

References 31 publications

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“…It is well known that the presence of rarely used codons is detrimental to efficient heterologous protein expression (Kim et al, 2006;Rosano et al, 2009). The data presented in Table 3 suggest that, based on the total number of codons in Gram negative PDC genes with less than 10% usage or 10 to 20% usage, they should be poorly expressed in the Gram positive host G.…”

Section: Assessing Codon Usage and Predicting A Gene Sequence For Harmentioning

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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Section: Assessing Codon Usage and Predicting A Gene Sequence For Harmentioning

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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“…Furthermore, this strategy has also been utilized on other retroelements, such as HIV-1, to optimize expression and to remove negative cis-acting sequence elements such as the Rev response element (Kotsopoulou et al 2000;Nguyen et al 2004). Despite these successes, synthetic biology's use of silent mutations also has the potential to introduce negative cisacting sequence elements and to generate synthetic genes with reduced expression and/or decreased RNA stability (Kim and Lee 2006;Kudla et al 2009;Welch et al 2009).In this study, we designed a synthetic element on the basis of Ty1-H3 with GeneDesign (Richardson et al 2006) to recode Ty1-H3 with codons optimized for Saccharomyces cerevisiae expression, introducing 1209 base changes. The resulting synthetic codon-optimized Ty1 (CO-Ty1) was defective for Ty1 transposition and had reduced Ty1 protein and fulllength RNA levels even though the known Ty1 cis-sequences had not been altered, suggesting identification of a novel cisacting sequence capable of regulating Ty1 expression.…”

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confidence: 99%

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Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

et al. 2012

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Extensive mutagenesis via massive recoding of retrotransposon Ty1 produced a synthetic codon-optimized retrotransposon (CO-Ty1). CO-Ty1 is defective for retrotransposition, suggesting a sequence capable of down-regulating retrotransposition. We mapped this sequence to a critical 20-bp region within CO-Ty1 reverse transcriptase (RT) and confirmed that it reduced Ty1 transposition, protein, and RNA levels. Repression was not Ty1 specific; when introduced immediately downstream of the green fluorescent protein (GFP) stop codon, GFP expression was similarly reduced. Rap1p mediated this down-regulation, as shown by mutagenesis and chromatin immunoprecipitation. A regular threefold drop is observed in different contexts, suggesting utility for synthetic circuits. A large reduction of RNAP II occupancy on the CO-Ty1 construct was observed 39 to the identified Rap1p site and a novel 39 truncated RNA species was observed. We propose a novel mechanism of transcriptional regulation by Rap1p whereby it serves as a transcriptional roadblock when bound to transcription unit sequences. SACCHAROMYCES cerevisiae Ty1 is the best-characterized and most abundant of five retrotransposon families present in the genome. Ty1 shares many similarities with retroviruses, such as HIV-1 and makes similar use of an RNA intermediate in its propagation. Unsurprisingly, this Ty1 RNA intermediate is essential for Ty1 retrotransposition. Not only does this "mRNA" code for the GAG and POL proteins required for retrotransposition, but it also serves a critical function as the genetic material for replication. Both Ty1 mRNA and its cDNA product contain cis-acting nucleotide determinants required for retrotransposition.Mutagenesis of Ty1 and screening for cis-acting nucleotide determinants has been previously performed with various strategies, including in-frame linker insertion mutagenesis Devine and Boeke 1994;Monokian et al. 1994), analysis of synthetic Ty1 DNA fragments to determine minimal requirements for the integration reaction (Eichinger and Boeke 1990;Braiterman et al. 1994;Devine and Boeke 1994;Sharon et al. 1994), deletion analysis and PCR-mediated mutagenesis of mini-Ty1 elements (Xu and Boeke 1990;Bolton et al. 2005), and comparative sequence analysis followed by targeted mutagenesis and/or generation of complementary mutations in RNA stem regions (Chapman et al. 1992;Heyman et al. 1995;Lauermann et al. 1995;Friant et al. 1998;Cristofari et al. 2002;Bolton et al. 2005). These studies provided insights into mechanisms underlying Ty1 retrotransposition and revealed several cis-acting sequences, including the Met i -tRNA:primer binding site (PBS) interaction (Chapman et al. 1992; Friant et al. 1998), LTRs (Eichinger andBraiterman et al. 1994;Devine and Boeke 1994;Sharon et al. 1994), polypurine tracts (PPTs) (Heyman et al. 1995;Lauermann et al. 1995), the GAG-POL frameshift region (Belcourt and Farabaugh 1990; Lawler et al. 2001), CYC5 and CYC3 (Cristofari et al. 2002), and an extensive 59 RNA structure required for efficient ini...

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“…This is due to the foreign DNA sequence containing codons that are rarely used in the host, a situation that leads to low levels of translational efficiency and protein expression (Kane, 1995;Kim & Lee, 2006;Rosano & Ceccarelli, 2009) due to a reduced translation elongation rate caused by the imbalance between the codons used in the target gene sequence and the available pool of charged tRNA in the host. These expression problems are then compounded by any incompatibility between the host translation machinery and the mRNA secondary structure due to changes in GC content from alternate codon usage patterns (Kim & Lee, 2006;Wu et al, 2004).…”

Section: Codon Usage Biasmentioning

confidence: 99%

Expression of Non-Native Genes in a Surrogate Host Organism

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et al. 2012

Genetic Engineering - Basics, New Applications and Responsibilities

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Rare codon clusters at 5′-end influence heterologous expression of archaeal gene in Escherichia coli

Cited by 55 publications

References 31 publications

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

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

Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

Expression of Non-Native Genes in a Surrogate Host Organism

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