Canadian Journal of Microbiology
Codon usage bias affects α‐amylase mRNA level by altering RNA stability and cytosine methylation patterns in Escherichia coli
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2019-0624.R2
Manuscript Type: Article
Date Submitted by the 01-Apr-2020 Author:
Complete List of Authors: Xing, Yanzi; East China University of Science and Technology, Bioengineering Gong, Ruiqing; East China University of Science and Technology, BioengineeringDraft Xu, Yichun; East China University of Science and Technology Liu, Kunshan; East China University of Science and Technology Zhou, Mian; East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering
Keyword: codon usage bias, DNA cytosine methylation, RNA stability
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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1 Codon usage bias affects α‐amylase mRNA level by altering RNA stability and cytosine methylation
2 patterns in Escherichia coli
3 Yanzi Xing1, Ruiqing Gong1, Yichun Xu1, Kunshan Liu1, Mian Zhou1*
4
5 1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
6 Shanghai 200237, China
7 Yanzi Xing: [email protected]
8 Ruiqing Gong: [email protected]
9 Yichun Xu: [email protected]
10 Kunshan Liu: [email protected] Draft
11 *Correspondence:
12 Mian Zhou, State Key Laboratory of Bioreactor Engineering, East China University of Science and
13 Technology, Shanghai, China.
14 Email: [email protected]
15 Telephone: 86-21-64252257
16 Author Contributions:
17 Conceptualization: Mian Zhou, Yanzi Xing. Methodology: Mian Zhou, Yanzi Xing, Yichun Xu.
18 Investigation and data curation: Yanzi Xing, Ruiqing Gong, Kunshan Liu. Writing – original draft: Mian
19 Zhou, Yanzi Xing. Writing – review and editing: Mian Zhou, Yanzi Xing. Funding acquisition: Mian
20 Zhou.
21
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1 Abstract:
2 Codon usage bias exists in almost every organism, and has been reported to regulate protein translation
3 efficiency and folding. Besides the translational level, the preliminary role of codon usage bias on gene
4 transcription has also been revealed in some eukaryotes such as Neurospora crassa. Here we took the
5 α‐amylase coding gene (amyA) as an example, and examined the role of codon usage bias in regulating
6 gene expression in the typical prokaryote Escherichia coli. We confirmed the higher translation
7 efficiency on codon optimized amyA RNAs, and found that RNA level itself was also affected by codon
8 optimization. The decreased RNA level was at least partially contributed by altered mRNA stability at
9 the post-transcriptional level. Codon optimization also altered the number of cytosine methylation sites.
10 Examinations on dcm knockouts suggestedDraft that cytosine methylation may be a minor mechanism adopted
11 by codon bias to regulate gene RNA levels. More studies are required to verify its global effect and the
12 detailed mechanism on transcription.
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14 Key words: codon usage bias, DNA cytosine methylation, RNA stability
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1 1.Introduction:
2 Codon degeneracy enables 20 amino acids to be encoded by 61 triplet nucleotide codons, which allows
3 the same protein sequence to be synthesized by a variety of mRNAs composed of synonymous codons.
4 The synonymous codons are often used at different frequencies, such phenomenon is known as codon
5 usage bias, a universal feature of genomes in both prokaryotes and eukaryotes (Sharp et al. 1986;
6 Comeron 2004; Plotkin and Kudla 2011). Experimental studies have demonstrated that codon usage
7 regulates translation elongation rate (Sørensen and Pedersen 1991; Presnyak et al. 2015; Yu et al. 2015),
8 translation efficiency (Ikemura 1981; Mitarai et al. 2008; Zarai et al. 2016) and protein folding (Komar
9 et al. 1999; Zhou et al. 2013). The advent of monitoring translation in real time (Iwasaki and Ingolia n.d.;
10 Chekulaeva and Landthaler 2016) providesDraft additional option to investigate codon usage’s impacts on
11 translation elongation rates. It has been reported that in a cell-free translation system, the rate of a codon-
12 optimized mRNA for completing translation was 1.5 minutes faster than a non-optimized one (Yu et al.
13 2015). A more recent study revealed that the translation rate of mRNAs after codon optimization was 4.9
14 codons per second, however, the rate of non-optimized mRNA was 3.1 codons per second (Yan et al.
15 2016; Hanson and Coller 2018). The hypothesis that codon usage could influence translation efficiency
16 (Ikemura 1981) was later underpinned by the observation that codon usage bias was more prominent in
17 highly expressed genes and its striking evolutionary conservation (Akashi 1994; Dong et al. 1996; Powell
18 and Moriyama 1997; Yang and Nielsen 2008). More recent in vitro translation assays suggested that
19 codon usage directly affected translation efficiency (Boël et al. 2016). Because of the important roles of
20 optimal codons in facilitating translation, codon optimization becomes a routine technique during
21 recombinant protein expression. However, optimal codon usage does not always guarantee good protein
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1 yield, which has promoted more research on solving detailed regulatory mechanisms of codon bias
2 towards transcription and translation.
3 Experimental evidence in both eukaryotes and prokaryotes showed that codon optimization on certain
4 genes might result in alterations on protein folding and conformation (Spencer et al. 2012; Zhou et al.
5 2013). Recently, the role of codon usage bias on gene transcription has attracted further attention. Studies
6 in Neurospora crassa suggested that codon choices regulated gene transcription mostly through altering
7 histone modification patterns instead of mRNA stability (Zhou et al. 2016). Besides, synonymous codon
8 replacement may result in pre-mature transcriptional termination through polyadenylation in coding
9 region (Zhou et al. 2018). However, codon usage has been considered to be a critical determinant of
10 mRNA stability in Saccharomyces cerevisiaeDraft (Presnyak et al. 2015; Boël et al. 2016). The presence of
11 rare codons in certain a subset of genes results in a significant decrease in mRNA stability (Hoekema et
12 al. 1987; Caponigro et al. 1993; Hu et al. 2009; Sweet et al. 2012). As for prokaryotes, how codon usage
13 bias regulates mRNA transcription remains obscure since histone modification and poly A tailing related
14 mechanisms should not exist. Some reports claimed that the mRNA decay process might be coupled to
15 codon-dependent variations in ribosomal elongation dynamics (Boël et al. 2016). However, more
16 evidence is needed to reveal further mechanisms.
17 DNA methylation is an important epigenetic mark that regulates gene expression. In bacteria,
18 methylation was previously considered to be a part of restriction-modification (R-M) systems, protecting
19 bacteria against invasion of foreign DNA including phages (Rambach and Tiollais 1974). However, later
20 discovery of solitary (or orphan) DNA methyltransferases indicated other possible biological functions
21 of DNA methylation (Palmer and Marinus 1994). The most well-studied solitary methyltransferase in
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1 E.coli is DNA adenine methyltransferase (Dam) encoded by dam, which targets the GmATC motif (Wion
2 and Casadesús 2006). Dam methylation has a well-known function for strand discrimination during
3 replication repair, however, later this methyl mark has been proven to regulate gene expression by
4 altering binding ability of transcription factors to methylated sites of DNA sequences (Waldron et al.
5 2002; Peterson and Reich 2008). Cytosine DNA methyltransferase (Dcm) is another methyltransferase
6 in E. coli which methylates the CmCWGG motif. None of this modification could be detected in the dcm
7 knock-out strain (Marinus and Løbner 2013). Regulation of gene expression by cytosine methylation has
8 been well reported in mammals (Deaton and Bird 2011). Microarray analysis of gene expression in the
9 E.coli Δdcm strain identified a role of cytosine methylation in controlling gene expression, especially
10 during stationary phase (Kahramanoglou Draftet al. 2012). Since DNA methylation is sequence specific, it is
11 likely that codon bias may affect gene expression through regulating DNA methylation patterns.
12 In this paper, we analyzed the role of codon bias on heterologous gene expression in E. coli in vivo. We
13 selected the amyA gene as an example to conduct codon optimization. Both protein and RNA levels were
14 measured to reveal the effect of codon usage bias at the translational and pre-translational levels. We
15 then investigated whether codon bias relied on RNA stability or DNA methylation to regulate mRNA
16 levels.
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1 2. Materials and Methods:
2 2.1 Strains and growth condition.
3 In this study, transgenic strains were made on the background of E. coli K-12 MG1655. The E. coli DH5α
4 strain was used for molecular cloning and plasmid amplification. The Luria-Bertani (LB) (1% NaCl, 1%
5 tryptone [Oxoid], 0.5% yeast extract [Oxoid]) broth and agar (2%) were used for routine growth. Bacteria
6 cultivated in the liquid LB were grown on a rotatory shaker at 200 rpm and 30℃ or 37℃. Bacteria
7 cultivated on agar plates were grown at 30℃ or 37℃ overnight. When necessary, ampicillin, kanamycin,
8 chloramphenicol and rifampicin were used at final concentrations of 100, 50, 34, and 250 μg/mL,
9 respectively.
10 2.2 Plasmid construction and strain generation.Draft
11 The original sequence of amyA was amplified from existing constructs in our group (Huang et al. 2017)
12 and codon optimized sequences were synthesized by Genewiz. The OmpA signal sequence was fused at
13 N terminus to facilitate secretion. For plasmid mediated expression, amyA sequences were inserted into
14 the expression cassette of pET-28a(+) and positive transformants were selected. For integration mediated
15 expression, pUC18 was utilized to build vectors containing the integration cassette. The integration
16 cassette was comprised of homologous sequence of upstream ptsG (100 bp, amplified from MG1655
17 genome), sequence containing lac promoter (320 bp, amplified from pUC18), amyA gene (optA or ori),
18 kanamycin resistance gene (1585 bp, amplified from pKD4) and homologous sequence of downstream
19 ptsG (100 bp, amplified from MG1655 genome). All primer sequences used in this study are listed in
20 Table S1. The vector with integration cassette was linearized by PCR and purified for later use.
21 To integrate amyA sequences into the ptsG locus of E. coli K12 MG1655, plasmid pKD46 encoding λ-
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1 Red recombination system was electroporated into MG1655. Positive transformants were selected on
2 ampicillin LB plate at 30℃, and inoculated into fresh liquid LB medium with 100 μg mL−1 ampicillin
3 (1:100 dilution) and grown to OD600 of 0.2 on a rotatory shaker at 200 rpm at 30℃. 10 mM L-arabinose
4 was added and cells were grown to the final OD600 of 0.4-0.6. Bacteria cells were then washed twice by
5 10% glycerol and re-suspended in 10% glycerol (100 μL/100 mL cultivation) to make electro-competent
6 cells. The cells were then transformed with the linearized vector containing integration cassette
7 (approximately 4.5 μg DNA into 100 μl freshly prepared competent cells). Positive transformants were
8 selected on LB plate with 50 μg mL−1 kanamycin at 30℃. The incubation should be strictly controlled at
9 30℃ in order to maintain pKD46, which was required later to make dcm knock-outs. Successful
10 chromosomal integrations were verified Draftby colony PCR and DNA sequencing. The transgenic strains
11 were named MG1655-amyA-ori and MG1655-amyA-optA.
12 Knock out of dcm was also achieved by the λ-Red recombination system. Chloramphenicol resistance
13 gene from pCP20 was amplified by PCR and fused with upstream and downstream sequences of dcm.
14 Then fragments were gel purified, and transformed into electro-competent cells of MG1655-amyA-ori
15 and MG1655-amyA-optA, which were prepared as described previously. Positive transformants were
16 selected on LB plate with 34 μg mL−1 chloramphenicol at 37℃ and verified by colony PCR.
17 2.3 RNA extraction and RT-PCR.
18 RNA was extracted using Trizol reagent (Solarbio). Briefly, bacteria cells were inoculated into fresh
19 liquid LB medium with 50 μg/mL kanamycin (1:100 dilution) and grown to OD600 of 0.6 on a rotatory
20 shaker at 200 rpm at 37℃. Then, 1 mM IPTG was added. Samples were taken at 12h and 36h after
21 induction. RNA was extracted following the manufacturer’s instruction. Finally, the total RNA was
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1 dissolved in 30 μl RNase-free water. RNA concentration was determined by Nanodrop 300 (Allsheng
2 Company). cDNA was generated from 1 μg total RNA using FastKing gDNA Dispelling RT SuperMix
3 Kit (TIANGEN BIOTECH) following the manufacturer’s guidelines. A total of 1 μl from a 1/50 dilution
4 of the cDNA was mixed with SYBR Green (TIANGEN BIOTECH) and primer sets for real-time PCR
5 (Bio-Rad CFX96). The PCR cycling consisted of a pre-denaturing step at 95°C for 10 min, followed by
6 40 cycles at 95°C for 10 s and 60°C for 32 s. 16S rRNA was used as control. The primers used in RT-
7 PCR are listed in Table S1. Due to the lack of identical sequence between amyA-optA and amyA-ori,
8 different primer sets were used. Therefore we additionally performed primer efficiency assay to adjust
9 RT-PCR results. In the primer efficiency assay, pUC18-amyA-ori and pUC18-amyA-opt constructs were
10 used as templates for RT-PCR. The kanamycinDraft resistance gene (kan) sequence was chosen as control.
11 Since the ratio of kan and amyA was 1:1, the relative levels of amyA-ori/amyA-opt to kan were used to
12 calculate primer efficiency. The result of primer efficiency assay was showed in Fig S2.
13 2.4 Bisulfite conversion.
14 Bacteria cells were grown in LB liquid medium in shake flasks at 37℃ until OD600 reached 2.0. The
15 genomic DNA was extracted using TIANamp Bacteria DNA Kit (TIANGEN BIOTECH). Bisulfite
16 conversion was performed using DNA Bisulfite Conversion Kit (TIANGEN BIOTECH) following the
17 manufacturer’s guidelines. The putative methylated regions were amplified by PCR using BSP-primers
18 and verified by sequencing.
19 2.5 The mRNA decay assay.
20 Bacteria cells were grown in LB liquid medium in shake flasks at 37℃ to OD600=0.8. Samples were
21 immediate harvested at different time points after the addition of rifampicin (final concentration of 250
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1 μg mL−1) which can initiate transcription inhibition. All RNA isolation procedures were accomplished
2 as described in 2.3, and RT-PCR was performed to quantify RNA levels.
3 2.6 Enzyme activity assay and western blot analysis.
4 Bacteria cells were cultured in LB liquid medium with 50 μg/mL kanamycin overnight and inoculated
5 into a fresh liquid LB medium with 50 μg/mL kanamycin (1:100 dilution) on a rotatory shaker at 200
6 rpm at 37℃. 1 mM IPTG was added to induce amylase expression when OD600 reached 0.6. 1 mL aliquot
7 was harvested at 12h and 36h time points, respectively. Supernatant was obtained by centrifugation and
8 enzyme activity of α‐amylase was measured by DNS method (Gosh.T.K 1987). For western blot,
9 appropriate amount of supernatant normalized by OD600 was loaded to ensure comparability. Western
10 blot was performed as described previouslyDraft (Zhou et al. 2013).
11 2.7 Codon optimization.
12 The codon usage frequency table of E. coli MG1655 (Fig. S1) was calculated from information on Codon
13 Usage Database (http://www.kazusa.or.jp/codon/). During codon optimization, every codon was
14 replaced by the synonymous one with highest usage frequency.
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1 3. Results:
2 3.1 Codon optimization enhances heterologous α‐amylase protein level while decreasing its mRNA level
3 As a representative recombinant protein, the deep sea Geobacillus sp 4j sourced α‐amylase coding gene
4 (amyA) (Huang et al. 2017) was studied here. Sequence codon optimization was designed according to
5 the codon usage frequency table (Fig. S1) generated from highly expressed genes in the E. coli MG1655
6 strain. Codon adaptation Index (CAI) plots of original, N-terminal codon optimized, C-terminal codon
7 optimized and full length codon optimized sequences were shown in Fig. 1a. Original and codon
8 optimized amyA sequences were expressed under lac promoter and induced by IPTG. The expression
9 cassettes were firstly transformed into E. coli MG1655 cells in the form of plasmids. Extracellular
10 enzyme activities were measured in strainsDraft bearing original, N-terminal codon optimized, C-terminal
11 codon optimized and full-length codon optimized sequences, respectively. As shown by Fig. 1b, enzyme
12 activities were not altered significantly by codon optimization, neither partial nor full-length. Plasmid
13 expression may not be a reasonable way to compare amylase expression under different codon
14 optimization strategies, since the copy number cannot be accurately controlled. Even though it can be
15 determined by experiment, the copy number of plasmids may vary during cultivation. Therefore, in order
16 to obtain the stable expression of foreign DNA, we then constructed transgenic E. coli strains with single
17 copy of the original amyA (amyA-ori) or full-length optimized amyA (amyA-optA) gene integrated into
18 the genome at the ptsG locus by Lambda Red recombinase-mediated homologous recombination.
19 Amplification of DNA across the junction of original genomic DNA and the amyA gene was used to
20 verify successful integration into the ptsG locus of E. coli MG1655 genome (Fig. 1c). Then the
21 extracellular enzyme activity was measured and compared in strains bearing original and codon
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1 optimized amylase sequences. As shown by Figure 1d, codon optimization resulted in 3-4 folds elevation
2 of extracellular enzyme activity. Elevated enzyme activity was due to higher protein level, confirmed by
3 western blot results (Fig. S5). Surprisingly, the trend in enzyme activity was opposite with intracellular
4 mRNA levels (Fig. 1e). Normalized by primer efficiency (Fig. S2), the amyA-optA mRNA level was
5 only 15% and 40% of amyA-ori at 12h and 36h after induction, respectively. This suggests that codon
6 usage bias works not only at the translational level, but also at the transcriptional or post-transcriptional
7 level.
8 3.2 Codon usage bias influences amyA mRNA stability
9 mRNA levels are usually determined by two factors: RNA synthesis and degradation. It has been
10 suggested that mRNA stability is related Draftto codon contents in Schizo-saccharomyces pombe (Harigaya
11 and Parker 2016) and zebrafish (Bazzini et al. 2016; Mishima and Tomari 2016). Non-optimal codons in
12 mRNA has been suggested to destabilize mRNA in yeast (Presnyak et al. 2015). However, in other
13 organism such as Neurospora, codon usage does not consistently influence mRNA stability (Zhou et al.
14 2016). To study the situation in E. coli, we then measured mRNA decay rates of amyA-optA and amyA-
15 ori in indicated strains after rifampicin treatment which blocked transcription by binding to the β subunit
16 of RNA polymerase (Campbell et al. 2001). mRNA levels amyA-optA/amyA-ori and 16S rRNA which
17 served as control were detected at 0, 5, 10, 15, 25 and 30 minutes after rifampicin addition. At each time
18 point, we calculated the ratio of amyA-optA/amyA-ori to 16S rRNA. Considering the different primer
19 amplification efficiency between amyA sequences and 16S rRNA, the initial ratio at time pint 0 was
20 normalized to 1. As shown by Fig. 2, the codon-optimized amyA-optA mRNA was obviously degraded
21 faster than 16S rRNA (Fig. 2b), while the amyA-ori mRNA was more stable (Fig. 2a). This piece of data
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1 is consistent with the amyA mRNA level data (Fig. 1e), suggesting that decreased amyA mRNA level in
2 codon optimized strain is at least partially caused by decreased stability.
3 3.3 DNA methylation pattern at amyA locus is altered by codon optimization
4 It has been reported in eukaryotes that codon bias regulated gene transcription events by affecting histone
5 modification. Although prokaryotic DNA does not bind histones, DNA methylation is an important
6 epigenetic mark that regulates gene expression. According to the sequence, there are 8 putative 5-
7 methylcytosine (5-mC) sites located within CCWGG motifs in amyA-optA (Fig. S4) while only 1 in
8 amyA-ori (Fig. S3). In order to confirm the cytosine methylation patterns, we performed the bisulfite
9 conversion assay. Bisulfide treatment converts cytosine to uracil, while 5-mC is resistant to this treatment.
10 Then a following PCR and sequencing analysisDraft are able to tell whether the cytosine is methylated or not.
11 As a result, all putative 5-mC sites were indeed methylated in both amyA-ori and amyA-optA (Fig. 3).
12 We then continued to figure out whether altered cytosine methylation pattern by codon optimization
13 contributed towards different RNA levels of amyA. The λ-Red recombination system was used again to
14 knock out the dcm gene encoding DNA cytosine methyltransferase (Dcm) in MG1655-amyA-optA and
15 MG1655-amyA-ori strains (Fig. 4a). Dcm knock out did not affect cell growth (Fig. 4b). Abolishment of
16 cytosine methylation slightly decreased amyA mRNA levels in both strains (Fig. 4c), suggesting that
17 cytosine methylation may have a minor effect to boost its transcription. Besides, the RNA level was
18 reduced by half in Δdcm-amyA-opt but by only 10%-30% in Δdcm-amyA-ori. This extent happen to
19 correlate with the number of 5-mC sites in amyA-opt and amyA-ori, suggesting that 5-mC sites may
20 contribute to regulate amyA RNA levels. However, more gene candidates should be tested to verify
21 whether this is a global effect or not, and experiments on transcription activity should be performed to
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1 tell the detailed mechanism.
2
Draft
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1 4. Discussion:
2 Here we took amyA gene as an example to study the role of codon usage bias on transcription in E. coli.
3 By codon optimization in vivo, we showed that codon bias affected both mRNA and protein levels.
4 Further examination showed that mRNA stability was indeed weakened by codon optimization,
5 contributing at least partially to the decreased RNA level. Since codon optimization resulted in sequence
6 change, cytosine methylation pattern was altered as well. Cytosine methylation may be a minor
7 mechanism brought by codon usage bias to regulate gene mRNA levels in E. coli, but more studies are
8 needed to confirm this. Besides, future researches on transcription activity may be able to localize the
9 detailed regulatory pattern.
10 Codon usage is considered as a translationalDraft mechanism to regulate protein expression for a long time.
11 Codon optimization is well known to improve protein expression by elevating translation rates (Lavner
12 and Kotlar 2005; Yu et al. 2015). This is a possible explanation for why AmyA protein level was
13 significantly higher although its mRNA was low (Fig.1 d&e). However, with recent discoveries, codon
14 bias seems also to be an important regulatory mechanism at the transcriptional level. Our research here
15 is a preliminary study to reveal the possible role of codon bias to regulate gene mRNA levels in
16 prokaryotes.
17 According to the codon usage frequency table of E. coli MG1655, G/C is enriched at the third position
18 of preferred codons. The overall GC content is 54.3% in codon optimized amyA sequence and 50.6% in
19 original sequences. RNA with a higher GC content was usually predicted to be more stable, however, an
20 opposite phenotype was observed here (Fig. 2a). It is likely that optimized codons may result in special
21 RNA structures, or entrance into degradation pathways.
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1 In the absence of histones, DNA modifications become major epigenetic marks in prokaryotes. Although
2 in most cases, DNA methylation regulating transcription is reported to reside within the promoter region,
3 its importance in coding regions cannot be omitted (Morris and Geballe 2000; Hood et al. 2009). Cytosine
4 methylation was also reported to alter the mechanical properties of DNA (Severin et al. 2011), and this
5 might have important impacts on transcriptional regulation. Overall abolishment of cytosine methylation
6 resulted slight decrease on amyA mRNA levels in E. coli. This sounds different with the most notable
7 effect of cytosine methylation in eukaryotes: 5-mC in promoter region always leads to gene silencing
8 (Bestor and Verdine 1994; Suzuki and Bird 2008). However, the phenotypes from prokaryotes and
9 eukaryotes may not have comparability, since in eukaryotes cytosine methylation occurs in CpG
10 dinucleotide context by DNA methyltransferasesDraft (DNMTs) instead of CCWGG (Castillo-Aguilera et al.
11 2017). The boost of amyA transcription by cytosine methylation may be gene specific, since different
12 genes have been reported to have different performance after knocking out dcm in E. coli
13 (Kahramanoglou et al. 2012). Of course further research is needed to reveal the detailed mechanisms.
14 Acknowledgements:
15 This work was sponsored by the National Key Research and Development Program of China
16 (2018YFA0900300), National Natural Science Foundation of China (31600056), Shanghai Science and
17 Technology Committee Rising-Star Program (19QA1402600) and Research Program of State Key
18 Laboratory of Bioreactor Engineering. The MG1655 stain and pKD constructs were kindly provided by
19 Dr. Shu Quan from East China University of Science and Technology.
20 Conflict of interest:
21 The authors declare that there is no conflict of interest.
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1 important determinant of gene expression levels largely through its effects on transcription. Proc. 2 Natl. Acad. Sci. U. S. A. 113(41): E6117–E6125. doi:10.1073/pnas.1606724113. 3 4
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1 Figure Legend
2 Fig.1 Codon Optimization Enhances Heterologous α‐amylase Protein Level While Decreasing its mRNA
3 Level in E. coli MG1655. (a) The Codon Adaptation Index (CAI) curves of amyA original sequence
4 (AmyA-ori), N-terminal codon optimized (AmyA-optN), C-terminal codon optimized (AmyA-optC) and
5 full length codon optimized (AmyA-optA) sequences (window size=20). (b) Extracellular enzyme activity
6 and specific activity of amylase under plasmid mediated expression. Enzyme activities were measured
7 24h after induction. (c) PCR performed with forward primer upstream of ptsG and reverse primer inside
8 amyA gene. Lanes: DNA Marker III; 2-16, positive transformants; WT, MG1655. Successful integration
9 results in an 800bp amplicon. (d) Specific extracellular amylase activity in MG1655-amyA-ori and 10 MG1655-amyA-optA strains. Enzyme activitiesDraft were measured 12h and 36h after induction. Error bars 11 denote ± s.e.m. (n = 3). ***, P < 0.001; *, P < 0.05. (e) Quantitative RT-PCR results showing the relative
12 amyA mRNA levels in MG1655-amyA-ori and MG1655-amyA-optA strains. Error bars denote ± s.e.m.
13 (n = 3). ***, P < 0.001; **, P < 0.01.
14
15 Fig.2 Stability of amylase mRNA was influenced by codon optimization. The relative ratio of amyA-ori
16 mRNA to 16S rRNA (Fig. 2a) and amyA-optA mRNA to 16S rRNA (Fig. 2b) are shown at the indicated
17 time points after addition of rifampicin. Error bars denote ± s.e.m. (n = 3). Considering the different
18 primer amplification efficiency between amyA sequences and 16S rRNA, the initial ratio at time point 0
19 is normalized to 1.
20
21 Fig.3 Schematic diagram of Dcm mediated 5-mC methylation sites in amyA-ori (a) and amyA-opt (b)
22 sequences. Complete DNA sequences are listed in Fig S2 and Fig S3. Original sequence has 1 while
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1 codon optimized sequence has 8 putative 5-mC sites. Line A, sequence after bisulfite conversion if 5-
2 mC is not methylated; Line B, sequence after bisulfide conversion if 5-mC is methylated; Line C, DNA
3 sequencing results confirming that all putative 5-mC sites are indeed methylated in the transgenic strains
4 we built.
5
6 Fig.4 Eliminating cytosine methylation affects amyA mRNA level more severely in the codon optimized
7 strain. (a) A schematic diagram showing the dcm locus and the region deleted in this study. In the Δdcm
8 strains, 1356 bp of dcm gene was deleted, leaving 43 bp at the 3’ end of the gene. This leaves the vsr
9 gene intact. (b) Growth curves of MG1655-amyA-optA, MG1655- amyA-ori, MG1655-Δdcm- amyA-optA
10 and MG1655-Δdcm- amyA-ori strains. (c)Draft Quantitative RT-PCR results showing the relative mRNA
11 levels of amyA gene in these strains. Error bars denote ± s.e.m. (n = 3). **, P <0.01.
12
13
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Draft
Fig.1 Codon Optimization Enhances Heterologous α‐amylase Protein Level While Decreasing its mRNA Level in E. coli MG1655. (a) The Codon Adaptation Index (CAI) curves of amyA original sequence (AmyA-ori), N- terminal codon optimized (AmyA-optN), C-terminal codon optimized (AmyA-optC) and full length codon optimized (AmyA-optA) sequences (window size=20). (b) Extracellular enzyme activity and specific activity of amylase under plasmid mediated expression. Enzyme activities were measured 24h after induction. (c) PCR performed with forward primer upstream of ptsG and reverse primer inside amyA gene. Lanes: DNA Marker III; 2-16, positive transformants; WT, MG1655. Successful integration results in an 800bp amplicon. (d) Specific extracellular amylase activity in MG1655-amyA-ori and MG1655-amyA-optA strains. Enzyme activities were measured 12h and 36h after induction. Error bars denote ± s.e.m. (n = 3). ***, P < 0.001; *, P < 0.05. (e) Quantitative RT-PCR results showing the relative amyA mRNA levels in MG1655-amyA-ori and MG1655-amyA-optA strains. Error bars denote ± s.e.m. (n = 3). ***, P < 0.001; **, P < 0.01.
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Draft
Fig.2 Stability of amylase mRNA was influenced by codon optimization. The relative ratio of amyA-ori mRNA to 16S rRNA (Fig. 2a) and amyA-optA mRNA to 16S rRNA (Fig. 2b) are shown at the indicated time points after addition of rifampicin. Error bars denote ± s.e.m. (n = 3). Considering the different primer amplification efficiency between amyA sequences and 16S rRNA, the initial ratio at time point 0 is normalized to 1.
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Fig.3 Schematic diagram of Dcm mediated 5-mC methylation sites in amyA-ori (a) and amyA-opt (b) sequences. Complete DNA sequences are listed in Fig S2 and Fig S3. Original sequence has 1 while codon optimized sequence has 8 putative 5-mC sites. Line A, sequence after bisulfite conversion if 5-mC is not methylated; Line B, sequence after bisulfide conversion if 5-mC is methylated; Line C, DNA sequencing results confirming that all putative 5-mC sites are indeed methylated in the transgenic strains we built.
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Fig.4 DNA methylation pattern at amyA locus is altered by codon optimization. (a) A schematic diagram showing the dcm locus and the region deleted in this study. In the Δdcm strains, 1356 bp of dcm gene was deleted, leaving 43 bp at the 3’ end of the gene.Draft This leaves the vsr gene intact. (b) Growth curves of optA, ori, Δdcm-optA and Δdcm-ori strains. (c) Quantitative RT-PCR results showing the relative indicated mRNA levels of ori, opt Δdcm-optA and Δdcm-ori strains. Error bars denote ± s.e.m. (n = 3). **P <0.01.
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