The work was jointly supported by the National Natural Science Foundation of China (grant no. 21676026), the National Key R&#38;D Program of China (grant no. 2017YFD0201400), and the China Postdoctoral Science Foundation (grant no. 2017M620643). Works in the UCLA Institute of Advancement (Suzhou) were supported by the internal grants from Jiangsu Province and Suzhou Industrial Park.

1. Construction of the plasmids expressing the rare-codon-rich marker genes
<ol>
	<li>Select a marker gene that contains an appropriate number of the common codons for the targeted amino acid. 
	NOTE: For L-leucine, the kanamycin resistance gene kanR, which contains 29 leucine codons, of which 27 are common codons, is used for the construction of the selection system13. The gfp gene, which contains 17 common codons out of 19 leucine codons, or the purple protein...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectinomycin+resistance+at+site+1192+in+16S+ribosomal+RNA+of+E.+coli:+an+analysis+of+three+mutants.">Spectinomycin resistance at site 1192 in 16S ribosomal RNA of E. coli: an analysis of three mutants.</a> Biochimie. 69 (8), 885-889 (1987).</li><li>Feng, L., Tumbula-Hansen, D., Toogood, H., S&#246;ll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+tRNA+recognition+of+a+tRNA+synthetase+by+a+single+amino+acid+change.">Expanding tRNA recognition of a tRNA synthetase by a single amino acid change.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (10), 5676-5681 (2003).</li><li>Naganuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+selective+tRNA+aminoacylation+mechanism+based+on+a+single+G&#8226;+U+pair.">The selective tRNA aminoacylation mechanism based on a single G&#8226; U pair.</a> Nature. 510 (7506), 507 (2014).</li><li>Hoesl, M. 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The number of rare codons in the marker genes and the selection or screening medium are critical to inhibit protein expressions from the rare-codon-modified marker genes. If no significant difference can be detected between protein expressions from the wild-type marker genes and their derivatives, increasing the number of rare codons or using a nutrient-limited medium may amplify the differences. However, if the inhibition effect is too strong, the protein expressions may not be recovered even by extra feeding of the cor...

The current production of amino acids relies heavily on fermentation. However, the titers and yields for most amino acid production strains are below the rising demands of the global amino acid market that is worth billions of dollars1,2. Obtaining high-performance amino acid overproducers are critical for the upgrade of the amino acid industry.
Traditional strategy to identify amino acid overproducers exploits the competitions between amino acids and their analogs in protein synthesis3,4. These analogs are able to charge the tRNAs that recognize the corresponding amino acids and thus inhibit the elongations of the peptide chains, leading to arrested growth or cell death5. One way to resist the analog stresses is to increase the concentrations of intracellular amino acids. The enriched amino acids will outcompete the analogs for the finite tRNAs and ensure the correct synthesis of functional proteins. Therefore, strains that survive the analogs can be selected and are likely the overproducers of the corresponding amino acids.
Although proved successful in selecting overproducers for amino acids such as L-leucine6, the analog-based strategy suffers from severe drawbacks. One major concern is the analog resistance originated from the process of mutagenesis or through spontaneous mutations. Strains with resistance can escape the selection by blocking, exporting, or degrading the analogs5. Another concern is the toxic side effects of the analogs on other cellular processes7. As a consequence, strains that survive the analog selection may not be the amino acid overproducers, while the desired overproducers could be falsely exterminated due to the negative side effects.
Here, a novel strategy based on the law of codon bias is presented in order to achieve accurate and rapid identifications of amino acid overproducers. Most amino acids are encoded by more than one nucleotide&#160;triplet that is favored differently by the host organisms8,9. Some codons are rarely used in the coding sequences and are referred to as the rare codons. Their translations into amino acids rely on the cognate tRNAs that carry the corresponding amino acids. However, the tRNAs that recognize rare codons usually have much lower abundances than the tRNAs of the common codons10,11. Consequently, these rare tRNAs are less likely to capture the free amino acids in the competitions with other isoacceptors, and translations of the rare-codon-rich sequences begin to decelerate or even are terminated when the amounts of amino acids are limited10. The translations could, theoretically, be restored if there is an amino acid surplus after charging the synonymous common tRNAs due to overproductions or extra feedings of the corresponding amino acids12. If the rare-codon-rich gene encodes a selection or screening marker, strains exhibiting the corresponding phenotypes can then be readily identified and are likely the overproducers of the targeted amino acids.
The above strategy is applied to establish a selection and a screening system for the identification of amino acid overproducers. The selection system uses antibiotic resistance genes (e.g., kanR) as markers while the screening system uses the genes encoding fluorescent (e.g., green fluorescent protein [GFP]) or chromogenic (e.g., PrancerPurple) proteins. The marker genes in both systems are modified by replacing defined numbers of the common codons for the targeted amino acid with its synonymous rare alternative. Strains in the mutation library that harbor the rare-codon-rich marker gene are selected or screened under proper conditions, and the overproducers of the targeted amino acids can be readily identified. The workflow begins with the construction of the rare-codon-rich marker gene system, followed by the optimization of the working conditions, and then the identification and verification of the amino acid overproducers. This analog-independent strategy is based on the dogma in protein translation and has been practically verified to enable accurate and rapid identifications of amino acid overproducers. Theoretically, it could be directly employed to amino acids with rare codons and to all microorganisms. In all, the rare-codon-based strategy will serve as an efficient alternative to the conventional analog-based approach when proper analogs for specific amino acids are unavailable, or when a high false positive rate is the major concern. The protocol below uses leucine rare codon to demonstrate this strategy in identifying Escherichia coli L-leucine overproducers.

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Thermo</td>
			<td>51101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure HiPure Plasmid MiniPrep Kit</td>
			<td>Transgen</td>
			<td>EM111-01</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure Quick Gel Extraction Kit</td>
			<td>Transgen</td>
			<td>EG101-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson assembly master mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Solarbio</td>
			<td>I8070</td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Biotek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>n-hexane</td>
			<td>Thermo</td>
			<td>H3061</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenyl isothiocyanate</td>
			<td>Sigma</td>
			<td>P1034</td>
			<td></td>
		</tr>
		<tr>
			<td>PrancerPurple CPB-37-441</td>
			<td>ATUM</td>
			<td>CPB-37-441</td>
			<td></td>
		</tr>
		<tr>
			<td>TransStar FastPfu Fly DNA polymerase</td>
			<td>Transgen</td>
			<td>AP231-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-high performance liquid chromatography</td>
			<td>Agilent</td>
			<td>1290 Infinity II</td>
			<td></td>
		</tr>
		<tr>
			<td>Wild type C. glutamicum</td>
			<td>ATCC</td>
			<td>13032</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-Gold E. coli competent cell</td>
			<td>Agilent</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>ZORBAX RRHD Eclipse Plus C18 column</td>
			<td>Agilent</td>
			<td>959759-902K</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying amino acid overproducers using rare-codon-rich markers

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally identified by harnessing the competitions between amino acids and their analogs. However, this analog-based method is of low accuracy, and proper analogs for specific amino acids are limited. Here, we present an alternative strategy that enables an accurate, sensitive, and high-throughput screening of amino acid overproducers using rare-codon-rich markers. This strategy is inspired by the phenomenon of codon usage bias in protein translation, for which codons are categorized into common or rare ones based on their frequencies of occurrence in the coding DNA. The translation of rare codons depends on their corresponding rare transfer RNAs (tRNAs), which cannot be fully charged by the cognate amino acids under starvation. Theoretically, the rare tRNAs can be charged if there is a surplus of the amino acids after charging the synonymous common isoacceptors. Therefore, retarded translations caused by rare codons could be restored by feeding or intracellular overproductions of the corresponding amino acids. Under this assumption, a selection or screening system for identifying amino acid overproducers is established by replacing the common codons of the targeted amino acids with their synonymous rare alternatives in the antibiotic resistance genes or the genes encoding fluorescent or chromogenic proteins. We show that the protein expressions can be greatly hindered by the incorporation of rare codons and that the levels of proteins correlate positively with the amino acid concentrations. Using this system, overproducers of multiple amino acids can be readily screened out from mutation libraries. This rare-codon-based strategy only requires a single modified gene, and the host is less likely to escape the selection than in other methods. It offers an alternative approach for obtaining amino acid overproducers.

For the selection system, a sharp decrease in OD600 for strains harboring the rare-codon-rich antibiotic resistance gene should be observed in comparison to the strain harboring the wild-type antibiotic resistance gene when cultured in a suitable medium (Figure 1a). Under the same conditions, the decrease in cell OD600 becomes more obvious as the number of rare codons in the antibiotic resistance gene increases (Figu...

Watch this Scientific Journal Video about Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers at JoVE.com

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

This study presents an alternative strategy to the conventional toxic analog-based method in identifying amino acid overproducers by using rare-codon-rich markers to achieve accuracy, sensitivity, and high-throughput simultaneously.

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

This method provides an efficient alternative to the conventional analog-based approach in obtaining amino acid overproducers. This technique outcompetes the traditional analog-based method by providing accuracy, sensitivity, and high throughput simultaneously. This method sheds new light on understanding amino acid overproduction strengths and the translation of rare codons and could, theoretically, be applied to all microorganisms.This protocol relies mostly on basic molecular experimental operations which are easy to follow but may require multiple trials in order to achieve the suitable selection of straining stringencies. A visual demonstration of this technique offers a direct appreciation on the performance of the selection and a screening system in identifying amino acid producers. To begin this procedure, add the vector in the marker fragments, on ice, in a molar ratio of one to one to seven point five microliters of assembly mix to a total volume of ten microliters.Incubate at 50 degrees Celsius for one hour. Transform five microliters of the assembly product into 50 microliters of component cells at 42 degrees Celsius for 30 seconds. Recover the cells in Super Optimal broth with Catabolite repression medium at 37 degrees Celsius for one hour.Then, plate them on LB Agar medium and incubate overnight at 37 degrees Celsius. The next day, use a preferred commercial kit to isolate the plasmid. First, transform 50 microliters of the competent cells with one microliter of plasmid that carries the wild-type KanR.Transform a second set of the competent cells with the plasmid containing KanR RC 29 with all leucine codons replaced by the rare codon CTA. Plate and incubate the cell culture as outlined in the text protocol. Next, transfer the colonies that harbor the wild-type selection marker gene in the rare-codon-rich derivative individually into five milliliters of five-fold diluted LB medium containing kanamycin at a concentration of 50 micrograms per milliliter.Incubate the samples in a shaker at 37 degrees Celsius while shaking at 250 rpm. Then, transfer 200 microliters of each of the cell cultures into a 96 well plate in triplicate at defined time points. Use a plate reader to measure the OD600.Inoculate the stains harboring the KanR RC 29 marker gene in two sets of five milliliters of the 0.2 x LB medium that contains kanamycin, with or without a supply of L-leucine. Add L-leucine to one of the media to a final concentration of one gram per liter. Incubate the samples in a shaker at 37 degrees Celsius with shaking at 250 rpm and measure the OD600 for each culture at defined time points.To begin, transform 50 microliters of the parent strain used for mutagenesis with one microliter of plasmid that carries the wild-type GFP or the wild-type PPG. Plate and incubate the cell cultures as outlined in the text protocol. Next, transfer the colonies individually to five milliliters of the properly diluted LB medium.Incubate the samples in a shaker at 37 degrees Celsius with shaking at 250 rpm. For fluorescence markers, transfer 200 microliters of the cell cultures into a 96 well clear bottom back plate in triplicate at defined time points. Measure the OD600 in the fluorescence and calculate the fluorescence intensity.For chromogenic markers, measure the color development of the cell cultures. Perform the feeding assay as previously described and measure the fluorescence intensity or the color development at defined time points. First, transform 50 microliters of the mutant cells with one microliter of the plasmid carrying the selection marker KanR RC 29, or the screening markers GFP RC or PPG RC.Next, centrifuge the cell cultures at 4000 times G for five minutes.For selection, discard the supernatant and add five milliliters of 0.2 x LB medium containing kanamycin to cells carrying the selection marker. Incubate the sample overnight in a shaker at 37 degrees Celsius. The next day, plate the overnight culture onto 0.2 x LB agar medium containing kanamycin and incubate at 37 degrees Celsius for 12 hours.For screening, plate the appropriate number of cells harboring the screening marker onto LB agar medium containing the appropriate antibiotic. Incubate at 37 degrees Celsius for eight hours. After this, inoculate the LB medium containing the appropriate antibiotic with each single colony from the plate.Incubate the samples in a shaker at 37 degrees Celsius while shaking at 250 rpm. Measure the OD600 and the fluorescence and calculate the fluorescence intensity if GFP RC is used. Measure the color development of the cell cultures if PPG RC is used.To verify the amino acid productivities of the candidate strains, prepare seed culture by inoculating five milliliters of LB medium with each of the candidate strains and let the cells grow overnight in a shaker at 250 rpm and at 37 degrees Celsius. The next day, harvest the cells from one milliliter of the cell culture by centrifuging at 4000 times G for two minutes. Discard the supernatant and resuspend the palette with one milliliter of sterile water.Inoculate 20 milliliters of M9 medium containing four percent glucose with 200 microliters of the cell suspension and incubate in a 250 milliliter shaker at 250 rpm and at 37 degrees Celsius for 24 hours. The next day, centrifuge one milliliter of the culture medium at 4000 times G for five minutes. Transfer 200 microliters of the supernatant into a clean 1.5 milliliter tube.Prepare L-leucine standard solutions at the concentrations shown here. In a fume hood, add 100 microliters of one millimolar triethylamine and 100 microliters of one molar phenyl isothiocyanate to both the supernatant and the standards. Mix the solutions gently and incubate them at room temperature for one hour.Then, add 400 microliters of n-hexane to the same tube and vortex it for ten seconds. The lower phase contains the amino acid derivatives and is used for HPLC analysis. Filter the lower phase through 0.2 microliter polytetrafluoroethylene membranes.After this, run one microliter of the sample on an ultra HPLC equipped with a C18 column with a flow rate of 0.42 milliliters per minute and at column temperature for 40 degrees Celsius. Use a diode array detector to detect the targeted amino acids at 254 nanometers and calculate their concentrations by mapping the peak areas under the standard curve. In this study, amino acid overproducers are identified by using rare codon-rich markers to simultaneously achieve accuracy, sensitivity, and high-throughput.For the selection system, a sharp decrease in OD600 for strains harboring the rare codon-rich antibiotic resistance gene should be observed in comparison to the strain harboring the wild-type. The inhibition of rare codon on protein expressions mostly takes place under starved conditions. After extra feeding of the corresponding amino acid, the OD600 for the strain harboring the rare codon-rich antibiotic resistance gene increases significantly.For the screening system, the fluorescence intensity in the number of fluorescent cells are significantly lower for the strain that expresses the fluorescent protein from the rare codon-rich gene than from the wild-type gene. Feeding of the corresponding amino acid will restore protein expressions from the rare codon-rich genes. When using the purple protein, the color developed from the rare codon-rich PPG is lighter than that from the wild-type gene.The undiluted LB medium allows slow expression of the rare codon-rich PPG without L-leucine feeding in the expressed purple protein becomes visible once the cells are pelleted. However, this does not conceal the fact that gene expression from the rare codon-rich PPG is enhanced by feeding of the L-leucine. The rare codon-based strategy could identify overproducers of the targeted amino acids from the mutation library and these mutants should produce higher amounts of the targeted amino acids than the parent strains.When attempting this procedure, it is important to adjust rare codon frequency to achieve a clear discrimination in the OD or color between cells harboring the wild-type markers and the rare codon-rich derivatives. Transcriptome analysis and the genome sequencing could be applied to the selected strains to investigate the mechanism related to amino acid productions. Using this technique, amino acid overproducers could be efficiently identified and analysis of their genetic information would offer new insights into their mechanisms behind amino acid overproductions.Amino acid derivatization could be harmful. Make sure to wear gloves and the protective clothing and perform these steps in a fume hood.

identifying-amino-acid-overproducers-using-rare-codon-rich-markers

Research

JoVE Journal

Biochemistry

7.4K visualizações.  Beijing Institute of Technology. Este método fornece uma alternativa eficiente à abordagem convencional baseada em analógico na obtenção de superprodutores de aminoácidos. Esta técnica compensa o método tradicional baseado em analógico, fornecendo precisão, sensibilidade e alto rendimento simultaneamente. Este método lança uma nova luz sobre a compreensão dos pontos fortes da superprodução de aminoácidos e a tradução de códons raros e poderia, teoricamente, ser aplicado a todos os microrganismos.Est...