Name: identifying amino acid overproducers using rare-codon-rich markers
Uploaded: 2019-06-24T13:30:00
Description: Watch this Scientific Journal Video about Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers at JoVE.com

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...

<ol>
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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 ...

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