Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

Summary

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.

Abstract

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.

Introduction

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 dollars^1,2. 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 synthesis^3,4. 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 triplet that is favored differently by the host organisms^8,9. 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 codons^10,11. 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., kan^R) 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.

Protocol

1. Construction of the plasmids expressing the rare-codon-rich marker genes

1. 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 kan^R, 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-encoding gene prancerpurple (ppg), which harbors 14 leucine common codons, is used for the screening system (Supplementary Table 1).
2. Replace the common codons in the marker genes with the synonymous rare codon. For L-leucine, replace its codons in kan^R, gfp, or ppg with the rare codon CTA, generating kan^R-RCs, gfp-RC, or ppg-RC, respectively¹³ (Supplementary Table 1).
   NOTE: The frequency of the rare codon in the marker genes will affect the stringency of the selection or screening system. In general, increasing the number of rare codons will increase the stringency of the selection or screening system. To achieve the appropriate selection or screening strength, design a series of marker genes that harbor different numbers of rare codons and compare their effects.
3. Generate building blocks of the rare-codon-rich marker genes using tools such as GeneDesign¹⁴ (http://54.235.254.95/gd/) for gene synthesis. Alternatively, order the marker genes from commercial gene synthesis services.
   1. On the GeneDesign page, choose Building block design (constant length overlap).
   2. Paste the sequences of the rare-codon-rich marker genes in the Sequence box.
   3. Define the overlap length between the assembly oligos; keep in mind that the default 40 bp works fine for most sequences.
      NOTE: See the online manual for more instructions on the settings of the other parameters.
   4. Click the Design building blocks button and order the oligonucleotides listed on the page.
4. Synthesize the rare-codon-modified genes by polymerase chain reaction (PCR)-based accurate synthesis¹⁵.
5. Ligate the rare-codon-rich kan^R-RC to vector pET-28a, gfp-RC to pSB1C3, and ppg-RC to CPB-37-441¹⁶.
   NOTE: The pET-28a and pSB1C3 plasmid maps are available on the SnapGene online plasmid database (Supplementary Table 1); the CPB-37-441 plasmid map is available on the ATUM chromogenic protein website.
   1. On ice, add the vector and the marker fragments in a molar ratio of 1:1 to 7.5 μL of assembly mix (see Table of Materials) to a total volume of 10 μL. Incubate the sample at 50 °C for 1 h.
   2. Transform 5 μL of the assembly product into 50 μL of competent cells (see the Table of Materials) at 42 °C for 30 s.
   3. Recover the cells in SOC (Super optimal broth with catabolite repression) medium at 37 °C for 1 h, plate them on LB (lysogeny broth) agar medium, and incubate them at 37 °C overnight.
   4. Inoculate the colony into LB medium and incubate at 37 °C for 8 h.
   5. Isolate the plasmid using a preferred commercial kit.

2. Optimizing the selection conditions

1. Make the parent strain used for mutagenesis into competent cells¹⁷.
2. Transform 50 μL of the competent cells with 1 μL of plasmid that carries the wild-type kan^R, and transform another set of the competent cells with the plasmid containing kan^R-RC29 with all leucine codon replaced by the rare codon CTA.
3. Add 950 μL of SOC medium and incubate the sample in a shaker at 250 rpm at 37 °C for 1 h.
4. Plate 100 μL of the cell culture onto the LB agar medium containing 50 μg·mL^-1 kanamycin, and incubate it at 37 °C for approximately 8 h until colonies appear.
5. Pick the colonies that harbor the wild-type kan^R and the leucine rare-codon-rich kan^R-RC29 and use each to inoculate 5 mL of fivefold diluted LB medium (0.2x LB) containing 50 μg·mL^-1 kanamycin. Incubate the samples in a shaker at 250 rpm at 37 °C.
   NOTE: The medium is crucial for the selection system. It should contain just enough carbon and nitrogen to allow expression of the antibiotic resistance protein from the wild-type gene rather than from the rare-codon-modified derivatives. In this case, the L-leucine concentration in 1x LB medium is too high to completely inhibit the protein expression from the rare-codon-rich kan^R. Thus, a diluted LB medium with a dilution factor such as 5 is used to generate a clear difference in protein expressions from the wild-type and the rare-codon-rich genes.
6. Transfer 200 μL of each of the cell cultures to a 96-well plate in triplicate at defined time points (e.g., 8 h, 16 h, and 24 h). Measure the OD₆₀₀ (optical density at 600 nm) using a plate reader.
   NOTE: If a decrease in the cell OD₆₀₀ cannot be detected for strains harboring the rare-codon-rich marker genes in comparison to the OD₆₀₀ of the strain harboring the wild-type marker genes, try to increase the amount of rare codon in the marker genes or use a more diluted medium.
7. Perform the amino acid feeding assay to test if the expressions of the rare-codon-rich marker genes (e.g., kan^R-RC29) can be restored by increasing the concentration of the targeted amino acids.
   NOTE: For the selection of L-leucine overproducers, L-leucine is used for feeding.
   1. Inoculate the strains harboring the kan^R-RC29 marker gene into 5 mL of the 0.2x LB (containing 50 μg·mL^-1 kanamycin) with or without the supply of 1 g·L^-1 L-leucine. Inoculate another 5 mL of the 0.2x LB with strains that harbor the wild-type kan^R as control. Incubate the samples in a shaker at 250 rpm at 37 °C.
   2. Measure the OD₆₀₀ for each culture at defined time points (e.g., 15 h, 17 h, 19 h, and 22 h).

3. Optimizing the screening conditions

1. Transform 50 μL of the parent strain used for mutagenesis with 1 μL of plasmid (~50 ng·μL^-1) that carries the wild-type gfp or the wild-type ppg. Also, transform the parent strain with the rare-codon-rich derivatives of the marker genes.
2. Add 950 μL of SOC medium and incubate the sample in a shaker at 250 rpm at 37 °C for 1 h.
3. Plate 100 μL of the cell culture onto the LB agar medium containing the appropriate antibiotics (25 μg·mL^-1 chloramphenicol for the gfp plasmid carrying a cm^R marker, or 50 μg·mL^-1 kanamycin for the ppg plasmid carrying a kan^R marker) and incubate overnight at 37 °C.
4. Pick one colony that harbors the wild-type gfp or ppg and one colony that harbors the corresponding rare-codon-rich derivative, and transfer them to 5 mL of the properly diluted LB medium individually. Incubate the samples in a shaker at 250 rpm at 37 °C.
   NOTE: For E. coli strains harboring the leucine rare-codon-rich gfp-RC or ppg-RC, the undiluted LB medium (1x LB) can be used to create significant differences in the expressions of the wild-type and the rare-codon-rich marker genes. Also, inducible promoters are used to drive the expressions of the screening marker genes. Begin the induction when the cells enter the exponential phase to achieve better discriminations.
5. For fluorescence markers, transfer 200 μL of each of the cell cultures into a 96-well clear-bottomed black plate in triplicate at defined time points (e.g., 2 h, 4 h, and 6 h). Measure the OD₆₀₀ and the fluorescence and calculate the fluorescence intensity (the ratio of fluorescence to OD₆₀₀). For chromogenic markers, measure the color development of the cell cultures.
   NOTE: If lower fluorescence intensities cannot be detected for strains harboring the gfp-RC in comparison to that of the strains harboring the wild-type gfp, or if a lighter color cannot be detected for cells expressing the purple protein from the ppg-RC than from the wild-type gene, try to increase the number of rare codon on the marker genes or use a more diluted medium.
6. Perform the feeding assay (see steps 2.7.1 and 2.7.2). Measure the fluorescence intensity or the color development at defined time points (e.g., 12 h, 18 h, and 24 h).

4. Identification of the amino acid overproducers

1. Inoculate 100 μL of the culture of the mutants into 5 mL of LB medium (2% v/v) and incubate it in a shaker at 250 rpm at 37 °C until the values of OD₆₀₀ reach 0.4.
2. Make the mutants into competent cells¹⁷.
3. Transform 50 μL of the mutant cells with 1 μL of the plasmid (~50 ng·μL^-1) carrying the selection marker kan^R-RC29 or the screening markers gfp-RC or ppg-RC. Add 950 μL of SOC medium and rotate the sample in a 37 °C shaker for 1 h.
4. Select amino acid overproducers.
   1. Centrifuge the cell culture at 4,000 x g for 5 min, discard the supernatant and add 5 mL of 0.2x LB medium containing 50 μg·mL^-1 kanamycin. Incubate the sample in a 37 °C shaker overnight.
   2. Plate the overnight culture (e.g., 100 μL, depends on the cell density of the culture) onto 0.2x LB agar medium containing 50 μg·mL^-1 kanamycin and incubate at 37 °C for 12 h.
      NOTE: The colonies developed are the candidates of the targeted amino acid overproducers and, in this case, the L-leucine overproducers.
5. Screen the amino acid overproducers.
   1. Plate the appropriate number of cells (e.g., 100 μL) harboring the screening marker (as described in step 4.3) onto the LB agar medium containing the appropriate antibiotic (25 μg·mL^-1 chloramphenicol for screening with gfp-RC and 50 μg·mL^-1 kanamycin for screening with ppg-RC) and incubate at 37 °C for 8 h.
   2. Inoculate the LB medium containing the appropriate antibiotic (see step 4.5.1) with each single colony from the plate. Incubate the samples in a shaker at 250 rpm at 37 °C.
   3. 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. Note that the strains exhibiting a higher fluorescence intensity or a deeper color than that of the parent strain are the candidate amino acid overproducers and, in this case, the L-leucine overproducers.
      NOTE: The fluorescence-activated cell sorting is also suitable for identifying single-cell high-producers when the rare-codon-rich fluorescent protein genes are used for screening.
6. Verify the amino acid productivities of the candidate strains.
   1. Inoculate 5 mL of LB medium with each of the candidate strains and let the cells grow overnight in a shaker at 250 rpm at 37 °C.
   2. Harvest the cells from 1 mL of the cell culture by centrifugation at 4,000 x g for 2 min. Discard the supernatant and resuspend the pellet with 1 mL of sterile water.
   3. Inoculate 20 mL of M9 medium containing 4% glucose with 200 μL of the cell suspension and incubate in a 250 mL shaker at 250 rpm at 37 °C for 24 h.
   4. Centrifuge 1 mL of the culture medium at 4,000 x g for 5 min. Transfer 200 μL of the supernatant to a clean 1.5 mL tube. Prepare L-leucine solutions (HPLC (high-performance liquid chromatography) grade) of 0.01, 0.05, 0.1, 0.5, and 1 g·L^-1 as standards.
   5. Add 100 μL of 1 mM triethylamine and 100 μL of 1 M phenyl isothiocyanate to the supernatant and the standards, mix them gently, and incubate them at room temperature for 1 h¹⁸.
      CAUTION: Triethylamine and phenyl isothiocyanate can cause severe skin burns and eye damage and is harmful if inhaled. Wear gloves and a mask and, if possible, perform this step in a fume hood.
   6. Add 400 μL of n-hexane to the same tube and vortex it for 10 s. Filter the lower phase containing the amino acid derivatives through a 0.2 μm polytetrafluoroethylene membrane.
      CAUTION: n-hexane can cause skin irritation. Wear gloves and protective clothing. If it comes in contact with skin, rinse the skin with plenty of water.
   7. Prepare mobile phase A by mixing 0.1 M sodium acetate (pH 6.5) and acetonitrile in a 99.3:0.7 volumetric ratio. Prepare acetonitrile (80% v/v) as mobile phase B. Filter all mobile phases through 0.2 μm polytetrafluoroethylene membranes.
      CAUTION: Acetonitrile is harmful if inhaled and can cause skin and eye irritations. Wear gloves and protective clothing and perform this step in a fume hood.
   8. Run 1 μL of the sample on an ultra-HPLC equipped with a C18 column according to the elution program in Table 1 with a flow rate of 0.42 mL·min^-1 and a column temperature of 40 °C. Detect the targeted amino acids at 254 nm with a diode array detector and calculate their concentrations by mapping the peak areas to the standard curve.

Time (min)	Mobile phase A (%)	Mobile phase B (%)
0	98	2
3.5	70	30
7	43	57
7.1	0	100
11	98	2

Table 1: Elution program for the quantification of amino acids.

Results

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...

Discussion

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...

Materials

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