The structure of a metal site in metalloenzymes critically influences the fine-tuning of some of the most complicated reactions in the chemistry of life processes.
We study the structure-function of the intracellular iron-sulfur world in aerobic and thermophilic archaea, and engineer new Escherichia coli auxotrophic expression host strains for deeper metalloenzyme analyses.LinkIcon

New Escherichia coli auxotrophic expression strains

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This is a collaborative project with Dr. Gennis research group at University of Illinois at Urbana-Champaign, U.S.A., supported in part by the JSPS-NSF International Collaborations in Chemistry Project Grant.

IMPORTANT NOTICE:

(Almost) ALL Escherichia coli auxotrophic expression strains listed in Table 1 (see below) are available through the public strain bank, LinkIconAddgene, USA - note that the item "Plasmid" in the table and heading in this link <https://www.addgene.org/Toshio_Iwasaki/> refers to a "Bacterial strain" and not a plasmid (as a result of the default setting of this website), as specified on the individual strain page.
These strains are also available from LinkIconRIKEN BRC, Japan <https://dnaconda.riken.jp/search/depositor/dep006963.html>.

LinkIcon If you have any questions about the auxotrophic expression strains listed in Table 1, please let me know by e-mail .

International shipment dates: on Mondays and Tuesdays (our local time) from Tokyo, Japan.

See "Lin, M. T., Fukazawa, R., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Iwasaki, T., and Gennis, R. B. (2015) Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins. Methods Enzymol. (Isotope Labeling of Biomolecules - Labeling Methods), 565, 45-66" Pubmed
and "Iwasaki, T., Miyajima-Nakano, Y., Fukazawa, R., Lin, M. T., Matsushita, S., Hagiuda, E., Taguchi, A. T., Dikanov, S. A., Oishi, Y., and Gennis, R. B. (2021) Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships. J. Biochem. (Review), 169, 387-394." Pubmed


Last update: December 20, 2021

Novel Escherichia coli C43(DE3) and BL21(DE3) Auxotrophic Expression Strains - an Overview


Amino acid-selective isotope labeling is an extremely powerful method to elucidate specific contributions of particular residues in the reaction mechanisms and/or folding of a target protein by magnetic resonance (e.g., nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR)) and vibrational (e.g., resonance Raman (RR) and Fourier transform infrared (FTIR)) spectroscopies, often aided by the X-ray crystal structure. These techniques can provide detailed information about protein–protein and protein–ligand interactions and dynamics.

One of the most convenient and cost-effective procedures for selective isotope labeling of proteins is to employ amino acid auxotrophic bacteria as the host strains for the overproduction of target proteins. A wild-type Escherichia coli strain has the ability to synthesize all 20 amino acids, whereas an E. coli auxotroph, having an essential gene involved in the biosynthesis of an amino acid disrupted, requires that particular amino acid for growth. However, no suitable auxotrophic strains are commercially available for high-level expression of the foreign genes coding for metalloenzymes from extremophilic archaea and bacteria, because (i) their high-level expression, e.g., in E. coli, often requires extra copies of tRNA genes for the cognate rare codons and (ii) specific growth conditions must be set for effective overproduction of holoproteins in a form suitable for biophysical studies.

To overcome these problems, Lin et al. have reported the construction of a set of cost-effective, high-yield auxotrophs in commonly used E. coli expression strain C43(DE3) (available from Lucigen Inc., Middleton, WI, USA), which is a derivative of the BL21(DE3) strain optimized for the successful overproduction of membrane proteins (called ML strains in Table 1). We adapted some of these auxotrophic strains by incorporation of a pACYC-based plasmid harboring tRNA genes (argU, ileY, and leuW) for the E. coli rare codons (Agilent Technologies) (step 5 in Fig. 3), and developed heterologous expression procedures suitable for site-specific isotope labeling of iron-sulfur proteins from extremophilic archaea and bacteria.

Table 1 summarizes the current set of new C43(DE3) and BL21(DE3) auxotrophic expression host strains of E. coli designed to facilitate the labeling of either membrane proteins or water-soluble proteins with selected amino acid types enriched with stable isotopes such as 2H, 13C and 15N (Methods Enzymol. 565, 45-66 (2015); J. Biochem. (Review) 169, 387-394 (2021)). The use of a suitable auxotrophic expression strain with the corresponding input isotope labeled amino acid(s) in the growth medium ensures high levels of efficiency as well as selectivity in stable isotope labeling, and is expected to solve many selective labeling problems.

PCR primer sequences for verification of these strains are given as DL (doc) files on the Supporting Information page .


Table 1. New E. coli amino acid auxotrophic host strains used for selective isotope labeling

Strain Precursor strain Genotype Selective amino acid labeling (and/or requirement)
(see Table 3)
ML2
CLY (Genotype cyo::kan)++ cyo::kan++ ilvE
 
Ile, Leu*
ML3 CLY (Genotype cyo::kan)++ cyo::kan++ hisG His
ML6 ML2 (Genotype cyo::kan)++ cyo::kan++ ilvE avtA Ile, Leu*, Val
ML8 CLY (Genotype cyo::kan)++ cyo::kan++ argH Arg
ML12 ML6 (Genotype cyo::kan)++ cyo::kan++ ilvE avtA aspC (Ala#), Ile, Leu*, Tyr#, Val
ML14 C43(DE3) tyrA+++ (Tyr)
ML17 C43(DE3) glnA Gln
ML21 ML14 tyrA hisG (Tyr), His
ML24 ML23 cyo ilvE avtA aspC hisG asnA (Ala#), Ile, Leu*, Tyr#, Val, His
ML25 ML24 cyo ilvE avtA aspC hisG asnA asnB (Ala#), Ile, Leu*, Tyr#, Val, His, Asn
ML26 ML23 cyo ilvE avtA aspC hisG argH (Ala#), Ile, Leu*, Tyr#, Val, His, Arg
ML31 ML26 cyo ilvE avtA aspC hisG argH metA (Ala#)Ile, Leu*, Tyr#, Val, His, Arg, Met
ML36 ML23 cyo ilvE avtA aspC hisG metA (Ala#), Ile, Leu*, Tyr#, Val, His, Met
ML40 ML31 cyo ilvE avtA aspC hisG argH metA lysA (Ala#), Ile, Leu*, Tyr#, Val, His, Arg, Met, Lys
ML41 ML40 cyo ilvE avtA aspC hisG argH metA lysA thrC (Ala#), Ile, Leu*, Tyr#, Val, His, Arg, Met, Lys, Thr
ML42 ML41 cyo ilvE avtA aspC hisG argH metA lysA thrC asnB (Ala#), Ile, Leu*, Tyr#, Val, His, Arg, Met, Lys, Thr
ML43 ML42 cyo ilvE avtA aspC hisG argH metA lysA thrC asnA asnB (Ala#), Ile, Leu*, Tyr#, Val, His, Arg, Met, Lys, Thr, Asn
ML45 ML44 cyo ilvE avtA aspC hisG  metA thrC lysA Ile, Leu*, Tyr#, Val, His, Met, Thr, Lys
       
YM138 C43(DE3) cysE Cys
YM154 C43(DE3) cysE   Cys (containing pACYC-based plasmid harboring argUileY, and leuW)
MS1 YM138 cysE hisG Cys, His
RF11 C43(DE3) metA Met
       
RF1 BL21 CodonPlus (DE3)-RIL glyA Gly
RF2 BL21 CodonPlus (DE3)-RIL thrC Thr** 
RF3  BL21 CodonPlus (DE3)-RIL aspC N.A. (Not applicable)
RF4++++ RF3 aspC tyrB Asp++++, Tyr, (Phe)
RF5++++ RF4 aspC tyrB hisG Asp++++, Tyr, (Phe), His
RF6 BL21 CodonPlus (DE3)-RIL proC Pro
RF8 BL21 CodonPlus (DE3)-RIL asnA asnB Asn 
RF10 BL21 CodonPlus (DE3)-RIL lysA
Lys
RF12 BL21 CodonPlus (DE3)-RIL trpA trpB 
Trp##
RF13++++ RF4 aspC tyrB trpA trpB 
Asp++++, Tyr, Trp, (Phe) 
RF14++++ RF13 aspC tyrB trpA trpB serB Asp++++, Tyr, Trp, (Phe), Ser+++++
RF15++++ RF14 aspC tyrB trpA trpB glyA serB Asp++++, Tyr, Trp, (Phe), Gly, Ser+++++
RF16++++ RF15++++ aspC tyrB trpA trpB glyA serB cysE Asp++++, Tyr, Trp, (Phe), Gly, Ser, Cys++++++, Ala++++++
RF17### RF4 aspC tyrB ilvE Asp++++, Tyr, Phe, Ile, Leu
RF18#### RF17 aspC tyrB ilvE avtA Asp++++, Tyr, Phe, Ile, Leu, Val####
RF21#### RF18 aspC tyrB ilvE avtA yfbQ(alaA) yfdZ(alaC)
Asp++++, Tyr, Phe, Ile, Leu, Val####
RF22 RF18 aspC tyrB ilvE avtA asnA asnB
Under investigation
RF23##### RF21 aspC tyrB ilvE avtA serB yfbQ(alaA) yfdZ(alaC)
Asp++++, Tyr, Phe, Ile, Leu, Val####, Ser#####
EH1 RF2 thrC ilvA Thr**, Ile




Notes:
Yellow, C43(DE3)-based auxotrophic expression strains. Cyan, BL21(DE3)-based auxotrophic expression strains.
These strains are NOT competent cells and one needs to make them competent before use.

++Note that CLY strain was derived from the C43(DE3)strain by transferring the cyo deletion (with kanamycin resistance cassette kanR) from an E. coli B strain to C43(DE3) strain by phage P1 [J. Biol. Chem. 282, 8777-8785 (2007)]. Thus, the kanR casette in ML3 strain and all other C43(DE3)-based auxotrophic expression strains derived from the CLY strain (cyo::kanR) CANNOT be removed by pCP20 because there are NO FRT sites flanking kanR (see Fig. 3).

+++ML14 without any cat cassette is available (as December 9, 2014). Note that earlier version of ML14 (before December 8, 2014) contained the cat cassette in the chromosome (genotype: tyrA::cat). ML14 requires L-Tyr for growth [J. Biochem. (Review) 169, 387-394 (2021)].

*In the presence of 0.4-1 mM Tyr, tyrB is repressed and Leu is required for growth in minimal medium (see Table 3). This strategy can only be applicable for a short-term cultivation but not suitable for a long-term cultivation for heterologous expression of foreign genes [Methods 55, 370-378 (2011); J. Biochem. (Review) 169, 387-394 (2021)].

#In the presence of 0.4-1 mM Tyr, tyrB is repressed and Tyr is required for growth in minimal medium. Under these conditions, this strain can also be used for selective labeling of the input Tyr and/or Ala label(s), although minor diffusion of the input label(s) can occur (see Tables 2, 3). Note that this strategy can only be applicable for a short-term cultivation but not suitable for a long-term cultivation for heterologous expression of foreign geness [Methods 55, 370-378 (2011); J. Biochem. (Review) 169, 387-394 (2021)].

**Minor diffusion and mixing of the input Thr 15Nα label to Gly and Ser can occur [J. Biochem. (Review) 169, 387-394 (2021)]. Further deletion of a L-threonine dehydrogenase (tdh) gene in the delta-thrC/ilvA strain EH1 (Table1) is planned, which would be more suitable for selective labeling of proteins with 13C-Thr.

##RF12 strain requires Trp for growth in M63 minimal medium [Methods Enzymol. 565, 45-66 (2015); J. Biochem. (Review) 169, 387-394 (2021)]. For effective Trp labeling of a recombinant protein, the presence of 0.4-1 mM Tyr in growth medium to repress the tyrB gene is recommended (see Table 3). This strategy can only be applicable for a short-term cultivation but not suitable for a long-term cultivation for heterologous expression of foreign genes [Methods 55, 370-378 (2011)].

++++RF4, RF5, RF13, RF14, RF15, RF16, RF17, RF18, RF21, RF22, and RF23 strains have knockouts in both aspC and tyrB genes and therefore require the presence of L-Asp (but not L-Glu) for growth in M63 minimal medium [Methods Enzymol. 565, 45-66 (2015); J. Biochem. (Review) 169, 387-394 (2021)]. These strains can be used for selective labeling of selected aromatic amino acid(s) such as Tyr (RF4, RF5, RF13, RF14, RF15, (RF16, RF17, RF18, RF21, RF22, RF23)) and/or Trp (RF13, RF14, RF15, (RF16)) (see Table 1 [J. Biochem. (Review) 169, 387-394 (2021)]). Of these, RF15, RF16, RF17, RF18, RF21, RF22, and RF23 strains strains grow only slowly in M63 minimal medium in the presence of the L-amino acids specified in Table 1 [J. Biochem. (Review) 169, 387-394 (2021)]. We have NOT tested if these strains could also be used for selective Asp labeling studies (under investigation).

+++++RF14 strain has knockouts in aspC, tyrB, trpA, trpB, and serB genes and requires the presence of L-Asp, L-Tyr, L-Trp plus L-Ser for growth in M63 minimal medium, but it is not considered to be ideal to selectively label L-serine [J. Biochem. (Review) 169, 387-394 (2021)](see Table 1). RF15 strain has knockouts in aspC, tyrB, trpA, trpB, glyA and serB genes and requires the presence of L-Asp, L-Tyr, L-Trp, L-Gly plus L-Ser for growth in M63 minimal medium, but it does NOT grow in the presence of L-Asp, L-Tyr, L-Trp, L-Gly, L-Ser plus L-Cys (either in the presence or absence of L-Ala) (i.e., L-Cys inhibits the growth of RF15 [J. Biochem. (Review) 169, 387-394 (2021)], see Table 1). These results suggest that appropriate settings for the bacterial growth are required for the "reverse labeling" experiments [e.g., see Methods Enzymol. 565, 45-66 (2015)]. We have NOT tested if RF15 could be used for selective Ser labeling studies (under investigation).

++++++Like RF15, RF16, which was previously considered as an "ideal genotype" to selectively label L-serine and grows slowly in the LB medium, does NOT grow in M63 minimal medium in the presence of L-Asp, L-Tyr, L-Trp, L-Gly, L-Ser plus L-Cys [J. Biochem. (Review) 169, 387-394 (2021)]. We found that RF16 (but not RF15) grows very slowly in M63 minimal medium in the presence of L-Asp, L-Tyr, L-Trp, L-Gly, L-Ser, L-Cys plus L-Ala [J. Biochem. (Review) 169, 387-394 (2021)] (see Table 1). These results suggest that appropriate settings for the bacterial growth are required for the "reverse labeling" experiments [e.g., see Methods Enzymol. 565, 45-66 (2015)]. We have NOT tested if RF16 could be used for selective Ser labeling studies (under investigation).

###RF17 strain has knockouts in aspC, tyrB, and ilvE genes and requires the presence of L-Asp, L-Tyr, L-Phe, L-Ile plus L-Leu for (slow) growth in M63 minimal medium [J. Biochem. (Review) 169, 387-394 (2021)].

####RF18 and RF21 strains have knockouts in the four general transaminase genes of E. coli (aspC, tyrB, ilvE, and avtA) and are found to require the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Val for slow growth in M63 minimal medium [J. Biochem. (Review) 169, 387-394 (2021)]. Although RF21 strain has further knockouts in yfbQ (alaA) and yfdZ (alaC) genes, it is NOT an L-Ala auxotroph, either (requiring the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Val for slow growth in M63 minimal medium, like RF18) [J. Biochem. (Review) 169, 387-394 (2021)]. We have NOT tested if these strains could also be used for selective Ala labeling studies.

#####RF23 stock strain should be used with caution. This strain has knockouts in aspC, tyrB, ilvE, avtA, alaA, alaC, and serB genes and requires the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Ser for slow growth in M63 minimal medium (i.e., it is NOT an L-Ala auxotroph) [J. Biochem. (Review) 169, 387-394 (2021)]. Although most of their target genes in Table 1 were disrupted as planned, serB (and possibly tyrB) gene(s) was deleted but not in a way originally designed, for reasons unclear to us (this apparently occurred during and/or after removal of pCP20 from the cells). We have NOT tested if this strain could also be used for selective Ala or Ser labeling studies. For selective Ser labeling studies, RF15 (and/or RF16) strain is more suitable than RF23.

ML41 and ML45 stock strains should be used with caution. Although most of their target genes in Table 1 were disrupted as planned, one or two genes (marked in red in Table 1) were deleted in a way not originally designed, for reasons unclear to us. LinkIconFollow the link to learn more.

ML43 stock strain in Table 1can be used as expression host cells although the recombinant protein production level (yield) in ML43 is significantly lower than normal strain (~10-20%), for reasons unclear to us (we could not produce 15Namide-Asn labeled TthNEET protein in a significant amount using this strain). For selective Asn isotope labeling study, RF8 strain can be used.

C43(DE3) (Lucigen) based cysteine auxotrophic strains, YM138, YM154, and especially MS1, grow slowly in Luria-Bertani medium, and even more poorly in nonlabeled algal CHL medium (Chlorella Industry Co. Ltd., Fukuoka, Japan) with negligible amount of L-cysteine [J. Am. Chem. Soc. 134, 19731-19738 (2012); Inorg. Chem. 57, 741-746 (2018)].

BL21-CodonPlus(DE3)-RIL (Stratagene) based strain RF3 is a precursor without any resistance cassette and was used for construction of the RF4 strain [J. Am. Chem. Soc. 134, 19731-19738 (2012)].

ML40, YM138, MS1, RF4, RF5, RF6, and RF8 strains containing a pACYC-based plasmid harboring tRNA genes (argU, ileY, and leuW) for the E. coli rare codons (Agilent Technologies) (called ML40K1, YM154 (see Table 1), MS1RIL, RF4RIL, RF5RIL, RF6RIL, and RF8RIL, respectively), are available upon request (to T.I.). These strains can be used for overexpression of foreign genes with rare codons [J. Am. Chem. Soc. 134, 19731-19738 (2012)].


LinkIconFollow the link to learn more about these auxotrophic expression strains here.

Primary references to be cited:

Lin, M. T., Sperling, L. J., Frericks Schmidt, H. L., Tang, M., Samoilova, R. I., Kumasaka, T., Iwasaki, T., Dikanov, S. A., Rienstra, C. M., and Gennis, R. B. (2011) A rapid and robust method for selective isotope labeling of proteins. Methods 55, 370-378. Pubmed

Iwasaki, T., Fukazawa, R., Miyajima-Nakano, Y., Baldansuren, A., Matsushita, S., Lin, M. T., Gennis, R. B., Hasegawa, K., Kumasaka, T., and Dikanov, S. A. (2012) Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin. J. Am. Chem. Soc. 134, 19731-19738. Pubmed

Lin, M. T., Fukazawa, R., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Iwasaki, T.,* and Gennis, R. B. (2015) Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins. Methods Enzymol. (Isotope Labeling of Biomolecules - Labeling Methods), 565, 45-66. Pubmed

Iwasaki, T.,* Miyajima-Nakano, Y., Fukazawa, R., Lin, M. T., Matsushita, S., Hagiuda, E., Taguchi, A. T., Dikanov, S. A., Oishi, Y., and Gennis, R. B. (2021) Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships. J. Biochem. (Review), 169, 387-394. Pubmed

  • All ML strains were engineered by Dr. Gennis research group (Myat T. Lin, Robert B. Gennis) at University of Illinois at Urbana-Champaign, U.S.A.
  • YM, MS, EH, and RF strains were engineered by our research group (Yoshiharu Miyajima-Nakano, Risako Fukazawa, Emi Hagiuda, Shin-ichi Matsushita, Toshio Iwasaki) at Nippon Medical School, Japan, in collaboration with Dr. Gennis research group.


(Almost) ALL these E. coli auxotrophic expression strains listed in Table 1 are available through either LinkIconAddgene, USA (note that the item "Plasmid" in the table and heading in this link <https://www.addgene.org/Toshio_Iwasaki/> refers to a "Bacterial strain" and NOT a plasmid as a result of the default setting of this website, as specified on the individual strain page), LinkIconRIKEN BRC, Japan <https://dnaconda.riken.jp/search/depositor/dep006963.html>, or upon request to T.I. (for all ML, YM, MS, EH, and RF strains listed inTable 1, Nippon Medical School, Japan) or R.B.G. (for all ML strains only, University of Illinois at Urbana-Champaign, U.S.A.).

Note that these strains are NOT competent cells and one needs to make them competent before use.

  • This strain bank project was supported in part by the International Collaborations in Chemistry Grant from JSPS (T.I.) and NSF (CHE-1026541 to S.A.D.), the JSPS Grant-in-aid 24659202 (T.I.), the Nagase Science and Technology Foundation Research Grant (T.I.), the DE-FG02-87ER13716 (R.B.G.) and DE-FG02-08ER15960 (S.A.D.) Grants from US DOE, NIH & NIGMS Roadmap Initiative (R01GM075937), and NIH grant GM062954 (S.A.D.).


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Background

Amino-acid selective isotope labeling of proteins represents a general approach in which a defined set of amino acid types in proteins is enriched with stable isotopes such as 2H, 13C and 15N. Biophysical analyses using such selectively labeled samples can provide detailed information about protein–protein and protein–ligand interactions and dynamics.

There are several ways to selectively incorporate isotope labels at selected amino acid residue types for protein samples. Residue-specific labeling can sometimes be performed without the use of auxotrophic host strains by the addition of excess unlabeled amino acids (Table 2) or enzyme inhibitors that block the interconversions between different amino acids. Although the isotopic dilution and scrambling cannot be completely eliminated, labeling specificity can be improved with such an approach in some cases.

Sample preparations by the in vitro cell-free protein synthesis system have several advantages over in vivo biosynthesis, particularly for selective labeling of Asp or Glu residues with proteins, which is extremely difficult or impossible by other means. However, this in vitro labeling approach is usually limited to small and medium size proteins with relatively simple subunit assembly and maturation processes, and are not yet suitable for producing huge membrane protein machineries, for example. For such proteins, in vivo expression in biological hosts such as E. coli is the only practical option for selective isotopic labeling.


Table 2. Possible diffusion and/or dilution of the input 15N-labeled materials

 Extensive scrambing without selective labeling of the input materials:  Asp, Glu.

 Selective labeling of the input without significant diffusion or dilution:  Ala, His, Lys, Leu, Thr.

Minor diffusion of the input: Ile, Gln, Val, Tyr.

Gly can be labeled as Gly and/or (under certain conditions) Ser. Input serine labels usually diffused very fast primaily into the 15N-amide side chain of Gln or Asn without labeling of Ser.

Notes:
*Diffusion and/or dilution of several input label materials can be minimized when a wild-type (non-auxotrophic) expression strain of E. coli BL21(DE3) is grown in a medium containing the appropriate unlabeled amino acid mixture. The results reported by Chae YK [J. Korean Mag. Res. Soc. 4, 133-149 (2000)] may be used as a short guideline when selective labeling strategy is considered.




A wild-type E. coli strain can synthesize all 20 amino acids in vivo. Note that the bacterial biosynthetic and degradation pathways of many amino acids significantly overlap, and many amino acids serve as either precursors or breakdown products for other amino acids (Fig. 1). Moreover, E. coli also has at least four general transaminases (or aminotransferases), encoded respectively by ilvE, avtA, aspC and tyrB genes, which catalyze the final (reversible) amino transfer reactions in biosynthesis of Ile, Leu, Val, Tyr, Phe, and Asp, and share many common substrates (Figs. 1, 2; see the Supporting Information page).

Figure 1. Schematic view of amino acid biosynthesis (main pathways) in Escherichia coli. This scheme can only be used as a general guide with precaution for some amino acids. Adapted from DS Waugh [J. Biomol. NMR 8, 184-192 (1996)]. The knocked-out genes in Table 1 are shown in red [J. Biochem. (Review) 169, 387-394 (2021)].


Figure 2. Schematic view of selected amino acid biosynthesis pathways in E. coli, catalyzed by four general transaminases (or aminotransferases) (adapted from Fig. 1). They are the products of the ilvE, avtA, aspC and tyrB genes, respectively, and catalyze the interconversion of amino acids and ketoacids by transfer of amino groups, with the overlapping specificities: except for the avtA gene product, the other three general transaminases can use multiple substrates. The reversible transfer reactions and the overlapping specificities of these four transaminases pose a major problem for selective labeling with certain amino acids. Ideally, the best option in such cases is to use a strain with defects in all four general transaminases (ilvE, avtA, aspC and tyrB). Such an "ideal" strain is recently available with E. coli BL21(DE3) (Table 1), although the ilvE / tyrB double deletion from the C43(DE3) chromosome has been unsuccessful so far, for reasons unclear to us (Table 1) [J. Biochem. (Review) 169, 387-394 (2021)]. This scheme can only be used as a general guide with precaution for some amino acids.




In contrast, an E. coli auxotrophic strain, which typically has an essential gene(s) involved in the biosynthesis of an amino acid(s) disrupted, requires that particular amino acid(s) for growth. Thus, the use of a suitable auxotrophic strain with the corresponding isotope labeled amino acid supplied in the growth medium is expected to ensure high levels of efficiency as well as selectivity in selective isotope labeling.


Table 3. Ideal E. coli genotypes for selective isotope labeling of proteins with 15N-amino acids

Amino acid Target genes
Ideal E. coli genotypes 
New E. coli auxotroph strains listed in Table 1
Arg. argH argH ML8, ML26, ML31, ML40, ML42, ML43 
Cys cysE cysE YM138, YM154, MS1, (RF16++++)
Gln glnA, gltB, ybaS, yneH glnA** ML17** 
Gly glyA glyA RF1, RF15++++, (RF16++++) 
His hisG hisG ML3, ML21, ML25, ML26, ML31, ML36, ML40, ML42, ML43, MS1, RF5 
Ile ilvE ilvE ML2, ML6, ML12, ML24, ML25, ML26, ML31, ML40, ML42, ML43
(EH1 requires L-Thr and L-Ile for growth in M63 minimal medium) 
Lys lysA lysA ML40, ML42, ML43, RF10
Met metA metA ML31, ML36, ML40, ML42, ML43, RF11
Pro proC proC RF6
Thr thrC thrC+ ML42, ML43, RF2+, EH1+ 
Ala# aspC, avtA, ilvE, tyrB aspC avtA ilvE tyrB# ML12#, ML24#, ML25#, ML26#, ML31#, ML36#, ML40#, ML42#, ML43# (#only in the presence of 0.4-1 mM Tyr);  RF16++++ (very slow growth and complicated amino acid requirements); RF18###, RF21###, RF22, and RF 23#### (##, ###, ####slow growth and complicated amino acid requirements)
Asn asnA, asnB asnA asnB ML25, ML43, RF8 
Asp++ asd, asnA, asnB, aspC, tyrB asd asnA asnB aspC tyrB# Not available++ (RF4+++, RF5+++, RF13+++, RF14+++, RF15+++, RF16+++, RF17##, RF18###, RF21###, RF22, and RF 23#### require L-Asp for growth in M63 minimal medium)
Glu++ argH, aspC, gdh, glnA, gltB, ilvE, proC, tyrB argH aspC gdh glnA gltB ilvE proC tyrB# Not available++
(In view of the crucial roles in E. coli central amino acid metabolism and nitrogen assimilation, as well as requirement of so many genetic defects to guard against scrambling of the input label, labeling with 15N-Glu is not recommended.) 
Leu* ilvE, tyrB ilvE tyrB* ML2*, ML6*, ML12*, ML24*, ML25*, ML26*, ML31*, ML36*, ML40*, ML42*, ML43* (*only in the presence of 0.4-1 mM Tyr), RF17##, RF18###, RF21###, RF22, and RF 23#### (##, ###, ####slow growth and complicated amino acid requirements)
Phe aspC, ilvE, tyrB aspC ilvE tyrB# ML12#, ML24#, ML25#, ML26#, ML31#, ML36#, ML40#, ML42#, ML43# (#only in the presence of 0.4-1 mM Tyr); (RF4, RF5, RF13?, RF15?, RF16?);  RF17##, RF18###, RF21###, RF22, and RF 23#### (##, ###, ####slow growth and complicated amino acid requirements)
Ser cysE, glyA, serB, trpB cysE glyA serB trpB (RF14++++, not recommended), RF15++++, (RF16++++, very slow growth), (RF 23####, slow growth and complicated amino acid requirements)
Trp trpB, tyrB trpB tyrB# RF13+++, (RF14++++), RF15++++, (RF16++++, very slow growth), RF12# (#tyrB repressed in the presence of 0.4-1 mM Tyr)
Tyr aspC, tyrB aspC tyrB# ML12#, ML24#, ML25#, ML26#, ML31#, ML36#, ML40#, ML42#, ML43# (#only in the presence of 0.4-1 mM Tyr); RF4, RF5, RF13+++, (RF14++++)RF15++++, (RF16++++, very slow growth), RF17##, RF18###, RF21###, RF22, and RF 23#### (##, ###, ####slow growth and complicated amino acid requirements)
Val avtA, ilvE avtA ilvE ML6, ML12, ML24, ML25, ML26, ML31, ML36, ML40, ML42, ML43, RF18###, RF21###, RF22, and RF 23#### (##, ###, ####slow growth and complicated amino acid requirements) 

Notes:
New E. coli auxotrophic strains in bold letters (right column) have been applied for selective isotope labeling of iron-sulfur proteins in our laboratory. Recombinant protein production yield is significantly lower (~10-20%) in ML43 strain than normal strains, for reasons unclear to us.

**This strain is suitable for selective labeling of proteins with Gln 15Nε. Minor diffusion and mixing of the input Gln 15Nα label can occur [Methods 55, 370-378 (2011)].

+ Minor diffusion and mixing of the input Thr 15Nα label to Gly and Ser can occur [J. Biochem. (Review) 169, 387-394 (2021)]. Further deletion of a L-threonine dehydrogenase (tdh) gene in the delta-thrC/ilvA strain EH1 (Table1) is planned, which would be more suitable for selective labeling of proteins with 13C-Thr.

*In the presence of 0.4-1 mM Tyr, tyrB is repressed and Leu is required for growth in minimal medium. This strategy can only be applicable for a short-term cultivation but not suitable for a long-term cultivation for heterologous expression of foreign genes [Methods 55, 370-378 (2011); J. Biochem. (Review) 169, 387-394 (2021)].

# In the presence of 0.4-1 mM Tyr in the growth medium, tyrB is repressed (like a tyrB-null strain). This strategy can only be applicable for a short-term cultivation but not suitable for a long-term cultivation for heterologous expression of foreign genes [Methods 55, 370-378 (2011)].

++ Note that Asp and Glu are central to the bacterial major metabolic pathways, and effective labeling of these amino acids is uncertain even after the ideal genotypes are available (not tested yet). Data adapted from DS Waugh [J. Biomol. NMR 8, 184-192 (1996)].

+++RF4, RF5, RF13, RF14, and RF 15 strains have knockouts in both aspC and tyrB genes and therefore require the presence of L-Asp (but not L-Glu) for growth in M63 minimal medium [J. Biochem. (Review) 169, 387-394 (2021)]. These strains can be used for selective labeling of selected aromatic amino acid(s) such as Tyr (RF4, RF5, RF13, RF14, RF15) and/or Trp (RF13, RF14, RF15) [Methods Enzymol. 565, 45-66 (2015); J. Biochem. (Review) 169, 387-394 (2021)] (see Table 1). RF16 strain has knockouts in aspC, tyrB, trpA, trpB, glyA, serB, and cysE genes, and grows very slowly in M63 minimal medium only in the presence of L-Asp, L-Tyr, L-Trp, L-Ser, L-Gly, L-Cys, plus L-Ala [J. Biochem. (Review) 169, 387-394 (2021)] (see Table 1). We have NOT tested if these strains could also be used for selective Asp labeling studies (under investigation).

++++RF15 strain has knockouts in aspC, tyrB, trpA, trpB, glyA, and serB genes, and requires the presence of L-Asp (but not L-Glu), L-Tyr, L-Trp, L-Gly, plus L-Ser for growth in M63 minimal medium, but further addition of L-Cys repressed its growth either in the presence or absence of L-Ala [J. Biochem. (Review) 169, 387-394 (2021)] (see Table 1). RF16, which was previously considered as an "ideal genotype" to selectively label L-serine and grows slowly in the LB medium, does grow very slowly in M63 minimal medium only in the presence of L-Asp, L-Tyr, L-Trp, L-Gly, L-Ser, L-Cys plus L-Ala [J. Biochem. (Review) 169, 387-394 (2021)] (see Table 1). We have NOT tested if these strains could also be used for selective Ser labeling studies (under investigation). RF14 grows faster than these two strains [in M63 minimal medium in the presence of L-Asp, L-Tyr, L-Trp, and L-Ser], but may NOT be suitable for selective Ser labeling studies.

##RF17 strain has knockouts in aspC, tyrB, and ilvE genes and requires the presence of L-Asp, L-Tyr, L-Phe, L-Ile plus L-Leu for (slow) growth in M63 minimal medium [J. Biochem. (Review) 169, 387-394 (2021)].

###RF18 and RF21 strains have knockouts in the four general transaminase genes of E. coli (aspC, tyrB, ilvE, and avtA) and are found to require the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Val for slow growth in M63 minimal medium [J. Biochem. (Review) 169, 387-394 (2021)]. Although RF21 strain has further knockouts in yfbQ (alaA) and yfdZ (alaC) genes, it is NOT an L-Ala auxotroph, either (requiring the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Val for slow growth in M63 minimal medium, like RF18) [J. Biochem. (Review) 169, 387-394 (2021)].

####RF23 stock strain should be used with caution. It has knockouts in aspC, tyrB, ilvE, avtA, alaA, alaC, and serB genes and requires the presence of L-Asp, L-Tyr, L-Phe, L-Ile, L-Leu plus L-Ser for slow growth in M63 minimal medium (i.e., this strain is NOT an L-Ala auxotroph) [J. Biochem. (Review) 169, 387-394 (2021)]. Although most of their target genes in Table 1 were disrupted as planned, serB gene was deleted but not in a way originally designed, for reasons unclear to us. For selective Ser labeling studies, RF15 (and/or RF16) strain is more suitable than RF23.




A major obstacle to selective isotopic enrichment of proteins with stable isotopes was the shortage of suitable auxotrophic host strains that are compatible with commonly used expression vectors. E. coli BL21(DE3) and C43(DE3) strains incorporate an inducible T7 RNA polymerase gene and represent one of the most popular hosts for protein production.

In this collaborative project, new auxotrophic C43(DE3) and BL21(DE3) strains, requiring the addition of a defined set of one or more selected amino acids in the growth medium (Table 1), were generated by genomic insertion/deletion mutagenesis techniques (Fig. 3). Genes were also targeted which encode enzymes, such as general deaminases, which would otherwise result in interconverting amino acids and scrambling the input isotope labels (Table 1). These auxotrophic strains are designed for the selective labeling of amino acids, and can be used for cost effective, high-yield production of any recombinant water-soluble or membrane protein that can be expressed in E. coli.

Figure 3. Schematic procedures used for the deletion of a target chromosomal gene with the λ-Red recombination system (steps 1-3). The resistance cassette was removed from the new knock-out strain by FLP recombinase expressed from pCP20 vector (step 4), and a pACYC-based plasmid harboring tRNA genes (argU, ileY, and leuW) for the E. coli rare codons was subsequently incorporated into the resulting cells (step 5) [J. Am. Chem. Soc. 134, 19731-19738 (2012)]. In addition to selective labeling of amino acids, the knockout procedures illustrated here can also be applicable to selectively label biological cofactors (such as hemes, flavins, or ubiquinone) and other metabolites by manipulating the biosynthetic pathways of these compounds. FRT, FLP recombinase recognition target.



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Selection of Appropriate E. coli Auxotrophic Expression Host Strains

In E. coli, the biosynthetic and degradation pathways of many amino acids significantly overlap, and many amino acids serve as either precursors or breakdown products for other amino acids (Fig. 1). The control over the network of metabolic pathways is strictly regulated at different levels (e.g., by feedback inhibition and/or by transcriptional attenuation) and can be highly complex. Such metabolic and regulatory information can be useful to select a suitable auxotrophic strain with the corresponding isotope-labeled amino acid supplied in the growth medium.

For any given amino acid biosynthesis pathway in E. coli, there are only a limited number of junctions that could lead to scrambling of the input label with other amino acids (Figs. 1, 2). Consideration of these branching points is critical to the basic design of auxotrophic strains with acceptably low levels of label scrambling for many amino acids, but in some cases only a single gene knockout is required: Arg, Cys, Gln, Gly, His, Ile, Lys, Met, Pro and Thr (Table 2). Within this set, for example, the C43(DE3) Cys auxotroph strains having the deletion of cysE (YM138 and MS1 in Table 1) grow very slowly but effectively eliminate most of the scrambling with media enriched with L-15N-Cys or L-3-13C-Cys (Inorg. Chem. 57, 741-746 (2018)). On the other hand, the deletion of thrC (previously considered to be an ideal genotype for threonine labeling; strains RF2, and EH1 in Table 1) eliminates most but not all of the scrambling of an input 15N-Thr label. In this case, metabolic scrambling to Gly and Ser may be reduced to lower levels by also deleting the threonine dehydrogenase (tdh) gene (see Fig. 1).

Auxotrophs for the remaining ten amino acids each require two or more genes to be deleted, but most of them share the requirement for deletions of the four common aminotransferase genes, aspC, avtA, ilvE and tyrB (Fig. 2). We have generated E. coli strains that are appropriate for selective labeling of eight other amino acids: Ala, Asn, Leu, Phe, Ser, Trp, Tyr, and Val (Table 1). In principle, the potential use of strains having knockouts of all four common aminotransferase genes (aspC, avtA, ilvE and tyrB) of E. coli should help minimize possible dilution and scrambling of the input amino acid label whenever available (J. Biomol. NMR 8, 184-192 (1996)) - in practice, some of these strains appear to exhibit complicated patterns of amino acid combination requirements for growth or they grow very slowly (J. Biochem. (Review) 169, 387-394 (2021)), so careful planning of experimental conditions is required (Tables 1, 3). The complicated patterns of amino acid requirements for bacterial growth in these strains clearly reflect the significant overlap in cognate biosynthetic and biodegradation pathways (Fig. 1), and our knowledge about the complicated regulation of the metabolic flow is still incomplete. Thus, appropriate growth conditions must be considered on a case-by-case basis.

While most of the auxotroph strains with multiple gene deletions (e.g. RF15, RF16 (very slow growth), RF17, RF18 and RF21) often exhibit slow growth in Luria–Bertani (LB) media, the almost normal growth of ML40 compared with the wild-type C43(DE3) strain is a remarkable exception. ML40 has knockouts in aspC, ilvE, avtA, hisG, argH, metA, lysA and cyo genes, and requires the presence of His, Arg, Met, Lys, Ile and Val (but not Asp or Ala) for growth in M63 minimal medium (J. Biochem. (Review) 169, 387-394 (2021)). In the presence of 0.4–1mM Tyr in the growth medium, tyrB gene is repressed, and the resulting ML40 grows very slowly with the requirement of His, Arg, Met, Lys, Ile, Val, Tyr, Phe and Leu (but not Asp or Ala) in the M63 minimal medium; under the latter tyrB-repressed conditions in the presence of 0.4–1mM Tyr, this strain can be used for selective labelling of L-15N-Ala, albeit only for short-term cultivations [after IPTG induction] for heterologous expression of foreign genes (Methods Enzymol. 565, 45-66 (2015); J. Biochem. (Review) 169, 387-394 (2021)).

Designing auxotrophs with acceptable levels of scrambling for each of the two remaining amino acids, L-Asp and L-Glu, remains challenging due to their central roles in metabolism (Figs. 1, 2), which means they are involved in networks of additional pathways to those described by DS Waugh (J. Biomol. NMR 8, 184-192 (1996)). This limits the ability for selective labeling of these amino acids. Deletion of the aspC and tyrB genes gives rise to L-Asp auxotrophs (strains RF4, RF5, RF13, RF15, and RF16), but this genotype is probably not sufficient to limit scrambling of a label within L-Asp.

One complication is that we have been unable to make the ilvE / tyrB double deletion from the C43(DE3) chromosome for unknown reasons. Thus, for selective labeling of L-Tyr, strains RF4 and RF5 (Table 1), both having the deletion of tyrB and aspC genes in BL21(DE3), can be used (J. Am. Chem. Soc. 134, 19731-19738 (2012)). Alternatively, L-Tyr is known to suppress expression of tyrB, and this can be useful in minimizing label scrambling using some of the C43(DE3) auxotrophic strains (Tables 1, 3; applicable only for the short-term cultivation (J. Am. Chem. Soc. 134, 19731-19738 (2012))). For example, by adding L-Tyr (0.4-1 mM) in the medium, we were able to selectively label Leu using strain ML6 (Methods 55, 370-378 (2011)). In another example, ML14 and ML21 strains have tyrA deletions and display auxotrophy for L-Tyr (Table 1). However, these strains are suitable only for the selective labeling of the Tyr side chain and should not be used for selective labeling of proteins with α-15N-Tyr due to the possible scrambling of 15N isotope by the transaminases encoded by aspC and tyrB (Fig. 2).



Table 1 summarizes the current set of new C43(DE3) and BL21(DE3) auxotrophic expression host strains of E. coli, all requiring the addition of a defined set of one or more amino acids in the growth medium. They are suitably designed for overproduction of selectively labeled membrane or water-soluble proteins with stable isotopes such as 2H, 13C and 15N, and deposited to public strain banks (Methods Enzymol. 565, 45-66 (2015); J. Biochem. (Review) 169, 387-394 (2021)). The bacterial metabolic and regulatory pathways are often worth considering to reduce the potential dilution and mixing of the input isotope labels.

Prior to the labeling experiment, these strains must be made competent first (they are not competent cells). Then, the expression efficiency of each target protein should be optimized, e.g. using LB medium first and finding out the optimal expression conditions for further experiments. One important note, here, is that the protein expression using the ML, YM138, MS1 and RF11 strains based on C43(DE3) (a derivative of the BL21(DE3) strain available from Lucigen Inc., Middleton, WI, USA, and optimized for the successful overproduction of membrane proteins) is strictly IPTG-controled, whereas using BL21(DE3)-based strains (RF and EH strains) is much more leaky, at least under our experimental conditions tested (e.g. (J. Am. Chem. Soc. 134, 19731-19738 (2012); Methods Enzymol. 565, 45-66 (2015); Inorg. Chem. 57, 741-746 (2018))). In other words, one might need longer cell growth times for effective expression of a target protein with C43(DE3) derivative strains. Therefore, one should decide carefully how to proceed with the growth conditions in each case (e.g. bacterial growth temperature, aeration, IPTG induction time for protein overproduction, and, when required for proper protein maturation processes, co-expression e.g. with groELS, trx or other cofactor biosynthesis gene(s)).

In the case of running selective amino acid isotope labeling, we strongly recommend adding the amino acid isotope label together with other non-labeled amino acids to reduce dilution and mixing of the input amino acid isotope label(s) (Fig. 1) - this is because some enzymes in the bacterial amimo acid biosynthesis/biodegradation pathways are often highly regulated by certain amino acids, either by feedback inhibition or repression of the cognate gene expression in the system J. Biochem. (Review) 169, 387-394 (2021)). One can make such a mixture, or if applicable, can use a commercially available algal medium containing extracts of amino acids and other compounds (Methods Enzymol. 565, 45-66 (2015)). Conversely, for the reverse labeling experiment, we recommend using a commercially available isotope-labeled (e.g. algae) medium and adding unlabeled amino acid(s) of interest prior to IPTG induction (J. Am. Chem. Soc. 134, 19731-19738 (2012)). The use of a suitable auxotrophic expression host strain with the corresponding isotopically labeled amino acid(s) in the appropriate growth medium will ensure high levels of effeciency as well as selectivity in the stable isotope labeling experiments, and is expected to solve many selective labeling problems.



For other examples and lab tips, see Lin et al. [Methods Enzymol. 565, 45-66 (2015)], Taguchi et al. [Inorg. Chem. 57, 741-746 (2018)], and Iwasaki et al. [J. Biochem. (Review) 169, 387-394 (2021)].


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