PCR primer sequences for verification of these strains are given as DL (doc) files on the Supporting Information page . Latest version: v202211.

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)] (Fig. 4). 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)] (Fig. 4). 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 and Fig. 4 [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 and Fig. 4). 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 and Fig. 4). 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 and Fig. 4). 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. Follow 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)].

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

(Almost) ALL these E. coli auxotrophic expression strains listed in Table 1 are available through either Addgene, 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), RIKEN 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.

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.

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.

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 and Fig. 4). 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 and Fig. 4). 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 and Fig. 4). 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 and Fig. 4). 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 (Fig. 4)], 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.

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.

Figure 4. Amino acid requirements of RF4 (#4, top), RF12 (#12, first middle left) and RF13 (first middle right) grown for ~20 h, RF14 (second middle), RF15 (third middle) and RF16 (fourth middle) grown for ~40-43 h, and ML40 (bottom right) grown for ~20 h in M63 minimal medium supplied with the specified L-amino acid(s) or in Luria-Bertani (LB) medium [Methods 55, 370-378 (2011); J. Biochem. (Review) 169, 387-394 (2021)]. RF4, having the deletions of aspC and tyrB and representing an ideal genotype for L-tyrosine labeling (Table 1), requires Asp for growth in M63 minimal medium; RF12 requires Trp for growth in M63 minimal medium, whereas RF13 requires Asp, Tyr, and Trp for growth in M63 minimal medium; RF14 requires Asp, Tyr, Trp and Ser for growth in M63 minimal medium; RF15 requires Asp, Tyr, Trp, Gly and Ser for growth in M63 minimal medium, but further addition of Cys inhibits growth (third middle and bottom left); RF16 unexpectedly appears to be a new Ala auxotroph of BL21(DE3) that requires Asp, Tyr, Trp, Gly, Ser, Cys and Ala for very slow growth in M63 minimal medium (fourth middle) and was not inihibited by the presence of Cys (bottom left). ML40 requires His, Arg, Met, Lys, Ile and Val for growth in M63 minimal medium; in the presence of 0.4-1 mM Tyr, ML40 requires His, Arg, Met, Lys, Ile, Val, Tyr, Phe and Leu for slow growth due to repression of tyrB under the applied confitions (bottom right). These examples illustrate that E. coli amino acid metabolic network is more complex than the simple sums of individual amino acid biosnthesis pathway catalogs (cf. Table 1). Concentration(s) of amino acid(s) added to M63 minimal medium (where they exist): 0.4 g/L L-aspartate, 0.17 g/L L-tyrosine, 0.1 g/L L-tryptophan, 2.1 g/L L-serine, 0.55 g/L glycine, 0.05 g/L L-cysteine, 0.5 g/L L-alanine, 0.1 g/L L-histidine, 0.4 g/L L-arginine, 0.25 g/L methionine, 0.42 g/L L-lysine, 0.23 g/L L-isoleucine, 0.23 g/L L-valine, 0.13 g/L L-phenylalanine and 0.23 g/L L-leucine. This figure can only be used as a general guide for the amino acid requirements of the selected strains, where precausion must be taken to optimize expression conditions for each target protein to be tested, e.g. by using LB medium first. Adapted from [Methods 55, 370-378 (2011)] and [J. Biochem. (Review) 169, 387-394 (2021)].