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

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>.

Last update: May 7, 2021

PCR Verification of Escherichia coli Auxotrophic Expression Strains (Supporting Information)


Table S1 summarizes the current set of new C43(DE3) and BL21(DE3) auxotrophic expression 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. 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. This page provides the supporting information about these auxotrophic expression strains.

LinkIconBack to the E. coli Auxotrophic Expression Strains Page.

Table S1. New E. coli amino acid auxotrophic host strains used for selective isotope labeling

Strain Precursor strain Genotype
ML2
CLY (Genotype cyo::kan) cyo::kan ilvE
 
ML3 CLY (Genotype cyo::kan) cyo::kan hisG
ML6 ML2 (Genotype cyo::kan) cyo::kan ilvE avtA
ML8 CLY (Genotype cyo::kan) cyo::kan argH
ML12 ML6 (Genotype cyo::kan) cyo::kan ilvE avtA aspC
ML14 C43(DE3) tyrA
ML17 C43(DE3) glnA
ML21 ML14 tyrA hisG
ML24 ML23 cyo ilvE avtA aspC hisG asnA
ML25 ML24 cyo ilvE avtA aspC hisG asnA asnB
ML26 ML23 cyo ilvE avtA aspC hisG argH
ML31 ML26 cyo ilvE avtA aspC hisG argH metA
ML36 ML23 cyo ilvE avtA aspC hisG metA
ML40 ML31 cyo ilvE avtA aspC hisG argH metA lysA
ML41 ML40 cyo ilvE avtA aspC hisG argH metA lysA thrC
ML42 ML41 cyo ilvE avtA aspC hisG argH metA lysA thrC asnB
ML43 ML42 cyo ilvE avtA aspC hisG argH metA lysA thrC asnA asnB
ML45 ML44 cyo ilvE avtA aspC hisG  metA thrC lysA
     
YM138 C43(DE3) cysE
YM154 C43(DE3) cysE  
MS1 YM138 cysE hisG
RF11 C43(DE3) metA
     
RF1 BL21 CodonPlus (DE3)-RIL glyA
RF2 BL21 CodonPlus (DE3)-RIL thrC
RF3  BL21 CodonPlus (DE3)-RIL aspC
RF4 RF3 aspC tyrB
RF5 RF4 aspC tyrB hisG
RF6 BL21 CodonPlus (DE3)-RIL proC
RF8 BL21 CodonPlus (DE3)-RIL asnA asnB 
RF10 BL21 CodonPlus (DE3)-RIL lysA 
RF12 BL21 CodonPlus (DE3)-RIL trpA trpB 
RF13 RF4 aspC tyrB trpA trpB 
RF14 RF13 aspC tyrB trpA trpB serB 
RF15 RF14 aspC tyrB trpA trpB serB glyA 
RF16 RF15 aspC tyrB trpA trpB serB glyA cysE
RF17 RF4 aspC tyrB ilvE
RF18 RF17 aspC tyrB ilvE avtA 
RF21 RF18 aspC tyrB ilvE avtA yfbQ(alaA) yfdZ(alaC)
RF22 RF18 aspC tyrB ilvE avtA asnA asnB
RF23 RF21 aspC tyrB ilvE avtA serB  yfbQ(alaA) yfdZ(alaC)
EH1 RF2 thrC ilvA




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

PCR Primers for Verification of Each Target Gene

List of PCR primers used for verification of each knocked-out gene in Table S1 (typical results are shown below).



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Colony PCR Data of Each Auxotrophic Strain in Table S1

Click to magnify each image (and either use browsers "back" button or double click to close the image window).
Left lane, standard marker; right lane, PCR-amplified target gene in the wild-type strain.

ML2

ML3

ML6

ML8

ML12

ML14

ML17

ML21

ML24

ML25

ML26

ML31

ML36

ML40K1


ML41 (see below)

ML42

ML43


ML45 (see below)

YM138

YM154

MS1

RF11

RF1

RF2

RF3 (non-auxotrophic strain)

RF4

RF5

RF6

RF8

RF10

RF12

RF13

RF15

RF16

RF17

RF18

RF21




RF23 (see below)

EH1

Auxotrophic stock strains having a few target genes deleted in an unexpected manner

The following stock strains should be used with caution! Colony PCR data (below) indicated that a few target genes (red) were probably deleted in an unexpected manner with the λ-Red recombination system (not tested by direct sequencing), whereas other target genes (black) were knocked out as originally designed. Click to magnify each image (and either use browsers "back" button or double click to close the image window).

ML41 (metA)

ML45 (aspC, his G)

RF23 (serB, tyrB)

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PCR Primers Used for Construction of the RF Auxotrophic Strains

List of PCR primers used for construction of the RF/YM/EH/MS/ML auxotrophic strains (see Table S1) in steps 1, 2, Fig. S1.




Figure S1. 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 recombination target.

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Four General Transaminases (or aminotransferases) of Escherichia coli


Figure S2. Schematic view of selected amino acid biosynthesis pathways in Escherichia coli, catalyzed by four general transaminases (see Fig. 1 [J. Biochem. (Review) 169, 387-394 (2021)]). 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. This poses a major problem for selective labeling with certain amino acids. This scheme can only be used as a general guide with precaution for some amino acids.


For selective labeling with certain amino acids, multiple genetic lesions are required because of the reversible transfer reactions and the overlapping specificities of four general transaminases (or aminotransferases) of E. coli (encoded respectively by ilvE, avtA, aspC and tyrB genes) (Fig. S2). In principle, the potential use of strains with defects in all four general transaminase genes (ilvE, avtA, aspC 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, S1):

  • (i) neither the tyrB nor the aspC deletion by itself confers amino acid auxotrophy on the BL21(DE3) cells (Table 1);
  • (ii) strains having knockouts in both aspC and tyrB genes (RF4, RF5, RF13, RF14, RF15, RF16, RF17, RF18 and RF21) require Asp (but not Glu) for growth in M63 minimal media, and some of them (RF15, RF16, RF17, RF18 and RF21) grow only slowly in M63 minimal media in the presence of the L-amino acids specified in Table 1;
  • (iii) strain RF17, having knockouts in the aspC, tyrB and ilvE genes, requires the presence of Asp, Tyr, Phe, Ile and Leu for (slow) growth in M63 minimal media;
  • (iv) strains RF18 and RF21, having knockouts in all four of the general transaminase genes (aspC, tyrB, ilvE and avtA ), appear to require the presence of Asp, Tyr, Phe, Ile, Leu and Val for slow growth in M63 minimal media - of these, although strain RF21 has further knockouts in genes yfbQ (alaA) and yfdZ (alaC), it requires the presence of Asp, Tyr, Phe, Ile, Leu and Val for slow growth in M63 minimal media like RF18, and is not an Ala auxotroph either [cf., RF16, having knockouts in aspC, tyrB, trpA, trpB, glyA, serB and cysE genes and representing an ideal genotype to selectively label Ser, unexpectedly represents a new BL21(DE3) derived Ala auxotroph, which requires the presence of Asp, Tyr, Trp, Gly, Ser, Cys and Ala for very slow growth in M63 minimal medium, but does not grow in M63 minimal medium in the presence of Asp, Tyr, Trp, Gly, Ser and Cys like RF15] (J. Biochem. (Review) 169, 387-394 (2021));
  • (v) considering also the regulation of the Asp degradation pathways by feedback inhibition by Thr and Lys, and some form of repression by Thr, Ile, Lys and Met, further deletions of asnA and asnB in RF18 would result in a BL21(DE3) derivative requiring the presence of Asp, Tyr, Phe, Ile, Leu and Asn for growth in M63 minimal media, which is expected to be applicable to selective labelling of Asp at least for short-term cultivations grown in medium supplied with sufficient amounts of Thr, Ile, Lys and Met (J. Biochem. (Review) 169, 387-394 (2021)).

Thus far, we have been able to obtain such "ideal" strains from E. coli BL21(DE3) (RF18 and RF21 in Tables 1, S1 (J. Biochem. (Review) 169, 387-394 (2021))) but not from C43(DE3) expression host cells, despite multiple attempts using the λ-Red recombination system (Fig. S1). For example, deletion of the ilvE, avtA and aspC genes from the C43(DE3) chromosome resulted in an auxotroph for Ile and Val (Tables 1, 3), and further knockout of the tyrB locus, which was not possible with the C43(DE3) cells for reasons unclear to us, would extend the auxotrophy to include Leu (J. Biochem. (Review) 169, 387-394 (2021)). For Leu auxotrophy of some C43(DE3) auxotroph strains in Table 1, it is therefore necessary to repress the tyrB gene expression by growing the cells in the presence of 0.4-1 mM Tyr in growth medium . 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 genes.

The complicated patterns of amino acid requirements for bacterial growth in these strains certainly reflect the significant overlap in cognate biosynthetic and biodegradation pathways (see Fig. 1), and our knowledge about the complicated regulation of the metabolic flow is still incomplete. It is therefore important to optimize the E. coli expression conditions for each target protein of interest before running selective amino acid isotope labeling experiments.

Practically speaking, as long as there is a high concentration of amino acids in the growth medium, the collection of the new auxotrophic C43(DE3) and BL21(DE3) expression host strains described here (Tables 1, S1) can solve many selective labeling problems, 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.

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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. Linn, 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 in Table 1, Nippon Medical School, Japan) or R.B.G. (for all ML strains only, University of Illinois at Urbana-Champaign, U.S.A.).

  • 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 Nagese 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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