by Dr. Tim Zeppenfeld, Dr. Harald Kranz and Gary Stevens
Biotechnological methods are being pushed forward for in use industrial production processes, termed white biotechnology, that include biotransformation, fermentation and metabolic engineering.
Metabolic engineering stands for targeted modifications of genes in the metabolism of microorganisms for optimizing their production efficiency. Possible modifications affecting production of strains are targeted deletions and the resultant inactivation of genes, selective variations in expression of single genes, or additional insertion of specific genes into the bacterial genome.
Among other things,
Escherichia coli is used for the production of aromatic amino acids such as L-tryptophan, L-phenylalanine or L-tyrosine,
(1) of carotenoids
(2) or for the production of shikimic acid used as starting material for chiral compounds.
(3) Metabolic pathways, which are to be modified for the production of different substances, affect not only the usability of different carbon sources but also the isoprenoid-2 and carotenoid
(4) biosynthetic pathways and the common metabolic pathway for the production of aromatic amino acids.
(5)  Figure 1. The central step in Red/ET recombination is the exchange of genetic information between two DNA fragments containing homology regions. Click to enlarge. |
The complexity of the necessary modifications requires a tool allowing precise alteration of different genes without leaving antibiotic resistance genes behind, genes which have been used for interim selection. The Red/ET recombination or recombineering, which is a Gene Bridges (Dresden, Germany) patent and was first published under the name ET cloning,
(6) is particularly suitable for such manipulation of the
E. coli genome. Thus rapid and easy modifications of DNA molecules can be carried out completely independent of the presence of suitable restriction sites or the size of the DNA molecule. In this publication we want to point out the possibilities arising from the application of the Red/ET recombination technique for strain optimization.
Whereas other systems for modification of the
E. coli genome based on group II introns of
Lactococcus lactis(7) or transposons like Tn7
(8) only allow modifications at specific positions of the genome, the Red/ET system allows manipulations at any position of the genome.
Targeted integration of new DNA is carried out by homologous recombination in
E. coli strains expressing either the phage proteins recE/recT of the Rac prophage or redα/redβ of the λ phage. In this way, the recombination is mediated through short homologous sequences on both recombining DNA molecules (Figure 1).
The Red/ET system needs 50 bp-long homology arms that can be synthesized in the form of oligonucleotides and added to a linear fragment by PCR. Since oligonucleotide synthesis can be carried out with any sequence for the homology arms, the technique allows the modification of any position in the genome.
 Figure 2. Application of suitable linear DNA fragments allows modifications such as gene knock-out through disruption (a) or deletion (b) of an open reading frame, insertion of a foreign gene (c) or change of a promoter (d). Selection markers (sm) are flanked by FRT sequences and are subsequently removed by expression of a site-specific recombinase (for example FLP); one single FRT sequence remains in the genome. Click to enlarge. |
Phage genes, essential for recombination, are provided by an expression plasmid that can be transferred into any
E. coli strain by transformation. The use of a temperature-sensitive replication origin for the construction of the expression plasmid allows rapid and easy removal of the plasmid after expiration of the reaction.
By using skillfully selected linear fragments, genes on the
E. coli chromosome can not only be disrupted through the insertion of a resistance gene, but can also selectively removed or inserted (Figure 2). Selection markers that are additionally flanked by recognition sequences of a sequence-specific recombinase (FLP or Cre) — so-called FRT or loxP sequences — can be subsequently removed in a rapid and safe way by short expression of the appropriate enzyme. Removal of the selection markers allows the modification of several genes, one after the other, whereas short FRT or loxP recognition sequences remaining in the genome are proved to be unproblematic. Therefore, up to seven genes could be consecutively modified in one strain through the application of the presented technique in customer projects.
Inactivation of the mannose transporter
Inactivation of the mannose transporter (manXYZ) in the
E. coli genome is to be cited as an example for efficiency and easy handling of the technique. Insertion of a resistance marker flanked by FRT sequences into the mannose transporter coding gene resulted in a selective disruption of this gene with the consequence that bacteria cannot grow with mannose as single carbon source. The resistance marker was removed from the genome by short expression of a sequence-specific recombinase (Figure 2a). A period of less than two weeks is required for the experimental procedure in the laboratory ranging from the first growth of bacteria to the control of the marker-free deletion strain. The insertion site of the selection cassette that was integrated into the genome by Red/ET recombination was checked by PCR. Correct insertion could be confirmed through the use of PCR primers binding partially the flanking sequences of the genome (Figure 3a; primer 1, 2, 5 and 6) and partially the inserted resistance gene (primer 3 and 4). In addition, removal of the antibiotic marker was confirmed by subsequent expression of sequence-specific FLP recombinase (Figure 3c).
 Figure 3. Check for correct insertion of the selection marker into the manX gene locus by PCR. Click to enlarge. |
Growth in mannose- and glucose-containing media was determined and documented with growth curves (Figure 4) serving as physiological test for effective inactivation of the mannose transporter.
The modified strain shows the expected phenotype: Bacteria no longer grow with mannose as single carbon source in contrast to the original strain indicating that the appropriate transporter was inactivated. In contrast the growth with glucose was not affected. Complete removal of the resistance marker was checked by streaking out cells on kanamycin-containing media. Furthermore, pulse field gel electrophoresis was used to confirm that no unspecific recombination event occurred (data not shown).
Conclusions
The Red/ET recombination provides a technique for rapid, easy and precise modification of the
E. coli genome in the field of white biotechnology. Because individual genes cannot only be selectively disrupted but also DNA fragments of other organisms can be selectively inserted into any desired position of the
E. coli genome, this technology facilitates not only the development of customized production strains for already existing biotechnological products but also targeted planning by combining DNA fragments from different organisms.
(9)
 Figure 4. Growth curves of the origin strain (wt) and the modified E. coli strain (DMan) with glucose (Glc) or Mannose (Man). Click to enlarge. |
Evidence suggests that in principle this technique is also applicable to other microorganisms, in addition to the application to E. coli, the organism most widely used by molecular biologists.(10-11)
About the authors Dr. Tim Zeppenfeld, Dr. Harald Kranz and Gary Stevens are with Gene Bridges GmbH. Corresponding author Dr. Harald Kranz can be reached via email at: Harald.Kranz@genebridges.com. Additional information about the company technology is available from:
References
1. Ikeda, M.
Appl. Microbiol. Biotechnol 69:615-626 (2006).
2. Yuan, L.Z., Rouviere, P.E., LaRossa, R.A. and Suh, W.
Metabolic Engineering 8:79-90 (2006).
3. Johansson, L., Lindskog, A., Silfversparre, G., Cimander, C., Nielsen, K.F. and Lidén, G.
Biotechnology and Bioengineering 92:541-552 (2005).
4. Lee, P.C. and Schmidt-Dannert C.
Appl. Microbiol. Biotechnol. 60:1-11 (2002).
5. Krämer, M., Bongaerts, J., Bovenberg, R., Kremer, S., Müller, U., Orf, S., Wubbolts, M. and Raeven, L.
Metabolic Engineering 5:277-283 (2003).
6. Zhang, Y., Buchholz, F., Muyers, J.P.P. and Stewart, A.F.
Nature Genetics 20:123-128 (1998).
7. Zhong, J., Karberg, M. and Lambowitz, A.M.
Nucleic Acids Research 31:1656-1664 (2003).
8. McKenzie, G.J. and Craig, N.L.
BMC Microbiology 6:39 (2006).
9. Wenzel, S.C., Gross, F., Zhang, Y., Fu, J., Stewart, A.F. and Müller, R.
Chemistry & Biology 12:349-356 (2005).
10. Bonifield, H.R and Hughes K.T.
J Bacteriology 185:3567-3574 (2003).
11. Metzgar, D., Bacher, J.M., Pezo, V, Reader, J., Döring, V. Schimmel, P., Malière, P. and De Crécy-Lagard, V.
Nucleic Acid Research 32:5780-5790 (2004).