High-level biosynthesis of norleucine in E. coli for the economic labeling of proteins.
The residue-specific labeling of proteins with non-canonical amino acids (ncAA) is well established in shake flask cultures. A key aspect for the transfer of the methodology to larger scales for biotechnological applications is the cost of the supplemented ncAAs. Therefore, we established a scalable bioprocess using an engineered host strain for the biosynthesis of the methionine analog norleucine at titers appropriate for the efficient and economic labeling of proteins. To enhance the biosynthesis of norleucine, which is a side-product of the branched chain amino acid pathway, we deleted all three acetolactate synthase isoforms of the methionine auxotrophic Escherichia coli expression strain B834(DE3). Additionally, we overexpressed leuABCD to boost the biosynthesis of norleucine. We systematically analyzed the production of norleucine under the conditions for its residue-specific incorporation in bioreactor cultures that had a 30-fold higher cell density than shake flask cultures. Under optimized conditions, 5g/L norleucine was biosynthesized. This titer is two times higher than the standard supplementation with norleucine of a culture with comparable cell density. We expect that our metabolically engineered strain for the improved biosynthesis of norleucine in combination with the proposed bioprocess will facilitate the efficient residue-specific labeling of proteins at a reasonable price in scales beyond the shake flask.
acib-Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria.
Niklaus
N
Anderhuber
acib-Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, A-8010 Graz, Austria.
Patrik
P
Fladischer
acib-Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria; Department of Organic Chemistry, Graz University of Technology, Stremayrgasse 9/4, A-8010 Graz, Austria.
Mandana
M
Gruber-Khadjawi
Department of Biotechnology, University of Natural Resources and Life Sciences, and Department of Biotechnology, Muthgasse 18, A-1190 Vienna, Austria; enGenes Biotech GmbH, Mooslackengasse 17, A-1190 Vienna, Austria.
Juergen
J
Mairhofer
acib-Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, and Department of Biotechnology, Muthgasse 18, A-1190 Vienna, Austria.
Gerald
G
Striedner
acib-Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria. Electronic address: birgit.wiltschi@acib.at.
Birgit
B
Wiltschi
D000095:Acetolactate Synthase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D009646:Norleucine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D009646:Norleucine; D000095:Acetolactate Synthase
Acetolactate synthase; Bioreactor; Biosynthesis; Deletion; Non-canonical amino acid; Norleucine