Metabolic engineering of Escherichia coli for high-level production of free lipoic acid.

Autor: Lennox-Hvenekilde D; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark; Biosyntia ApS, Fruebjergvej 3, 2100, Oesterbro, Denmark., Bali AP; Biosyntia ApS, Fruebjergvej 3, 2100, Oesterbro, Denmark., Gronenberg LS; Biosyntia ApS, Fruebjergvej 3, 2100, Oesterbro, Denmark., Acevedo-Rocha C; Biosyntia ApS, Fruebjergvej 3, 2100, Oesterbro, Denmark., Sommer MOA; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark., Genee HJ; Biosyntia ApS, Fruebjergvej 3, 2100, Oesterbro, Denmark. Electronic address: hjg@biosyntia.com.
Jazyk: angličtina
Zdroj: Metabolic engineering [Metab Eng] 2023 Mar; Vol. 76, pp. 39-49. Date of Electronic Publication: 2023 Jan 10.
DOI: 10.1016/j.ymben.2023.01.004
Abstrakt: L-Lipoic acid (LA) is an important antioxidant with various industrial applications as a nutraceutical and therapeutic. Currently, LA is produced by chemical synthesis. Cell factory development is complex as LA and its direct precursors only occur naturally in protein-bound forms. Here we report a rationally engineered LA cell factory and demonstrate de novo free LA production from glucose for the first time in E. coli. The pathway represents a significant challenge as the three key enzymes, native Octanoyltransferase (LipB) and Lipoyl Synthase (LipA), and heterologous Lipoamidase (LpA), are all toxic to overexpress in E. coli. To overcome the toxicity of LipB, functional metagenomic selection was used to identify a highly active and non-toxic LipB and LipA from S. liquefaciens. Using high throughput screening, we balanced translation initiation rates and dual, orthogonal induction systems for the toxic genes, LipA and LpA. The optimized strain yielded 2.5 mg free LA per gram of glucose in minimal media, expressing carefully balanced LipB and LipA, Enterococcus faecalis LpA, and a truncated, native, Dihydrolipoyllysine-residue acetyltransferase (AceF) lipoylation domain. When the optimized cell factory strain was cultivated in a fed-batch fermentation, a titer of 87 mg/L free LA in the supernatant was reached after 48 h. This titer is ∼3000-fold higher than previously reported free LA titer and ∼8-fold higher than the previous best total, protein-bound LA titer. The strategies presented here could be helpful in designing, constructing and balancing biosynthetic pathways that harbor toxic enzymes with protein-bound intermediates or products.
Competing Interests: Declaration of competing interest All authors are or have been employed at Biosyntia ApS, which has a financial interest in the cell factory production of vitamins and nutraceuticals, including lipoic acid.
(Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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