Metabolic engineering of cofactor F420 production in Mycobacterium smegmatis

Abstract

Cofactor F(420) is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F(420) has a direct and important role in archaeal energy metabolism whereas the role of F(420) in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F(420) has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F(420), M. smegmatis has previously been used to provide this cofactor for studies of the F(420)-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F(420) biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F(420) metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F(420) production. The results indicate that co-expression of the F(420) biosynthetic proteins can give rise to a much higher F(420) production level. This was achieved by designing and preparing a new T7 promoter-based co-expression shuttle vector. A combination of co-expression of the F(420) biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F(420) production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F(420) produced in this study provide a suitable source of this cofactor for studies of F(420)-dependent proteins from other microorganisms and for possible biotechnological applications.

Figure 1. The molecular structures of F₄₂₀, the flavins and NADP⁺.

(A) Schematic representation of F₄₂₀ showing different parts of the molecule, whereas (B) and (C) show the molecular structures of the flavins and NADP⁺, respectively. The atoms involved in oxidoreductive reactions are numbered in all structures.

Figure 2. The proposed biosynthetic pathway for cofactor F₄₂₀.

The FbiA, B and C are Mycobacterial proteins whereas the CofA, B, C, D, E, G and H are Archaeal proteins involved in the biosynthetic reactions. The pathway to formation of the activated phospholactate moiety (LPPG) is yet to be experimentally established in Mycobacteria. In some Archaeal species an α–linked terminal glutamate residue caps the γ–linked poly–glutamate tail, the addition of which is catalyzed by CofF.

Figure 3. A schematic representation of the vectors designed in this study.

The unique restriction sites in the multiple cloning sites of each vector are indicated.

Figure 4. FO/F₄₂₀ production by M. smegmatis cells expressing different recombinant proteins.

(A) comparative FO/F₄₂₀ production between wild type strain and six different recombinant strains expressing proteins involved in F₄₂₀ biosynthesis or metabolism. (B) FO/F₄₂₀ production using co–expression vector compared to the wild type strain. (C) F₄₂₀ production ratio by M. smegmatis cells expressing FbiABC construct over wild type strains for eight days. (D) Effect of iron/sulphur and L–glutamate/manganese additives on the F₄₂₀ production (FO is shown as black and F₄₂₀ as grey). In panels (A), (B) and (D) the error bars are derived from experiments carried out in triplicate.

Figure 5. The F₄₂₀ production profile from M. smegmatis cells over–expressing the FbiABC construct.

The F₄₂₀–6 and F₄₂₀–7 species constitute the main species as deduced using mass spectrometry.