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Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct.

Scheps D, Honda Malca S, Richter SM, Marisch K, Nestl BM, Hauer B - Microb Biotechnol (2013)

Bottom Line: As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction.The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l(-1) ω-hydroxy dodecanoic acid.We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.

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Bottlenecks in ω-hydroxy fatty acid production by recombinant E. coli in a two-phase system. (1) CYP153A improvements via engineering approaches to increase the activity and stability of the biocatalyst. (2) Optimization of the coupling efficiency of the applied self-sufficient fusion construct to prevent production of reactive oxygen species (ROS). (3a) Ensurance of sufficient supply of cofactor with an additional NAD(P)H regeneration system or increase of the flux of NAD(P)H-producing reactions. (3b) Restriction in the production of harmful acetate formation by the selection of a suitable C-source feeding strategy or by strain engineering approaches. (3c) Elimination of oxidoreductases responsible for the overoxidation of the formed ω-hydroxylated products to, e.g. α,ω-diacids. (4) Elimination of metabolic pathways to avoid substrate and product depletion (this figure was adapted from Grant et al., 2012).
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fig05: Bottlenecks in ω-hydroxy fatty acid production by recombinant E. coli in a two-phase system. (1) CYP153A improvements via engineering approaches to increase the activity and stability of the biocatalyst. (2) Optimization of the coupling efficiency of the applied self-sufficient fusion construct to prevent production of reactive oxygen species (ROS). (3a) Ensurance of sufficient supply of cofactor with an additional NAD(P)H regeneration system or increase of the flux of NAD(P)H-producing reactions. (3b) Restriction in the production of harmful acetate formation by the selection of a suitable C-source feeding strategy or by strain engineering approaches. (3c) Elimination of oxidoreductases responsible for the overoxidation of the formed ω-hydroxylated products to, e.g. α,ω-diacids. (4) Elimination of metabolic pathways to avoid substrate and product depletion (this figure was adapted from Grant et al., 2012).

Mentions: The ω-regioselectivity (including ω-OHFA and α,ω-DCA) of our system was higher than 98%. More than 91% of the formed hydroxylated products consisted of the ω-OHFA (Table S3). Although the product formation rate decreased after 4 h of reaction time (Fig. 3), a plateau was never reached as observed in previous shake flask experiments. The two-phase system configuration with the liquid FAME allows the permanent extraction of the intermediates and the target product, thus minimizing product inhibition and product toxicity. However, once more, hydrogen peroxide and acetate were accumulated in levels close or above the limit for causing a detrimental effect on the cells or on the protein biocatalyst. Hydrogen peroxide was detected in a concentration of up to 240 μM after 28 h, which might have prevented the system to obtain higher product titres (Fig. 4). Acetate levels reached more than 2.2 g l−1 after 12 h. The optimization of coupling efficiency and the coexpression of an enzymatic system to degrade reactive oxygen species like hydrogen peroxide to protect the biocatalyst and the metabolic machinery of the cell are essential (Huang et al., 2007; Fig. 5). Strategies to improve the production of ω-OHC12 include CYP153A engineering approaches, optimization of the coupling efficiency of the self-sufficient fusion construct, sufficient cofactor and C-source supply as well as elimination hampering oxidoreductases and metabolic pathways (Fig. 5).


Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct.

Scheps D, Honda Malca S, Richter SM, Marisch K, Nestl BM, Hauer B - Microb Biotechnol (2013)

Bottlenecks in ω-hydroxy fatty acid production by recombinant E. coli in a two-phase system. (1) CYP153A improvements via engineering approaches to increase the activity and stability of the biocatalyst. (2) Optimization of the coupling efficiency of the applied self-sufficient fusion construct to prevent production of reactive oxygen species (ROS). (3a) Ensurance of sufficient supply of cofactor with an additional NAD(P)H regeneration system or increase of the flux of NAD(P)H-producing reactions. (3b) Restriction in the production of harmful acetate formation by the selection of a suitable C-source feeding strategy or by strain engineering approaches. (3c) Elimination of oxidoreductases responsible for the overoxidation of the formed ω-hydroxylated products to, e.g. α,ω-diacids. (4) Elimination of metabolic pathways to avoid substrate and product depletion (this figure was adapted from Grant et al., 2012).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815936&req=5

fig05: Bottlenecks in ω-hydroxy fatty acid production by recombinant E. coli in a two-phase system. (1) CYP153A improvements via engineering approaches to increase the activity and stability of the biocatalyst. (2) Optimization of the coupling efficiency of the applied self-sufficient fusion construct to prevent production of reactive oxygen species (ROS). (3a) Ensurance of sufficient supply of cofactor with an additional NAD(P)H regeneration system or increase of the flux of NAD(P)H-producing reactions. (3b) Restriction in the production of harmful acetate formation by the selection of a suitable C-source feeding strategy or by strain engineering approaches. (3c) Elimination of oxidoreductases responsible for the overoxidation of the formed ω-hydroxylated products to, e.g. α,ω-diacids. (4) Elimination of metabolic pathways to avoid substrate and product depletion (this figure was adapted from Grant et al., 2012).
Mentions: The ω-regioselectivity (including ω-OHFA and α,ω-DCA) of our system was higher than 98%. More than 91% of the formed hydroxylated products consisted of the ω-OHFA (Table S3). Although the product formation rate decreased after 4 h of reaction time (Fig. 3), a plateau was never reached as observed in previous shake flask experiments. The two-phase system configuration with the liquid FAME allows the permanent extraction of the intermediates and the target product, thus minimizing product inhibition and product toxicity. However, once more, hydrogen peroxide and acetate were accumulated in levels close or above the limit for causing a detrimental effect on the cells or on the protein biocatalyst. Hydrogen peroxide was detected in a concentration of up to 240 μM after 28 h, which might have prevented the system to obtain higher product titres (Fig. 4). Acetate levels reached more than 2.2 g l−1 after 12 h. The optimization of coupling efficiency and the coexpression of an enzymatic system to degrade reactive oxygen species like hydrogen peroxide to protect the biocatalyst and the metabolic machinery of the cell are essential (Huang et al., 2007; Fig. 5). Strategies to improve the production of ω-OHC12 include CYP153A engineering approaches, optimization of the coupling efficiency of the self-sufficient fusion construct, sufficient cofactor and C-source supply as well as elimination hampering oxidoreductases and metabolic pathways (Fig. 5).

Bottom Line: As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction.The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l(-1) ω-hydroxy dodecanoic acid.We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.

Show MeSH
Related in: MedlinePlus