Limits...
Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor.

Zhou H, Vonk B, Roubos JA, Bovenberg RA, Voigt CA - Nucleic Acids Res. (2015)

Bottom Line: This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly).Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease.This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Show MeSH

Related in: MedlinePlus

The biosynthetic pathway to 6-ACA and associated genetic designs. (A) The four-step pathway involving six enzymes is shown along with the two known byproducts (dashed arrows). (B) The organization of the initial two-plasmid system with three operons (eAKP672 includes both plasmid pAKP444 and pAKP96) is shown. The full plasmid maps are shown in Supplementary Figure S4. (C) The genome integrated T7 RNAP controller cassette is shown as the box to the left (locus shown). The operons were maintained as in part b and only the promoters were changed. (D) The 6-ACA production titers are shown for the starting construct (part a) and different T7 promoters substituted into the red positions (PT7-X) in part b. The promoters were substituted simultaneously into both positions. Error bars were calculated as the standard deviation of three independent experiments performed on different days.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4666358&req=5

Figure 1: The biosynthetic pathway to 6-ACA and associated genetic designs. (A) The four-step pathway involving six enzymes is shown along with the two known byproducts (dashed arrows). (B) The organization of the initial two-plasmid system with three operons (eAKP672 includes both plasmid pAKP444 and pAKP96) is shown. The full plasmid maps are shown in Supplementary Figure S4. (C) The genome integrated T7 RNAP controller cassette is shown as the box to the left (locus shown). The operons were maintained as in part b and only the promoters were changed. (D) The 6-ACA production titers are shown for the starting construct (part a) and different T7 promoters substituted into the red positions (PT7-X) in part b. The promoters were substituted simultaneously into both positions. Error bars were calculated as the standard deviation of three independent experiments performed on different days.

Mentions: We selected a de novo pathway for 6-aminocaproic acid (6-ACA) biosynthesis in Escherichia coli (E. coli) for proof-of-principle experiments (unpublished results) (Figure 1A). 6-ACA is the linear form of caprolactam, which is the chemical building block of nylon-6. Nylon is the most highly produced synthetic fiber globally (about 4M tons/yr). The fossil-based chemical process for producing caprolactam leads to significant greenhouse gas emission, quantified by its global warming potential (GWP). Depending on the origin of the carbon and energy sources used, the GWP for bio-based production has the potential to be 91% lower than that of the chemical caprolactam route, which is considered to be a sustainable and green process (unpublished results).


Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor.

Zhou H, Vonk B, Roubos JA, Bovenberg RA, Voigt CA - Nucleic Acids Res. (2015)

The biosynthetic pathway to 6-ACA and associated genetic designs. (A) The four-step pathway involving six enzymes is shown along with the two known byproducts (dashed arrows). (B) The organization of the initial two-plasmid system with three operons (eAKP672 includes both plasmid pAKP444 and pAKP96) is shown. The full plasmid maps are shown in Supplementary Figure S4. (C) The genome integrated T7 RNAP controller cassette is shown as the box to the left (locus shown). The operons were maintained as in part b and only the promoters were changed. (D) The 6-ACA production titers are shown for the starting construct (part a) and different T7 promoters substituted into the red positions (PT7-X) in part b. The promoters were substituted simultaneously into both positions. Error bars were calculated as the standard deviation of three independent experiments performed on different days.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4666358&req=5

Figure 1: The biosynthetic pathway to 6-ACA and associated genetic designs. (A) The four-step pathway involving six enzymes is shown along with the two known byproducts (dashed arrows). (B) The organization of the initial two-plasmid system with three operons (eAKP672 includes both plasmid pAKP444 and pAKP96) is shown. The full plasmid maps are shown in Supplementary Figure S4. (C) The genome integrated T7 RNAP controller cassette is shown as the box to the left (locus shown). The operons were maintained as in part b and only the promoters were changed. (D) The 6-ACA production titers are shown for the starting construct (part a) and different T7 promoters substituted into the red positions (PT7-X) in part b. The promoters were substituted simultaneously into both positions. Error bars were calculated as the standard deviation of three independent experiments performed on different days.
Mentions: We selected a de novo pathway for 6-aminocaproic acid (6-ACA) biosynthesis in Escherichia coli (E. coli) for proof-of-principle experiments (unpublished results) (Figure 1A). 6-ACA is the linear form of caprolactam, which is the chemical building block of nylon-6. Nylon is the most highly produced synthetic fiber globally (about 4M tons/yr). The fossil-based chemical process for producing caprolactam leads to significant greenhouse gas emission, quantified by its global warming potential (GWP). Depending on the origin of the carbon and energy sources used, the GWP for bio-based production has the potential to be 91% lower than that of the chemical caprolactam route, which is considered to be a sustainable and green process (unpublished results).

Bottom Line: This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly).Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease.This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Show MeSH
Related in: MedlinePlus