Figure 1.
Depiction of the heterologous mevalonate pathway expressed in E. coli to produce amorpha-4,11-diene.
Genes in blue arrows are derived from E. coli, those in brown arrows from yeast, and ADS in red from A. annua. pMevT, pMBIS and pADS indicate the arrangement of genes on expression plasmids. Gene names and the enzymes they encode: atoB, acetoacetyl-CoA thiolase; ERG13, HMG-CoA synthase; tHMG1, truncated HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; MVD1, mevalonate pyrophosphate decarboxylase; idi, IPP isomerase; ispA, farnesyl pyrophosphate synthase. Pathway intermediates: Ac-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; HMG-CoA, hydroxymethylglutaryl-CoA; Mev-P, mevalonate 5-phosphate; Mev-PP, mevalonate pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FDP, farnesyl pyrophosphate; ADS, amorphadiene synthase.
Figure 2.
Feed, metabolite, production and cell density for restricted glucose feed (Process A) fed-batch fermentation of E. coli strain B32.
2a. Glucose and NH4 concentrations. 2b. Cell density, amorpha-4,11-diene production, and acetate concentration.
Table 1.
List of Strains.
Table 2.
List of Plasmids.
Figure 3.
Feed, metabolite, production and cell density for restricted glucose and nitrogen feed (Process B) fed-batch fermentation of E. coli strain B32.
3a. Comparison of cell density and amorpha-4,11-diene production in process A (glucose restricted) and process B (glucose and nitrogen restricted) fermentations. 3b. Glucose, acetate and ammonia concentrations.
Figure 4.
Schematic of operons in plasmids encoding the first 3 enzymatic activities of the synthetic mevalonate pathway.
ACT, E. coli acetoacetyl-CoA thiolase (atoB); HMGS, S. cerevisiae HMG-CoA synthase (ERG13); tHMGR, truncated S. cerevisiae HMG-CoA reductase (HMG1); E.f. mvaS, E. faecalis HMGS; E.f. mvaE, E. faecalis acetoacetyl-CoA thiolase / HMGR; S.a. mvaS, S. aureus HMG-CoA synthase; S.a. mvaA, S. aureus HMG-CoA reductase; (c.o), codon-optimized for E. coli expression.
Figure 5.
Production of amorpha-4,11-diene in shake-flask cultures by strains expressing different HMGR and HMGS enzymes.
5a: Production of amorpha-4,11-diene by shake-flask cultures of strains B64, B65 and B66. B64 (pAM25, codon-optimized pMevT: blue curve), B65 (pAM34, E. faecalis mvaE mvaS: red curve), B66 (pAM41, S. aureus mvaA: green curve). 5b: Production of amorpha-4,11-diene by shake-flask cultures of strains B66 and B86. B66 (pAM41, S. aureus mvaA green curve), B86 (pAM52, S. aureus mvaS mvaA: purple curve).
Figure 6.
Cell growth and amorpha-4,11-diene production for strains B32 and B86 in process A.
Figure 7.
Comparison of ammonia concentration, cell growth and amorpha-4,11-diene production in fed-batch processes A (restricted glucose), B (restricted glucose and nitrogen) and C (restricted glucose and nitrogen with NaOH pH control) for strain B86.
7a: Ammonium concentration. 7b: Cell growth. 7c: Production of amorpha-4,11-diene.
Figure 8.
Amorpha-4,11-diene production in triplicate fed-batch process C fermentations with strain B86.