pjbei-6410  (Addgene inc)

Journal: Metabolic engineering communications

Article Title: Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds.

doi: 10.1016/j.mec.2021.e00173

Figure Lengend Snippet: Fig. 1. ADP1 produces mevalonate, but plasmid instability reduces production in PCA-only condition. ADP1 was cultured in batch mode for 24 hours with expression of the mevalonate pathway. (A) The mevalonate pathway comprising atoB, ERG13, and HMG1, catalyzes the conversion of three molecules of acetyl-CoA into mevalonate. (B) Mevalonate was detected in all conditions, including in medium containing solely 10 mM PCA (p = 0.01, t-test, 2-tailed). The primary vertical axis shows mevalonate production. The secondary vertical axis shows OD600. Data is mean and error bars represent S.E.M. (n = 2). (C) Plasmid instability in the 10 mM PCA condition occurred for glucose-free medium. The top band represents the full pathway, with the second, third, and fourth bands being distinct mutants found in the 10 mM PCA-only condition cultures.

Article Snippet: The upper mevalonate pathway (AtoB, ERG1, and HMG1) was amplified from the pJBEI-6410 (Addgene #47049) (Alonso-Gutierrez et al., 2013) plasmid using primers ECA01/02 (Supplemental Table 1).

Techniques: Plasmid Preparation, Cell Culture, Expressing

Journal: Metabolic engineering communications

Article Title: Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds.

doi: 10.1016/j.mec.2021.e00173

Figure Lengend Snippet: Fig. 2. Aromatic substrates affect growth, titer, and yield. As PCA only condition resulted in low titers and mutations, we screened four aromatics to determine which was best tolerated. Glucose was included to repress mutations found in aromatic only experiments. (A) Mevalonate titers (left axis) and optical density (right axis) after 48 h. (B) Carbon-molar yield of mevalonate on substrate. Both glucose and aromatic carbon were included as substrate. Data reflect mean, and error bars are S.E. M. (n = 4).

Article Snippet: The upper mevalonate pathway (AtoB, ERG1, and HMG1) was amplified from the pJBEI-6410 (Addgene #47049) (Alonso-Gutierrez et al., 2013) plasmid using primers ECA01/02 (Supplemental Table 1).

Techniques:

Journal: Metabolic engineering communications

Article Title: Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds.

doi: 10.1016/j.mec.2021.e00173

Figure Lengend Snippet: Fig. 3. Mevalonate production is improved by wax ester knock-out and fed-batch cultivation. A. baylyi ADP1 pMev-LacI-trc (ECA10) and the wax ester knock- out, ADP1Δacr1 pMev-LacI-trc (ECA15) were cultivated in fed-batch. Cultures initially contained 20 mM POB and 10 mM glucose. Cultures were fed every 24 hours one bolus of 20 mM substrate, either p-hydroxybenzoate (POB) or glucose, or not fed, as indicated. (A) Mevalonate titers after 168 h of POB fed-batch. (B) Mevalonate titers over culture time for in ADP1Δacr1 pMev-LacI-trc (ECA15) with POB feeding. Data reflect mean and error bars represent S.E.M. (n = 6 for ADP1 pMev-LacI-trc POB, glucose, not fed, and for ADP1Δacr1 pMev-LacI-trc POB, and n = 2 for ADP1Δacr1 pMev-LacI-trc glucose and not fed).

Article Snippet: The upper mevalonate pathway (AtoB, ERG1, and HMG1) was amplified from the pJBEI-6410 (Addgene #47049) (Alonso-Gutierrez et al., 2013) plasmid using primers ECA01/02 (Supplemental Table 1).

Techniques: Knock-Out