Biosynthetic Pathways

Example 2

PAO1, the parent strain of PGN5, is a wild-type P. aeruginosa strain that produces relatively small amounts of alginate and exhibits a non-mucoid phenotype; thus, PGN5 is also non-mucoid when cultured (FIG. 3A). In PAO1, the alginate biosynthetic operon, which contains genes required for alginate production, is negatively regulated. Activation of this operon leads to alginate production and a mucoid phenotype. For example, over-expression of mucE, an activator of the alginate biosynthetic pathway, induces a strong mucoid phenotype in the PAO1 strain (e.g., P. aeruginosa strain VE2; FIG. 3B). The plasmid pUCP20-pGm-mucE, which constitutively over-expresses MucE, was used to test whether the genetically-modified PGN5 strain could produce alginate. Indeed, the presence of this plasmid in PGN5 (PGN5+mucE) induced a mucoid phenotype (FIG. 3B). To measure the amount of alginate produced by PGN5+mucE on a cellular level, a standard carbazole assay was performed, which showed that the PGN5+mucE and VE2 (i.e., PAO1+mucE) strains produce comparable amounts of alginate (FIG. 3C; 80-120 g/L wet weight).

To examine whether the alginate produced by PGN5+mucE was similar in composition to alginate produced by VE2, HPLC was performed to compare the M and G content of alginate produced by each strain. The chromatograms obtained from alginate prepared from VE2 and PGN5+mucE were identical (FIG. 3D), and the M:G ratios were comparable to a commercial alginate control (data not shown). To confirm that the physical properties of VE2 and PGN5+mucE alginates were also similar, alginate gels were prepared from alginate produced by each strain and the viscosity and yield stress was measured. The viscosities of VE2 and PGN5+mucE alginate gels were comparable at 73.58 and 72.12 mPa, respectively (FIG. 3E). Similarly, the yield stress of VE2 and PGN5+mucE alginate gels were comparable at 47.34 and 47.16 Pa, respectively (FIG. 3G).

Example 2

Expressed and purified dihydropteroate synthase (DHPS) from S. aureus (saDHPS) was cloned. DHPS is the enzyme that installs PABA (p-aminobenzoic acid) in the folate biosynthesis pathway (Scheme 2). It has been demonstrated that the PABA analog PAS (2-aminosalicylate) is incorporated into folic acid in M. tuberculosis (Chakraborty, S. et al. 2013), suggesting that PAS is a substrate for DHPS. Using a coupled assay, it was determined that the kinetic parameters for saDHPS with PABA, PAS and F-PABA. Importantly, all three compounds have similar kcat and Km values indicating that F-PABA is an alternative substrate for saDHPS. Since PAS is an antibacterial compound whose mechanism of action may be related for the ability of this compound to compete with PABA for DHPS, we determined the antibacterial activity and cytotoxicity of F-PABA for several bacterial species as well as Vero cells. In each case no growth inhibition was observed up to 200 μg/ml. Unlike PAA, 2-F-PABA has no antibacterial activity (Table 1).

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Example 1

Since the biosynthetic pathway of anatabine and its associated genes is not completely known, a novel genetic variation was created in a population of tobacco plants to identify plants that have a significantly reduced ability to biosynthesize anatabine. These plants very likely have a mutated non-functional gene, critical for anatabine biosynthesis.

A population of the Flue-cured variety “401” was used in these experiments. Approximately 5000 seeds were treated with 0.6% ethyl methane sulfonate and germinated. M1 plants were grown in the field and M2 seeds were collected. Fifteen hundred M2 seeds were germinated and grown in 4-inch pots. At 50% flowering stage, plants were topped. Leaf samples were collected 2 weeks after topping and the samples screened for anatabine levels using high performance thin layer chromatography (HP-TLC) and gas chromatography.

After screening for alkaloids, two Flue Cured (FC) 401 ultra-low anatabine (ULA) lines were selected for trait development. It is noted that the amount of nicotine in both ULA lines is unchanged.