Background Information on Folate
Folate, the generic term for the various forms of Vitamin B9, is an essential vitamin because it is heavily involved in amino acid synthesis as well as single-carbon transfer reactions. Folate deficiencies in women can result in birth defects such as neural tube defects and other spinal cord malformations. As important as folate is, humans are unable to produce folate, and so must obtain it from eating foods such as green leafy vegetables or folate-fortified cereals . An engineered strain of bacteria that would constantly release folate into the gut would reduce the need to fortify breads and cereals with folate, as well as reduce folate-related birth defects in regions with little access to folate-containing foods. In addition to all the reasons stated above, folate is an ideal vitamin to be produced in the gut because, unlike many other vitamins, it has been shown to be absorbed in physiologically relevant quantities in the large intestine.
Structurally, folate consists of three main parts: pteridine, p-aminobenzoic acid (pABA), and L-glutamate.
Folate Biosynthesis Pathway
Although folate is naturally produced in "E.coli", the folate biosynthesis pathway in the bacteria "Lactococcus lactis" has been more heavily characterized and studied. There are six major enzymes involved in folate synthesis, which, in "L.lactis", are contained in five genes: "folB", "folKE", "folP", "folC", and "folA". The first four, which we have chosen to focus on, are located in a gene cluster approximately 4.4kb long. We’ve chosen not to focus on "folA" for the time being because "folA" encodes an enzyme which turns one form of folate (dihydrofolate) into another form of folate(tetrahydrofolate). Since our assay would detect both types of folate as part of the total folate production, "folA" was not a prime target for overexpression of folate. In previous studies, this folate gene cluster has been successfully transformed into the folate-consuming bacteria "L.gasseri", turning the bacteria in to folate-producers. Therefore, we have chosen to also use the folate operon from L.lactis, which also offers the additional benefit of removing the operon from its natural regulatory context.
Our strategy is to clone the entire folate operon from the "L.lactis" genome and to transform the entire operon into "E.coli". However, because we are unsure of whether or not the ribosomal binding sites (RBS) within the "L.lactis" operon would work in E.coli, we are also going to unpack the operon by cloning each of the four genes individually, placing them behind "E.coli" RBSs, and then recombine them into a single empty BioBricks™ plasmid. In addition to the main folate operon, we will also be experimenting with overexpression of the para-aminobenzoic acid (pABA) synthesis pathway from chorismate. Wegkamp "et al." have shown that in order to increase overall total levels of folate, both the pABA synthesis genes and certain folate production genes need to be overexpressed. The pABA pathway involves three genes, "pabA", "pabB", and "pabC" – though in "L.lactis", "pabB" is actually a fusion gene encoding for both "pabB" and "pabC".
The overall system design for testing folate production in "E.coli" is to construct two plasmids – one for the folate biosynthesis pathway, and one for the pABA synthesis pathway. In addition to ensuring that the plasmids are complementary, each plasmid would need to contain a different variable copy origin of replication, which would be low copy by default, but can be switched to high copy via the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG)to the media. This will allow us to test overexpression of each plasmid separately. In addition, each plasmid will contain a constitutive promoter, since we would want folate to be produced constantly. The purple dots represent ribosomal binding sites (RBS), followed by the gene (green arrow), and eventually terminating in a double stop (TAATAA) sequence, as regulated by the Registry of Standard Biological Parts.
Folate Detection Methods
We will be detecting folate production, and thus the relative success of our engineering efforts, via a microbiological assay involving the folate-dependent strain "Enterococcus hirae". This assay involves first the characterization of a standard growth curve of "E. hirae" given known quantities of folate present in the growth media. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated. PABA concentrations will be measured via high performance liquid chromatography (HPLC) .