"
Team:Caltech/Project/Vitamins
From 2008.igem.org
|
People
|
In vivo Folate Production
Background Information on Folate Folate, the generic term for the various forms of Vitamin B9, is an essential vitamin involved in single-carbon transfer reactions, which are important for many pathways including amino acid synthesis. 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 {sybesma1}. 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 {asrar}. Structurally, folate consists of three main parts: pteridine (derived from GTP), p-aminobenzoic acid (PABA, derived from chorismate), and a poly-glutamyl tail (derived from linking glutamate) {sybesma1}. Folate Biosynthesis Pathway The folate gene cluster from L.lactis. Black arrows represent genes which have been tested in metabolic engineering studies, shaded arrows represent genes involved in folate biosynthesis, and white arrows represent genes not involved in folate synthesis. sybesma1 Although folate is naturally produced in ‘‘E. coli”, the folate biosynthesis pathway in the bacteria ‘‘Lactococcus lactis’’ has been more heavily characterized and studied. 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 {wegkamp1}. Using the folate gene cluster from ‘‘L. lactis’’ also offers the additional benefit of removing the operon from its natural regulatory context.There are six major enzymatic activities involved in folate synthesis, which, in ‘‘L. lactis’’, are contained in five genes: ‘‘folB’’, ‘‘folKE’’, ‘‘folP’’, ‘‘folC’’, and ‘‘folA’’ {sybesma1}. The first four, which we have chosen to focus on, are located in a gene cluster approximately 4.4kb long. We have chosen not to test overexpression of ‘‘folA’’ because it encodes for 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.  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 overexpressedwegkamp2. 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 pabCwegkamp2. System Design  The overall system design for testing folate production in ‘‘E. coli” is to construct several plasmids: one for each individual gene, one for the folate biosynthesis pathway, and one for the PABA synthesis pathway. In addition to the main folate operon, we worked 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 {wegkamp2}. The PABA pathway involves three genes, ‘‘pabA’’, ‘‘pabB’’, and ‘‘pabC’’ – though in ‘‘L. lactis’’, ‘‘pabB’’ is actually a fusion gene encoding for both the enzymatic activities of ‘‘pabB’’ and ‘‘pabC’’ {wegkamp2}. Each gene, or gene cluster, would be cloned in an inducible-copy plasmid, which would be low copy by default, but can be switched to high copy via the addition of Isopropyl $\beta$-D-1-thiogalactopyranoside (IPTG)to the media. This will allow us to test overexpression of each plasmid separately. In addition, each plasmid constains a constitutive promoter, to ensure the constant production of folate. Folate Detection Methods We measured folate production, and thus the relative success of our engineering efforts, via a microbiological assay involving the folate-dependent strain ‘‘Lactobacillus rhamnosus’’(ATCC 7469) {horne}. The assay uses growth of the folate-dependent strain ‘‘L.rhamnosus’’as an indicator of folate concentrations in the sample. Since the assay media specifically lacks folate, folate becomes the limiting factor in the growth of ‘’L. rhamnosus’’, with the only possible source being the bacterial lysates of the engineered ‘‘E. coli’’. In order to quantify the relative growth of the folate-dependent strain ‘‘L.rhamnosus’’, a standard growth curve must first be characterized using known quantities of folic acid in assay media. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated. Protocols are based off of BD Biosciences Folate Assay Medium datasheet {BD}. Growth was measured by taking the OD at 546nm. To prepare the ‘‘L.rhamnosus’’ inoculum, 5 mL of Lactobacilli Broth AOAC(BD Biosciences) was inoculated with ‘‘L.rhamnosus’’ and then incubated for 12-24 hours. Next the culture was centrifuged(13,000x g, 10 min, 20C) and the supernatant removed. The culture was washed twice with saline (0.9\% NaCl in H20) prior to being resuspended in folate assay media and adjusted to an OD (546nm) of approximately 1.0. The folic acid assay media was prepared by adding 100g of the dehydrated assay media to water (light sensitive), autoclaving, and then adding 0.4 mL/L of filter sterilized Tween 80 to the media. The addition of Tween 80, derived from oleic acid, improves aerobic growth rates in ‘’Lactobacilli’’{corcoran}.
Samples were prepared by first centrifuging the fully-grown 5mL cell culture at 13,000g for 10 minutes at 20 C. After centrifugation, both cells and supernatant were recovered. The supernatant was diluted 1:1 with 0.1M sodium acetate buffer (pH4.8) -1\% ascorbic acid; this deviates slightly from the protocol, which asks for sodium ascorbate. The cells were washed with the 0.1M sodium acetate-1\% ascorbic acid and resuspended in 5 mL of the same buffer. Folate was released from the cells by incubating the samples at 100C for 5 min, which is optimal for folate release and heat inactivation of the bacteria. The deconjugation reaction mixture (2.5\% vol/vol) was added to both supernatant and lysates, and incubated for 4h at $37^{\circ} C$. Samples were filter sterilized, and folate assay media was added 1:1, bringing the volume up to 5mL. 50$\mu L$ of prepared ‘‘L.rhamnosus’’inoculum was added to each assay tube, and the tubes are incubated for 16-20 at $37^{\circ}$C. The absorbances of the samples were read at 546nm on a plate reader (200$\mu L$/well). A standard curve was determined by using a range of folic acid dilutions from 0- 10 ng/mL, usually 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 8, 10ng samples with a total volume of 5 mL. Folate Microbiological Assay Protocol pABA Detection Methods para-Aminobenzoic Acid (PABA) can be detected using high performance liquid chromatography (HPLC). The PABA elution buffers used were 0.1\% formic acid in H2O (Buffer A), and 0.1\% formic acid in MeOH (Buffer B). Both cell lysates and supernatant were tested using the following HPLC method. Using an injection volume of 20$\mu$L and column temperature of 40°C, the starting eluent was 92\% A mixed with 8\% B. The percentage of B was then increased linearly to 50\% in 7 min, then to 100\% in 3 min. The mobile phase was then immediately brought back to its initial composition (92\%A) and held for 4 min in order to re-equilibrate the column {Zhang}. The retention time of PABA was found to be around 4.9-5.0 minutes, compared to a literature report of 6.0 minutes. Using a 14 minute protocol, we were able to detect PABA peaks coming off a C18 column at 4.9 min. A standard curve was made by running a series of 1:3 PABA dilutions starting at a 10ug/ml concentration. The PABA for the standard curve was spiked into wild type ‘‘E. coli’’ cell lysate, which by itself did not show any detectable PABA peaks. Samples were run using the same HPLC protocol as the standards, and both lysate and supernatant were tested for PABA peaks. para-aminobenzoic acid (pABA) HPLC protocol ResultsModifications to System Design We were able to successfully extract and clone the following four genes: ‘‘folB’’, ‘‘folKE’’, ‘‘pabA’’, and ‘‘pabB’’. We aimed to create individual constructs of each gene in the IPTG inducible-copy plasmid pSB2K3, as well as constructs with the two folate genes combined and with the two PABA synthesis genes combined. However, we had issues cloning our genes into the pSB2K3 + B0015 terminator construct, and so switched to completing the constructs in the high copy plasmid pSB1AK3 + B0015 terminator. Our final constructs, shown in, are ‘‘folB’’ in pSB2K3, and ‘‘folKE’’, ‘‘pabA’’, ‘‘folB’’+’‘folKE’’, and ‘‘pabB’’ in pSB1AK3. Previous studies on folate overexpression have shown that both folate synthesis and PABA synthesis genes need to be overexpressed simultaneously in order to increase total folate levels {wegkamp1}. Therefore, tests on ‘‘folB’’ and ‘‘folKE’’ were done with and without the addition of PABA during the ‘‘E. coli” inoculation; detection of higher folate levels on addition of PABA would indicate PABA as a limiting factor for folate production. We were unable to use PCR to extract several of the target genes: the entire folate gene cluster, ‘‘folP’’, and ‘‘folC’’. After analyzing the sequencing results of our other folate biosynthesis genes, we realized that the genetic sequences had several point mutations which were not `stop' codons. This indicated that we had a homologous, but not identical, genomic DNA than the one that we has used for PCR primer design. We believe that this homologous sequence could have resulted in too many differences between the primer and the genomic DNA, resulting in little to no binding. This may explain why we were unable to successfully extract ‘‘folP’’, ‘‘folC’’, and subsequently, the entire folate gene cluster (since ‘‘folP’’ and ‘‘folC’’ are the last two genes in the cluster) from the genomic DNA of ‘‘L. lactis’’. The revised target constructs are shown .
Folate Assay Results for folB, folKE, and folBKEThe experimental setup included running a standard curve with known amounts of folic acid (0-10ng) simultaneously with the samples, in order to have the same basis for comparison. The hope was that the standard curve growth OD values would correspond with known concentrations, and so sample concentrations could be interpolated based upon the standard. ‘‘folKE’’, which was cloned into a constitutive high-copy plasmid, was tested with and without the addition of 500 ng of PABA during inoculation. ‘‘folB’’, which is in an inducible copy plasmid, it was tested induced to high-copy as well as uninduced low-copy, with and without 500 ng of PABA during inoculation. We assayed both the supernatant and the cell lysate, though only the supernatant had measurable results. The standard curve is linear in the expected range from .1 to 1ng folate, though the actual absorbance values do not correspond to sample values. Although the reasons for this were unclear, the growth data for the samples are very encouraging since the relative folate levels match what we would expect. We see that the addition of 500 ng of PABA during inoculation dramatically increases overall folate levels for ‘‘folKE’’ relative to the samples without PABA. Furthermore, adding PABA to the wild type controls did not affect growth at all, suggesting that the assay bacteria ‘‘L.rhamnosus’’was not metabolizing the extra PABA. The extremely high OD values for all the samples were possibly the result of not completely washing out all of the culture media prior to adding the ‘‘L.rhamnosus’’to the assay samples.
When we repeated the assay in duplicate, this time on ‘‘folB’’ as well, we were able to observe the same trends in growth with and without the addition of PABA. Again, we see that for ‘‘folKE’’, the addition of PABA does increase total folate levels, though not as dramatically as before. The rampant growth of the wild type control is disconcerting, but wild type folate production again appears to be unaffected by the addition of PABA. Furthermore, folate levels for ‘‘folKE’’ are still higher than the control where ‘‘L.rhamnosus’’was added to only media without supernatant. And what of ‘‘folB’’? Recall that ‘‘folB’’ was the only gene to be successfully cloned into an inducible-copy plasmid, and so it was tested both induced and uninduced. The folate levels in the induced sample (high-copy) are higher than in the uninduced (low-copy) sample, which is consistent with what we would expect. The addition of PABA to both induced and uninduced increases relative levels of folate, which is also consistent. However, it is interesting to note that folate levels for the +PABA samples are the same for induced and uninduced. Of course, given the small sample size, this could just be due to variability, but it could also suggest that folate production reached an upper limit. This explanation seems even more likely if we reconsider the folate biosynthesis pathway, and we see that ‘‘folB’’ and ‘‘folKE’’ are both located upstream of actual integration of PABA into the molecule, accomplished by ‘‘folP’’. It is very possible that we are increasing the flux of both pathways going into the ‘‘folP’’ junction, but as we were unable to overexpress ‘‘folP’’ as well, we may have created a bottleneck.
 para-Aminobenzoic Acid (pABA) HPLC Assay Results for pabA and pabB
Using high performance liquid chromatography (HPLC) we were able to detect PABA peaks from overexpression of ‘‘pabA’’ and ‘‘pabB’’ individually compared to a control sample of wild type supernatant. Compared to the peak areas of the standard curve (Fig. \ref{pabastandard}), we were able to use a linear approximation to determine the concentration of PABA to be 703.2 ng/mL for ‘‘pabA’’ overexpression. Conclusions and Future WorkWe have shown, through very preliminary tests, the feasibility of overproduction of folate in ‘‘E. coli” using genes originally derived from ‘‘L. lactis’’. Our data have confirmed previous studies in the necessity of overexpressing both folate and PABA synthesis genes, and we have shown that folate appears to be mostly present in the supernatant. Our data also suggest that overexpression of ‘‘folKE’’ is the most effective, definitely more effective than ‘‘folB’’, and possibly more effective than overexpressing ‘‘folB’’ + ‘‘folKE’’. We have also shown that it is possible to overexpress para-aminobenzoic acid production in ‘‘E. coli’’ and that overexpression of either ‘’pabA’’ or ‘‘pabB’’ increases total levels of para-aminobenzoic acid (PABA). Further work on this project would include repeating the folate assay to generate more data, perfecting the assay in order to quantify folate levels with the standard curve, making the ‘‘pabA’’ + ‘‘pabB’’ and ‘‘folB’’ + ‘‘folKE’’ constructs, making a ‘‘folB’’ + ‘‘folKE’’ + ‘‘pabA’’ + ‘‘pabB’’ construct, and extracting and cloning ‘‘folP’’ from the ‘‘L. lactis’’ genome. Relevant PartsBasic Parts
Construction Intermediates
Composite Parts
References<biblio>
</biblio>   |
