PncB: nicotinic acid phosphorbosyl-transferase
Increasing the levels of NADH
Our goal was to boost electron production by increasing the concentration of electron carriers (i.e. NADH). To achieve this goal we decided to engineer E. coli with an enzyme that would provide more intracellular NAD+, and thus NADH.
PncB catalyzes one of the rate-limiting step in the NAD synthesis pathway. This gene is naturally found in E. coli and encodes for the enzyme NAPRTase (nicotinic acid phosphorbosyl- transferase) that catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD, from NA (nicotinic acid).
Biochemical pathway of NAD synthesis PncB gene encodes the transcription of the NARPTase which catalyzes the formation of nicotinate mono-nucleotide from nicotinic acid.
Unitn-Trento iGEM part 2015 (pncB) Upper: pncB without illegal site in pSB1C3, BBa_K1604030. Lower: pncB under the control of inducible promoter araC-pBAD, BBa_K1604031
Our device is controlled by an inducible arabinose promoter built by the Unitn iGEM team in 2012. PncB was extracted by E. coli genome, the illegal site PstI was removed, and it was placed in pSB1C3 (BBa_K1604030). Subsequently it was placed under the araC-pBAD promoter (BBa_K1604030).
Characterization
PncB is not toxic if overexpressed in E.coli
NEB10β transformed with BBa_K1604030 (araC-pBAD-pncB) or BBa_K731201 (i.e. araC-pBAD) were grown up to an OD of 0.6, splitted in two tubes of 23 mL each and induced with 5 mM of arabinose. Negative controls were not induced.
The OD (600 nm) was measured every 45 minutes for 5 hours. All measurements were done for 3 different biological samples and 3 technical measures.
Although the growth rate is slightly decreased, due to the cell stress when expressing pncB, the data indicate that this enzyme does not have toxicity effect on the cells.
Growth rate of BBa_K1604031 (aracpbad-pncb) and BBa_K731201 (i.e. aracpBad). Cells were grown up to an OD of 0.6 and splitted before induction with arabinose. BBa_K1604031 (Orange line) and BBa_K731201 (green line) induced with 5 mM arabinose. BBa_K1604031 (yellow line) and BBa_K731201 (blue line) not induced.
PncB enhances NAD production by ~2.5 fold
Our goal was to demonstrate that pncB increased intracellular levels of NAD+ and thus NADH. We quantified the levels of NAD+ by a colorimetric test that measures the levels of NAD indirectly by quantifying the concentration of NAD total (NAD + NADH) and NADH only. To make precise quantitation a standard curve with NADH was built.
The test provides the ratio of NAD/NADH: $\frac{NAD}{NADH} = \frac{NAD_{TOTAL} - NADH}{NADH}$
NADtotal = Amount of total NAD (NAD+NADH) in unknown sample (pmole) from standard curve.
NADH = Amount of NADH in unknown sample (pmole) from standard curve.
We started by looking at NAD+ after 6 hours of induction in LB. Colonies expressing pncB (BBa_K1604031) and the negative control (BBa_K731201) were grown in LB broth with chloramphenicol (CF). When an Optical Density (OD) of 0.6 (600nm) was reached, the cultures were centrifuged and resuspended in fresh LB medium, CF and 5mM of arabinose. After 6 hours of induction the OD (600nM) was measured and an aliquot of 1 mL of culture corresponding to 10^8 cells was centrifuged at 2000 rpm for 5 minutes and washed with cold PBS. The NAD and NADH levels were calculated with the Sigma NAD and NADH Quantification Kit (MAK037) following the technical bulletin.
Standard curve of NADH and NAD/NADH ratio in LB-Broth NAD+ and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037) following the instructions described in the technical bulletin. Panel A. Standard curve (0, 20, 40, 60, 80, 100 pmole/well of NADH)). Panel B. NAD/NADH levels for three biological samples of BBa_K1604030 (green) and one negative control BBa_K731201 (blue).
Colorimetric Assay for NAD/NADH ratio Lane B samples 2-7 calibration curve (0, 20, 40, 60, 80, 100 pmole/well of NADH). Lane C samples 2-9 NAD total levels; Lane D samples 2-9 NAD total repeated with a 2 fold concentrated sample; Lane E NADH only; Lane F NADH only, repeated with a 2 fold concentrated sample. In lanes C-F the order of the samples is: 2 technical replicates of the negative control, and 2 technical replicates of each of the 3 biological samples of BBa_K1604031. The plate was read with a Tecan Infinite M-200 pro instrument at 450 nm. The measurements were taken after 0.5, 1, 2, 3, 4 hours to allow color development. The data shown are representative of the best measurement at 2 hours.
BBa_K1604031 does increase NAD levels by 126% (2.5 fold) and NADH levels by 44% (1.4 fold) when expressed in NEB10β. Although we did see an enhancement in NAD levels, this did not correlate to a proportional boost in NADH levels. We plan in the future to add a NAD reducing enzyme and to give a medium able to enhance the cell metabolism to further increase NADH intracellular levels.
New Tests?
At this point we tested the performance of pncB in different conditions: as in Terrific Broth, with the addiction of Nicotinic Acid and in anaerobic condition. This time the cells transformed with pncB and araC-pBAD were grown and induced with arabinose for a longer time (20 hours). The test was repeated in aerobic and anaerobic conditions and with a rich culture media (Terrific Broth) in aerobic conditions. The effect of nicotinic acid, the precursor used by pncB was also analyzed in the presence of oxygen.
Why Terrific broth?
Terrific Broth was used to accelerate the E.coli metabolism giving a more nutrient broth as this, with the ultimate goal of producing more NAD+.
Why Nicotinic Acid?
Nicotinic Acid (NA) is the molecule precursor of NAD+ and was supplemented in the media to provide more substrate for the enzyme.
Why anaerobic condition?
Anaerobic conditions were used to mimic the conditions in the Microbial Fuel Cell. To reach anaerobic conditions, after 5 hours of induction in Thermoshaker (37°C at 190 rpm shaking) 2 samples of pncB in LB and one of araC-pBAD were taken and put in sealed glass bottles with a rubber septum under anaerobic work station to keep samples without oxygen.
Subsequently after 20 hours of growth in Thermoshaker an OD (600nm) measure was taken at the spectrophotometer. 0,5 mL of cells corresponding to 10^8 were centrifuged and the supernatant was discarded. The test was done as described before following the technical bulletin. Was obtained a NADH standard curve comparable with the standard curve represented in figure 4 Panel A. Was used again Formula 1.
The plate was read with a spectrophotometer instrument at absorbance 450 nm. The measurements were taken after 0.5, 1, 2, 3, 4 hours to allow color development. The data shown are representative of the best measurement after 1 hour. For aerobic condition every data was taken considering 3 biological replicates and with 3 technical replicates.
NAD/NADH ratio: aerobic condition, pncB and negative control in LB and Terrific Broth Panel A. NAD/NADH ratio between negative control and cells expressing BBa_K1604031, both samples were grown in LB medium. pncB does increase NAD+ levels by 2,5 fold. (p-value= 0,0078) Panel B. NAD/NADH ratio between negative control and cells expressing BBa_K1604031, both samples were grown in Terrific Broth medium. pncB does increase NAD+ levels by ~2,7 fold. (p-value=0,0024)
NAD/NADH ratio: aerobic condition, pncB + NA 10μM and negative control Panel C. NAD/NADH ratio between negative control and cells expressing BBa_K1604031 10 μM of Nicotinic Acid, both samples were grown in LB medium. pncB+NA does increase NAD+ levels by ~3,7 fold. (p-value=0,0001) Panel D. NAD/NADH ratio between pncB in LB + 10 μM NA and cells expressing BBa_K1604031 in LB broth. pncB+NA does increase NAD+ levels by ~1,5 fold. (p-value=0,0231)
NAD/NADH ratio: pncB and negative control in anaerobic condition (LB-Broth) NAD/NADH ratio between negative control and cell expressing BBa_K1604031, both samples were growth in LB medium. Both samples after 6 hours of induction in Thermoshaker (37° C, 190 rpm) were transferred in sealed glass bottles with a rubber septum under anaerobic work station to keep samples without oxygen. PncB does
To sum up...
Overexpression of the gene pncB enhances significantly the intracellular level of NAD+. The best data were obtained in anaerobic conditions where the increasing of NAD+ was of 13 fold. which are the conditions to be used in a Microbial Fuel Cell (Anode chamber is in anaerobic condition). When Nicotinic acid was added in the medium there was a significant enhancement of NAD+ levels (1,5 fold versus pncB and 3,7 fold versus negative control). Also in Terrific broth condition there was an increasing in the NAD level (2,7 fold versus negative control in the same condition) In the future it will be interesting to measures NAD+ in anaerobic conditions with the presence of high levels of Nicotinic Acid.