Part:BBa_K1604031

araC-pBAD + pncB

pncB encodes for the enzyme NAPRTase (nicotinic acid phosphorbosyl-transferase). It catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD, from NA (nicotinic acid). This device is controlled by an inducible arabinose promoter.

Usage and Biology

PncB catalyzes one of the rate-limiting step in the NAD+ synthesis pathway. PncB encodes for NAPRTase, the enzyme responsible for the formation of nicotinate mono-nucleotide (NAMN), a direct precursor of NAD+. The reaction is coupled with ATP hydrolysis using NA and a-d-5-phosphoribosyl 1-pyrophosphate (PRPP) as substrates. It has been shown that the overexpression of pncB in E.coli increases the intracellular level of NAD+.[1] [2] [3].

FIGURE 1. Biochemical pathway of NAD⁺ synthesis. PncB gene encodes the transcription of the NARPTase which catalyzes the formation of nicotinate mono-nucleotide from nicotinic acid.

FIGURE 2. pncB does not affect cells growth rate. Neb10β cells transformed with BBa_K1604031 were grown overnight in LB. The day after the cells were diluted 1:100 and grown until they reached an optical density (OD600) of 0.5. The cells were splitted in two different aliquotes of 23 mL each and induced with 5mM of arabinose. The OD was measured every 45 minutes for 5 hours. Negative control were cells transformed with an empty plasmid bearing the arabinose promoter from Unitn iGEM 2012 (BBa_K731201). 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.

FIGURE 3. PncB enhances NAD production by ~2.5 fold in aerobic condition after 6 hours of induction. Cells expressing pncB (BBa_K1604030) and the negative control (BBa_K731201) were grown as described before (figure 2) for a total of 5 hours. After 5 hours the OD was measured again for normalization. 1 mL of cells corresponding to 10⁸ were centrifuged and the supernatant was discarded. The cells were washed with PBS for three times. NAD+ and NADH levels were calculated with a colorimetric assay using the Sigma NAD /NADH quantification kit (MAK037) following the instructions described in the technical bulletin. Quantification was based on a standard curve made with 0, 20, 40, 60, 80 pmole/well of NADH standard. The kit provides the measures of NAD+ levels indirectly from total levels of NAD + NADH and NADH only. All samples were normalized by removing the blank.Panel A. Standard curve. Panel B. NAD/NADH levels for three biological samples of BBa_K1604031 and one negative control.

FIGURE 4. Colorimetric assay for NAD/NADH ratio. Colorimetric assay for NAD/NADH quantification. Cells expressing pncb and the negative control were grown as described in figure 3. NAD and NADH levels were calculated with the Sigma kit MAK037. Lane B samples 2-7 calibration curve (o, 20, 40, 60, 80, 100 pmol/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.

At this point was 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 the terrific broth, 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 (10μM) 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 OD (600nm) was taken at the spectrophotometer. 0,5 mL of cells corresponding to 10^8 were centrifuged and the supernatant was discarded. The cells were washed with cold PBS. NAD+ and NADH levels were calculated with a colorimetric assay using the Sigma NAD /NADH quantification kit (MAK037) following the instructions described in the technical bulletin.

FIGURE 5. Aerobic condition, pncB and negative control in LB broth and in 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)

FIGURE 6. Aerobic condition, pncB + NA 10 μM 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)

FIGURE 7. An overview of the NAD/NADH ratio between negative control (BBa_K731201) and cells expressing BBa_K1604031 in aerobic condition. The culture were grown in LB until an OD of 0.5 and induced with 5 mM arabinose in the presence of oxygen. Before inducing the cells, the media were changed with: fresh LB, LB supplemented with nicotinic acid or terrific broth. Panel A: BBa_K731201 in LB (blu), BBa_K1604031 in LB (green) and in LB with 10 uM nicotinic acid (yellow). Panel B: BBa_K731201 (orange) and BBa_K1604031 (red) both in terrific broth

FIGURE 8. PncB enhances NAD production by 13 fold in anaerobic condition in 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 increase NAD+ levels by ~ 13 fold. In this graphic there are no bars of standard deviation because test was made for an only biological sample for negative control araC-pBAD and two samples for pncB because we have limiting glass to put under anaerobic work station.

Conclusion:

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.

This device was built for the iGEM Trento 2015 project psolar MFC to boost NADH levels and therefore electricity production. 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.

For more updates on Unitn iGEM 2015 project check out our Wiki: Unitn iGEM wiki 2015

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1. Susana J. Berrıos-Rivera, K.-Y. San, and G. N. Bennett. (2002) Metabolic Engineering 4, 238–247

2. Marcel G. Wubbolts, Peter Terpstra, Jan B. van Beilen, Jaap Kingma, H. A. Rene Meesters, and Bernard Witholt. (1990) JBC, 265 (29), 17665-72

3. Ka-Yiu San, George N. Bennett, Susana J. Berríos-Rivera, Ravi V. Vadali, Yea-Tyng Yang, Emily Horton, Fred B. Rudolph, Berna Sariyar, and Kimathi Blackwood (2002) Metabolic Engineering, 4, 182–192

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 2317
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961