Team:UNITN-Trento/Results/pncB

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^[1].

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) ^[2] ^[3] ^[4].

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_K1604031).

Characterization

PncB enhances NAD⁺ production by 13 fold in anaerobic conditions

Our goal was to demonstrate that pncB increased intracellular levels of NAD⁺and thus NADH. We measured NAD⁺ levels under different growth conditions modifying the culture media conditions and the presence or lack of oxygen, which is the condition of the anodic chamber of a MFC.

Colonies expressing pncB (BBa_K1604031) and the negative control (BBa_K731201) were grown in LB broth. When an Optical Density (OD) of 0.6 (600nm) was reached, the cultures were centrifuged and resuspended in fresh medium and induced with 5mM of arabinose. The cultures after 5 hours of induction were transferred in sealed glass bottles with a rubber septum under an anaerobic work station. After additional 20 hours of induction 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.

NAD/NADH ratio: pncB and negative control in anaerobic condition NAD/NADH ratio of BBa_K731201 (araC-pBAD, blu) and cell expressing BBa_K1604031 (pncB, green), in LB medium after 20 hours post-induction in anaerobic conditions. NAD⁺ and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037). The concentration of NAD⁺ and NADH were calculated based on a standard curve.

In anaerobic conditions we obtained our best result, however we were able to repeat this measurement only one time due to technical difficulties with the anaerobic chamber. We also tested the performance of pncB in different conditions in LB, in a rich media (Terrific Broth), with the addition of Nicotinic Acid, the starting molecule used by pncB, in the presence of oxygen. For all these samples we were able to use 3 biological replicates and with 3 technical replicates. Also under the conditions described above pncB enhanced NAD⁺ intracellular levels.

NAD/NADH ratio between negative control BBa_K731201 and cells expressing BBa_K1604031.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.

We did not observe significant difference in LB versus terrific broth (2.5 and 2.7 fold, respectively), while nicotinic acid boost the NAD⁺ levels by 3.7 fold respect to araC-pBAD and 1.5 compared to pncB in LB only.

Colorimetric Assay for NAD/NADH ratio in aerobic conditions Left: 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. Right: Standard curve with 0, 20, 40, 60, 80, 100 pmole/well of NADH

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 (araC-pBAD - pncb) and BBa_K731201 (i.e. araC-pBAD). 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.

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 there was an increase 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. 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 Part!

We successfully developed and submitted a new functional part: BBa_K1604031

More NAD⁺ produced

NAD⁺ production was enhanced in our cells by overexpressing the pncB gene, thanks to our novel part.

Towards the pMFC

Bacteria equipped with pncB produce more electrons and are thus ideal for our Microbial Fuel Cell environment.
Check out our Solar pMFC results to know more!