Part:BBa_K5254018

Introduction: limited PAPS

Heparin biogenesis involves the sequential modification of a polysaccharide chain by specific enzymes to produce heparin, a highly sulfated glycosaminoglycan with significant anticoagulant properties. In this process, all four sulfotransferase enzymes—except for C5-epimerase—require phosphoadenosine phosphosulfate (PAPS) as a sulfate donor to add sulfate groups to the polysaccharide backbone. Escherichia coli (E. coli) can synthesize PAPS; however, its basal levels are typically low because the bacterium also expresses phosphoadenosine phosphosulfate reductase, an enzyme encoded by the cysH gene. This enzyme degrades PAPS by reducing it to adenosine phosphosphate (PAP), thereby limiting the availability of PAPS for sulfation reactions.

Figure 1: PAPS pathway.

Badri A et.al have demonstrated that deleting the cysH gene significantly increases basal PAPS levels. Building on this finding, we have removed the cysH gene from E. coli Nissle 1917—a probiotic strain known for its safety profile—to enhance intracellular PAPS concentrations. This genetic modification ensures a higher availability of PAPS for the sulfotransferase enzymes involved in heparin biosynthesis.

As a result, the cysH knockout in E. coli Nissle 1917 provides two essential components for heparin biogenesis:

Presence of Heparosan: Nissle 1917 is a non-pathogenic e.coli strain that naturally synthesizes Heparosan, the unsulfated precursor of heparin.

Increased PAPS Levels: The removal of the cysH gene elevates PAPS concentrations, supplying the necessary sulfate donor for sulfotransferase enzymes.

Assembly

Fig 1 Assembly: PCR for generation of the knock in cassette The knockout cassette was generated by PCR. PCR was performed using Super Fi II DNA polymerase, and an ~800 bp band was amplified. The band was cut out, and the PCR product was extracted using the Qiagen gel extraction kit.

Fig 2 Assembly: Recombinase pKD 46 map pKD46 is a plasmid carrying the lambda Red recombinase system under a promoter driven by arabinose. It is widely used for homologous recombination in bacterial genome editing (6).

Fig 3 Assembly: RE digestion confirmation of pKD46 pKD46 incorporation in Nissle 1917 was confirmed by RE digestion. Sac I is a unique site, so plasmid incorporation was confirmed by Sac I digestion, which produced a 6.3 Kb band.

Characterization

In order to confirm that the homologous recombination was successful, we needed to confirm the cysH knock out by genomic pcr and a functional assay.

Genomic PCR

Figure 5. CysH knock out confirmation by genomic pcr

Bacteria genome was extracted by SDS and isopropanol precipitation. 400 ul of bacterial culture was pelleted and lysed in 200 ul of 1% SDS for 5 min ambiently. 30 ul of 10 mM NaCl and 350 ul of isopropanol were added to form the precipitation. The precipitation was pelleted by centrifugation at 15000 g for 10 min. The pellets were resuspended with 200 ul water and used for the PCR reaction. Primers against 5’ UTR of CysH and mid portion of CysH were used for WT cysH gene detection. Primers against 3’UTR of CysH and mid portion of CMR were used for the detection of CMR cassette in CysH locus. Clone #7 has the band corresponding to CMR cassette integration and missing CysH gene.

Positive clone #7 signals a successful knockout because it lacks the wildtype cysH band but shows the presence of the cmR cassette band. The absence of a PCR product for the wildtype confirms that the cysH gene is no longer present at that location, while the presence of the cmR cassette band confirms its replacement at that site. These results indicate that the cysH gene has been successfully replaced by the cmR cassette.

Functional Assay

The second experiment we conducted to confirm our cysH knockout was a functional assay to measure the amount of PAPS present in our cells.

Experimental approaches​

Initially we wanted to incubate the bacterial cell lysate with PAPS and measure the production of PAP. However, we couldn't carry out this step because purchasing PAPS was too expensive. Instead, we measured the PAP levels in the bacterial lysate and compare the amounts.

Concerning: Free phosphate in lysate may mask PAP detection because the assay kit measure free phosphate induced by IMPAD1 (Inositol monophosphatase domain-containing protein 1)​

Wild-type Nissle 1917 has a higher PAP amount than CysH KO cells. The PAP amount is reflected by the difference in absorbance with and without IMPAD1. We did not have purified PAPS; therefore, cell lysates were used as the source of PAPS. We prepared a mixture of cell lysates from wild-type and CysH knockout Nissle 1917 to standardize the other background factors. The difference in absorbance in the presence of IMPAD1 increased as the wild-type Nissle cell lysate increased, while in the absence of IMPAD1, no increase was observed.

This assay does not directly measure the amount of PAPS but instead quantifies the amount of PAP generated from PAPS by CysH in E. coli. Therefore, in CysH knockout cells, we expected an accumulation of PAPS, which would correspond to a decrease in PAP levels. Notably, the assay measures the phosphate generated by IMPAD1 (added during the assay) from PAP, rather than PAP itself. Since free phosphate is present in the cell lysates from the start, we observed high background phosphate levels. To account for this, we calculated the difference in absorbance with and without IMPAD1, which reflects the phosphate newly generated by IMPAD1 and is correlated with PAP levels in the samples.

We anticipated that various factors under basal conditions could affect the measurements, so we mixed two different cell lysates (wild-type: high PAP, and CysH knockout: low PAP) in varying ratios to standardize the basal conditions for comparison. As shown in the figure, increasing the proportion of wild-type lysate led to a greater increase in phosphate after the addition of IMPAD1, while without IMPAD1, the increase in wild-type lysate did not affect the differences. These findings indicate that wild-type cell lysates contain more PAP compared to CysH knockout lysates, suggesting that CysH knockout cells have higher levels of PAPS.​​

Impact

Although our primary goal was the generation of heparin in vivo, the successful creation of the cysH knockout (Nissle 1917 E. coli) stands as a major breakthrough for both future iGEM teams and the broader field of glycobiology. By engineering high levels of sulfation in a non-pathogenic cell that carries the heparosan sulfation precursor, we’ve paved the way for significant advancements in the production of complex polysaccharides. The use of Nissle 1917, a safe and well-studied strain, adds an invaluable advantage in bringing these developments to real-world applications. For future iGEM teams, this breakthrough extends far beyond heparin synthesis. Our engineered strain offers a solution to a critical problem: sourcing PAPS, a key reagent for sulfation enzymes, which is often expensive for teams working out of a community lab like ours. By using this strain, teams can bypass the potential financial hurdles of obtaining PAPS, transforming it into a simple, self-sustaining resource.

Moreover, the success of our homologous recombination process sets the stage for even more ambitious genetic engineering. With the addition of FRT sites, this process can serve as a blueprint for teams attempting knock-ins of larger, more complex pathways.

In essence, the creation of the cysH KO strain doesn’t just achieve heparin production in vivo—it opens new frontiers in synthetic biology, positioning this tool as a versatile asset for future research and industrial applications. We believe this contribution will inspire and create an audible impact in the field of glycosaminoglycans.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 41
  Illegal XhoI site found at 900
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]