Team:Lubbock TTU/Description


The metabolic engineering of E. coli has significant potential to provide an accessible cellular factory for the in vivo production of essential chemicals during space exploration. Recognizing the versatility of using E. coli for bio-manufacturing during space travel, we investigate applications in polyamine production. In particular, a diamine known as putrescine with medicinal and materials applications. To expand on earlier improvements of the product yield for putrescine in E. coli, we explore modifying the W3110 strain of K-12 E. coli. Additionally, we explore the use of TX-TL cell-free synthetic biology to design transcription factor-based biosensors for the detection of improved putrescine yield and to monitor other small molecules of interest. With these strategies we hope to improve the yield of putrescine in E. coli and to expand the synthetic biology toolkit for metabolic engineering.

Metabolic Modifications

The production of putrescine is valuable because it has applications in polymer synthesis, pharmaceuticals, surfactant production, and agriculture. However, traditional synthesis of this compound by the chemical route involves expensive catalysts, flammable and toxic reactants, non-renewable starting materials, and relatively harsh conditions. For these reasons, biosynthesis is an attractive alternative to the chemical synthesis of putrescine.

We are focused on putrescine’s pharmaceutical application as a metabolic precursor to a group of plant-based compounds called tropane alkaloids. The biosynthesis of these compounds is relevant because they have a wide range of medicinal properties; for example, the tropane alkaloid scopolamine is used to treat motion sickness, postoperative nausea, and vomiting, while the tropane alkaloid atropine is used to treat spasms in the stomach, intestines, bladder, and other organs.

A total of 12 modifications were previously made to E. coli W3110 ΔlacI to increase putrescine yield. A combination of gene deletions and gene overexpression using plasmid-based recombination was deployed to produce these modifications.

These modifications include swapping the native promoter with the trc promoter for the argCBH, argD, and argE genes to prevent inhibition from arginine. The trc promoter is repressed by lacI and can only drive expression in the presence of IPTG or lactose; by using E. coli W3110 ΔlacI, we ensure the trc promoter is uninhibited and will therefore drive constitutive gene expression. The potE and speF genes also had their native promoter swapped with trc to increase gene expression, however their native promoters are not repressed by arginine (Table 1). Additionally, the speC gene was introduced on a low-copy plasmid under the tac promoter (Table 1).

Chromosomal inactivation of argI, speE, speG, puuP, and puuA was performed to limit putrescine flow to the degradation and utilization pathways, and to prevent putrescine uptake (Table 2). This was also done for rpoS to inhibit the stress response, thereby allocating more resources for putrescine production. We aimed to replicate these modifications and apply synthetic biology principles to increase putrescine yield.

Table 1- Overexpressed Genes:

Table 2-Chromosomal Gene Deletions:

Recombination Method

In order to produce the desired chromosomal modification to E. coli, we elected to utilize the recombination method described by Yang et. al. This method has three components: a bifunctional helper plasmid, a donor plasmid, and a linear fragment generated by PCR. The bifunctional helper plasmid encodes the yeast endonuclease I-sceI and recombination proteins from the phage lambda. I-sceI will cut at unique 18-bp sites, while the lambda proteins will integrate a linear DNA fragment into the genome by homologous recombination. The donor plasmid contains the desired chromosomal insert flanked with I-sceI sites. The linear PCR fragment contains homology arms to direct recombination, as well as an antibiotic gene for selection. The advantage of this technique over other recombineering methods is the lack of scar-sites, which can negatively impact the efficiency of multiple genome modification attempts.