First Thoughts

We first became obsessed with the protein lmrA - during our brainstorming process, we read about a kind of multidrug resistance factors that make bacteria nearly invincible to common antibiotics, and lmrA is one of them. Then we had a bizarre idea: what if we make use of this trait so that certain engineered bacteria strains can export their antibiotic products more easily? Doing so is like taking advantage of evolution’s ingenuity, so we feel compelled to explore more. To make our project sounds feasible, we then set out to find a proper context for lmrA to create our project.

Since lmrA is a membrane protein from Lactococcus lactis, we decided that it would be more appropriate to express it in a Gram-positive bacteria. We then chose Bacillus subtilis as our host since it is a well-studied Gram-positive bacteria and it’s also nonpathogenic, which restraints the potential danger in using lmrA. Then, to simplify the genetic system we would need to build, we started looking for useful xenobiotics that could be naturally synthesized by any common Bacillus subtilis strain and could be a potential substrate of lmrA. In this way, xenobiotics normally synthesized at a trace amount due to their toxicity could be efficiently translocated to extracellular space, thereby increasing the upper limit of production rate.

It has been discovered that Bacillus subtilis can produce more than two dozen antibiotics with varying chemical structures. Most antibiotics are ribosomally synthesized, post-transcriptionally modified, including lantibiotics, or produced by a nonribosomal pathway, leading to lipopeptides, polyketides or some aminosugars. Among those antibiotics, one especially attracted us - surfactin. It’s a cyclic heptapeptide with a lipid tail so it can act as a biosurfactant dut to its amphiphilic property. It’s biosynthesis, industrial production, purification, and economic values have been well characterized. Moreover, it can be used in bioremediation of polluted lands, which just fits in our plan of a project with ecological concerns.

Facilitate the Synthesis

We have agreed upon the fact that the rate of bacterial production is dependent upon the rate of synthesis and efflux, while all other cellular parameters are kept the same. Therefore, after choosing lmrA initially as the transporter, we start out to seek ways for overproducing surfactin. At first, we wanted to use CRISPRa(a for activation) to target the surfactin synthase operon. Doing so is relatively easy theoretically since we only need to express the dCas9-VP64 fusion protein and the guide RNA with an appropriate target sequence to achieve upregulation. However, we discussed with the team from Peking University and realized that CRISPRa may be cumbersome for Bacillus subtilis - even a basal level expression of the system can be a burden. And if we choose CRISPRa, we would have to spend lots of time to tweak it, making the plan less desirable.

Other plans such as overexpressing certain transcriptional factors proved to be unviable because of the pleiotropism of those genes, which means that the overexpression is bound to disturb other parts of cellular metabolism. Finally, we found sfp, encoding a 4’-phosphopantetheinyl transferase, that could specifically boost surfactin production by overriding a limiting step in surfactin synthesis: the transfer of the 4-phosphopantetheinyl moiety of coenzyme A to a serine residue in the peptidyl carrier protein(PCP) domain, allowing further “elongation” of the nonribosomal polypeptide surfactin. (More detail is discussed in the description part.) Later, we also modeled the PCP activation process to gain more insight into the effect of sfp.

Export the Product

The next step was to find a suitable way to export the newly synthesized surfactin from our engineered Bacillus subtilis. We first investigated the feasibility of using lmrA to export surfactin. Though we didn’t find any experimental data of lmrA exporting sfp, we did find papers describing lmrA’s affinity to similar drug molecules. We also did an in silico modeling to check if lmrA does form a binding pocket for surfactin so that the efflux process is possible theoretically. To make our project more feasible, we also found a secondary option that involves overexpressing YerP. This protein is a lipopeptide transporter-dependent on proton motive force as the energy source, and it can mediate surfactin efflux, already validated by previous research. Thus, we have two transporters, one putative and one that could work, and our plan was near completion.

Now, we decided to put sfp and YerP/ lmrA together in a single operon to obtain coordinated and controllable expression. Since we want an overexpression of surfactin, we placed a strong constitutive promoter pVeg, which is selected from iGEM registry, before sfp, the gene we’d like to have a stronger expression. We also used a characterized effective ribosome binding sequence gsiB in proximity to sfp and YerP/ lmrA in the same fashion described as previous studies. Some routine considerations in designing our genetic fragments include the addition of double stop codons, codon optimization and planned removal of illegal restriction sites.

Regulation, Degradation and the Killer Switch

Besides constructing an operon, we also took care of other necessities to make our Bacillus subtilis behave more intelligently in our project.