Team:RDFZ-China/Description

RDFZ-China

Overview

Soil contamination due to crude oil causes environmental and health-related problems. Our project engineers Bacillus subtilis that function as surfactin producing units to remediate contaminated soils. Surfactin is a biosurfactant that can emulsify hydrophobic organic compounds and, in turn, enhance the biodegradation process. To synthesize and export surfactin more efficiently, we overexpress sfp, the 4’-phosphopantetheinyl-transferase, and YerP, a surfactin efflux pump. In addition, lmrA, a multidrug resistance transporter from Lactococcus lactis, is mutated and tested for higher surfactin specificity. We want our product to provide a greener and safer alternative to methods such as heat treatment and leaching. Biosurfactants and the introduction of Bacillus subtilis should have fewer impacts on soil microbiome, and should be more effective than relying on bioremediation alone. We hope that our project can contribute to the use of B. subtilis as a chassis in synthetic biology, and explore new methods of utilizing multi-drug resistance factors.

Introduction

Oil contamination is a very severe type of environmental degradation. It is estimated that 20-30% of oilfields in China are already contaminated, with a total area that exceeds 48000 km2. The major culprit for such a hazardous problem in oilfields is usually credited to unprocessed solid wastes that contain oily sludge, which has been left to stagnate for years and amounts to over three million tons in total mass.

Leaking oil adheres to plant roots after its entry into the soil and causes root systems to rot, which drastically decreases crop yield in fields nearby. With excessive oil accumulating in the soil, even crops grown in the land become malodorous and unsafe to eat.

Various aromatic compounds in oil, like benzene and naphthalene, and aliphatic hydrocarbons are now clearly known to be carcinogenic, pathogenic and teratogenic even at concentrations as low as few ppm. When healthy soil, a natural breed ground for countless microscopic organisms and supplier of water and mineral for plants becomes contaminated by oils, toxic substances flow along the food chain and amplify in concentration through biomagnification. They finally enter human bodies and accumulate in adipose tissues to a life-threatening amount in the long run. Therefore, we feel urgent to deal with oil contamination for the sake of the health of our dear citizens.

One crucial step for solving the problem relies upon the remediation of damaged soil. While traditionally the issue is approached from a physical or chemical perspective, for example, by the mean of incineration and chemical leaching, both of which are deteriorative to the environment due to their potential of augmenting the greenhouse effect, polluting the air with sulfurous gases or leaving behind organic solvents in the soil, we are willing to face it biologically and adopt an eco-friendly solution.

Cleaning up oil leaks with bioremediation is becoming mature nowadays. By utilizing the metabolic power of microorganisms, not only toxic substances in soil and water bodies nearby can be decomposed to water and other harmless compounds, the overall integrity of soil ecosystem is also preserved to the most extent. Among all agents employed in the process of bioremediation, surfactants play a key role by emulsifying insoluble oil layers into tiny droplets that can be efficiently degraded by soil microbes.

It’s now well known that Bacillus subtilis, a well characterized gram-positive bacteria that is extensively used in protein production, and its related strains are able to produce surfactin, a biosurfactant that is originally used by the bacteria to reduce the surface tension of the substrate to achieve swarming behavior. As a replacement for chemically synthesized surfactants, surfactin has several advantages:

  1. It has lower toxicity and it’s totally biodegradable. Therefore it’s safe and eco-friendly in this application. A previous study on mice shows that it has no impact on several physiological functions when the dosage is below 47.5mg/kg.
  2. It’s effective even under extreme temperatures, pH conditions, and salinity levels.
  3. It efficiently facilitates the biodegradation of various carbohydrates: an 88-day long field study suggests that surfactin-enhanced soil has carbohydrate concentration decreased from 6981mg/kg to 1655mg/kg while the control group.

We believe that surfactin will be useful for emulsifying leaked oil, thus, it can greatly facilitate the bioremediation of oil-contaminated lands.

Our Idea

We now propose a synthetic biological device called Mobile Surfactant Factory to help clean up oil contamination more efficiently through bioremediation. That is, we wish to design a new microorganism that is able to produce surfactin in bulk and digest oil carbohydrates as well.

According to the goal, we choose Bacillus subtilis as our chassis and determined the biological parts we need to use to implement our ideas. The reason that we choose Bacillus subtilis is obvious: it’s a model organism for studying many biological processes; some strains are already known to be surfactin producers and the genetic components are well characterized. Another consideration is critical to our design yet less obvious: transporters capable of mediating surfactin efflux are scattered among Bacillus subtilis strains and other Gram-positive bacteria so we can further augment surfactin production by excreting it quickly using them.

Discovering LmrA

Multidrug resistance exhibited by pathogenic microorganisms and human cancer cells drastically reduces the effectiveness of clinically used drugs and threatens the quality of our lives. One major mechanism causing multidrug resistance has to do with xenobiotic efflux transporters on cell membranes that actively pumps out a plethora of toxic substances out of the cell. During the investigation on surfactin’s properties, we learned that besides being an outstanding biosurfactant, it’s also a wide-spectrum cyclopeptide antibiotic. Using a reverse logic, we decide that we can try to excrete surfactin with transporters that lead to multidrug resistance. Based on previous studies, we choose a well-characterized MDR transporter called lmrA, which origins from Lactococcus lactis(also a G+ bacteria) for our purpose because it’s sequence, structure, function and substrate diversity is already elucidated and it’s confirmed that lmrA confers resistance to 17 out of 21 clinically most used antibiotics and other drugs that belong to the classical MDR spectrum.(consider revising the final part!!!)

The Biochemistry of Surfactin Synthesis

As summarized by Sieber, SA and Marahiel, MA in their succinct mini review, the surfactin synthetase from Bacillus subtilis is a large multienzyme complex consisting of three enzymatic subunits, SrfA (402 kDa), SrfB (401 kDa), and SrfC (144 kDa), which consist of seven modules that comprise 24 catalytic domains. Each module is responsible for the specific incorporation of one dedicated substrate into the growing heptapeptide chain. The N-terminal module of an assembly line, the initiation module, specifically recognizes and activates the N-terminal amino acid of the peptide product. All chemical reactions necessary to incorporate and modify each substrate are mediated by a catalytically independent set of domains incorporated within the modules.

The first step in surfactin biosynthesis is the recognition and activation of a dedicated substrate by the adenylation domain. In the next step, the aminoacyladenylate intermediate is transferred to the free thio group of the cofactor phosphopantetheine, which is tethered to the thiolation domain located downstream of the A domain. The phosphopantetheine arm is attached to a serine residue of the apo-T domain by a dedicated 4 -phosphopantetheine (ppan) transferase that uses coenzyme A (CoA) as a substrate. This transferase is encoded by the gene sfp. The intermediates, tethered by the reactive thioester to the flexible cofactor phosphopantetheine (in each module), can be transferred to other domains for subsequent catalytic reactions. Peptide bond formation between two adjacent substrates is catalyzed by the condensation domain, which is located between the A and T domains of subsequent modules. The condensation domain catalyzes the nucleophilic attack of the amino acid bound to the downstream T domain with its free amino group on the activated thioester of the upstream T-domain-bound intermediate, as suggested by the three-site model for phosphopantetheine tethers described by T. Stein et al.

A common structural motif of nonribosomal peptides is the incorporation of D-amino acids, which is mediated by the epimerization domain. It catalyzes the racemization of the T-domain-bound amino acid to form an equilibrium between the L and D conformers.

All those catalytic domains contribute to the synthesis of a linear peptide molecule tethered to the multienzyme. In order to reactivate the multienzyme for a next synthesis cycle, the mature peptide must be cleaved once it reaches the end of the assembly line. This reaction usually is accomplished by a thioesterase domain(TE) fused to the C-terminal module. The peptide can be released either by hydrolysis as a linear acid or by an intramolecular reaction with an internal nucleophile to give a cyclic peptide.

sfp – The Master Regulator in Surfactin Biosynthesis

The Bacillus subtilis enzyme Sfp is required for production of the lipoheptapeptide antibiotic surfactin. It specifically acts on each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase by posttranslationally phosphopantetheinylating a serine residue to create docking sites for amino acid loading and peptide bond formation, as can be seen in the depiction for the three-site-model above.

Yerp - The Endogenous Surfactin Exporter

The following excerpt is cited from a research by Li, X et al. that validates our overexpression of YerP to enhance surfactin production:

“The gene of yerP was cloned from the genomic DNA of B. subtilis THY-7 and then overexpressed in THY-7 cells with a plasmid vector, forming a recombinant strain TS662. Through 1 mM IPTG induction, the surfactin production of TS662 increased remarkably after 12 h and reached 1.58 g L−1 (157.8 mg g−1 CDW) at 24 h, 1.45-fold higher than that of control (0.64 g L−1, 55.8 mg g−1 CDW). The highest surfactin production occurred at 36 h for both TS662 (1.67 g L−1, 219.3 mg g−1 CDW) and TS762 (0.75 g L−1, 99.7 mg g−1 CDW), the control strain containing the blank plasmid. Furthermore, cell growth of TS662 was not influenced by the overexpression of YerP.

Therefore, the promotional effect on surfactin efflux of YerP was greater than either YcxA (0.89-fold) or KrsE (0.52-fold). Using HPLC, we further compared the surfactin isoforms produced by different engineers with elevated producing yield, as shown in Fig. S7. Results indicated that overexpression of the three putative transporters (i.e. YcxA, KrsE and YerP) in THY-7 did not show selective effect towards speci c surfactin isoforms.”

References

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  2. Parthipan, Punniyakotti et al. “Biosurfactant and Degradative Enzymes Mediated Crude Oil Degradation by Bacterium Bacillus Subtilis A1.” Frontiers in Microbiology 8 (2017): 193. PMC. Web. 14 July 2017.
  3. Bolhuis, H., et al. "Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis.." Journal of Bacteriology 176.22 (1994): 6957-6964.
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