ReMixHD’s goal is to demonstrate a proof of concept for mixed plastic bioremediation using smart co-culture
of
Pseudomonas fluorescens. By engineering a helper strain capable of depolymerizing polyethylene and
polyethylene
terephthalate and a main strain utilizing the intermediates as a carbon source, a recombinant product can be
synthesized. The bacteria co-culture will be regulated by a custom-engineered operon capable of controlling
the
helper strains growth using an operon dependent on plastic degradation. For sensing the PE-breakdown
product,
alkanes, the alkane sensor, AlkS-V760E/PalkB, can be used to express a P. fluorescens growth factor
predicted by
our dry lab team. Using a novel engineered XylS-K38R-L224Q/Pm expression system, the PET-breakdown product,
terephthalic acid, is recognized and activates the sRNA repressor system, downregulating the growth of the
helper strain.
The main strain is capable of using terephthalic acid as a carbon source. Once the terephthalic acid levels
are high enough to maintain maximum main strain growth, the operon dynamically represses the helper strain
growth,
enabling synthesis of the recombinant product.
Overview of genetic construct of the final operon composite part
The PET degradation product terephthalic acid (TPA) is monitored by the XylS-K38R-L224Q (XylS-mt) transcription factor. Li et al. discovered two point mutations K38R and L224Q makes XylS sensitive to TPA in concentrations as low as 10 µM in E. coli (Li et al., 2022). Upon activation with TPA or the well described XylS inducer 3-methyl-benzoate (MBA), XylS-mt dimerizes and binds the Pm promoter (Gawin et al., 2017). Pm activation results in the expression of small regulatory RNAs (sRNAs), capable of blocking the translation of the GOI. A negative feedback loop is established, downregulating the GOI activity at high PET depolymerization rates.
The expression of XylS-mt itself is regulated through the Ps1/Ps2 promoter (Gallegos et al., 1996; Gawin et al., 2017). In the absence of TPA, a low baseline of XylS-mt is present in the cell through constitutive low expression from the Ps2 promoter. However, upon XylS-mt activation the transcription factor also binds the Ps1 promoter leading to high levels of induction (Gallegos et al., 1996). This is the first time a TPA sensor is characterized in P. fluorescens and in the iGEM parts registry.
We introduced a synthetic sRNAs repressor system in Pseudomonas fluorescens to dynamically adjust gene
expression dependent on changing plastic concentrations. While extensively characterized and capable of
tightly regulating expression, protein-based repressors pose a high metabolic burden on the host cell
and cannot be easily expanded for repression of genome-encoded genes (Na et al.,
2013). Regulatory small RNA (sRNA) molecules are a viable option for an expandable and
low-burden repression system as they can dynamically and reversibly control gene expression.
Previous research has explored the use of synthetic small regulatory RNA molecules (Kelly et al., 2018).
mRNA interference and degradation ubiquitous in all organisms can achieve repression (Modi et al., 2011). Prokaryotic Organisms natively regulate gene expression through small RNAs (sRNAs) and RNA chaperon protein hfq mediated sRNA-mRNA binding and degradation (Na et al., 2013; Gottesman, 2004; Storz et al., 2011; Modi et al., 2011; Møller et al., 2002), which is also present in Pseudomonas species (Trouillon et al., 2022; Wu et al., 2021) and confirmed in our sub-strain ATCC 50090 by BLAST. The sRNAs have two main regions: the scaffold and seed regions. The scaffold region forms the secondary structure, recruiting hfq and aiding in the RNA degradation (Na et al., 2013; Møller et al., 2002). The seed or target region is homologous to the mRNA and needed for sRNA-mRNA binding (Kelly et al., 2018). For our experimental design, three different sRNA scaffolds were tested that had previously been used for synthetic sRNAs and three seed regions in the 5’UTR or 5’ beginning of the CDS. The chosen scaffold included one previously used iGEM part (MicC, Part:BBa K1124005, (Na et al., 2013), an engineered scaffold, and its WT version (SgrS, SgrSmt (SgrS-S CUUU 6 nts stem)) (Na et al., 2013, Noh et al., 2019).
Along with testing different sRNA scaffolds, multiple RBS were tested as no standardized RBS is
available for P. fluorescens. Three synthetic RBS, including two previously used in iGEM and
taken from the Anderson library (RBS1 Part:BBa
J61100; RBS2 Part:BBa J61101, and one designed
using the Salis lab RBS calculator, were tested. The RBS designed by the Salis lab RBS calculator was
designed to maximize the expression of mKate2 mRNA with a predicted translation initiation rate of
516487.12 (Salis, 2023).
To test the gene expression, a C-terminal degradation tag was linked to the reporter gene mKate2. The
degradation tag was shown to work in Pseudomonas putida (Halvorsen et al.,
2022). The degradation tag reduced the halflife of the constitutive expression mKate2, which
was expressed using the promoter pEM7. This enabled us to observe the dynamic repression resulting from
the sRNAs.
An alkane sensor using the AlkS/pAlkB expression system creates a positive feedback loop promoting the expression of our GOI dependent on the PE degradation products n-alkanes. During depolymerization, PE’s carbon backbone fragments into n-alkanes, which are further metabolized by alkane monooxygenase AlkB (Pinto et al., 2022). The length of the n-alkanes resulting from the PE depolymerization is unknown, as the mechanism is yet to be determined. However, the length of the alkane chains is presumably below C17 as long-chain alkanes cannot pass through the cell membrane via diffusion, and there is no known transporter in P. fluorescens for alkanes longer than C16. The alkane, along with the transcription factor AlkS originally found in P. oleovorans, builds a complex leading to the activation of the AlkB promotor and the expression of the GOI (Yuste et at., 1998). The alkane sensing abilities of AlkS are well-researched. WT AlkS is only sensitive to short alkanes and is not sensitive to chain lengths between C12 and C16 (Wu et al., 2015). Dongdong Chen et al. identified AlkS with the mutation V760E, which can recognize alkanes with carbon chain lengths up to C17 (Chen et al., 2023). AlkS and the alkane bind to the AlkB promoter, leading to the expression of the GOI (Yuste et al., 1998). The constitutive promoter pEM7 or pS2 expressed AlkSmt, and pAlkB expressed mKate 2 in P. fluorescens. Alkanes needed to be introduced to the cell to test the alkane-responsive transcriptional regulatory system. As alkanes are not soluble in water, a biosurfactant is necessary to make them soluble. Multiple biosurfactants were tested, including rhamnolipids, tween 80, and DMSO (Chen et al., 2023). The growth rate of the cells and the fluorescence were then measured and used to determine the expression rate of AlkS and pAlkB.
The growing amount of plastic polymer pollution, like polyethylene terephthalate (PET) and low-density
polyethylene (LDPE), poses a formidable challenge that scientists are trying to solve bioremediation
using enzymatic degradation. Enzymes have been identified in nature capable of breaking down these
polymers.
PET is composed of alternating terephthalic acid and ethylene glycol molecules connected by ester bonds.
Esterases, such as WT-Petase from Ideonella sakaiensis or leaf-branch compost cutinase (LCC)
discovered through metagenomic analysis, can cleave the esters and, when secreted, lead to PET
degradation (Yoshida et al., 2012; Tournier et al., 2020). However,
optimizing these enzymes for P. fluorescens poses distinct challenges. Most PETases reach peak
activity at an alkaline pH and temperatures above 50°C (Yoshida et al.,
2012). At the optimal culture conditions for P. fluorescens (pH 7 and 28°C), the PETase
activity severely decreases. Even with recent breakthroughs, temperature- and pH-optimized mutants such
as the fast-PETase and LCC are still suboptimal for use within the ReMix framework (Lu et al., 2022; Tournier et al., 2020).
For enzymatic plastic degradation to occur, they need to be exported out of the cell using export tags.
One tag of two tags tested hijacks the naturally occurring gelatinase export system found in P.
fluorescence. The second tag utilizes the signal peptide SPpstu from the maltotetraose-formin
amylase Pseudomonas stutzeri MO-19 (Lu et al., 2022).
PE has a pure carbon backbone structure, making enzymatic degradation difficult as it lacks easily
hydrolyzable groups. Alkane monooxygenase (AlkB) found in various Pseudomonas species plays a role in
breaking down PE (Pinto et al., 2022). Expression of AlkB in E. coli
enabled growth on LDPE. AlkB oxidizes medium-length n-alkanes in the periplasm into their corresponding
primary alcohols, subsequently ferrying them across the inner membrane. The 1-alkanols are oxidized
intracellularly into fatty acids, feeding into the cell's metabolism via the beta-oxidation cycle (Callaghan et al., 2006). The exact PE depolymerization mechanism is not fully
understood. It is unlikely that AlkB alone facilitates the depolymerization into n-alkanes as the enzyme
resides in the inner membrane, unreachable for any alkane-like structures above C16 (Grant et al., 2014). AlkB cannot oxidize n-alkanes longer than n-dodecane,
which supports this hypothesis. For iGEM, we want to demonstrate that P. fluorescens can
functionally express AlkB as an additional depolymerizing gene for our helper strain.
We replaced the GOI with a fluorescent reporter protein to test our expression systems. As P. fluorescens is a naturally fluorescent organism, we must choose a reporter protein whose excitation and emission wavelengths are distinct from the naturally emitted wavelengths. mKate2 has an excitation wavelength of 588 nm and an emission wavelength of 633 nm (Shcherbo et al., 2009). Another criterion was the fast maturation time as we designed our operon to be dynamic. With a 20-minute maturation time, mKate2 fulfilled this criterion (Heppert et al., 2016).
The main strain's goal is to utilize the TPA as a carbon source and generate a recombinant product. The set of genes for TPA uptake were introduced to our organism using the pBAMD1-2 plasmid containing the tphII operon (Brandenberg et al., 2022). Brandenberg et al. (2022) presented four genes found in Comamonas sp. E6m needed to convert TPA into PCA, which can reach the central carbon metabolism via the ß-ketoadipate pathway. The mini-Tn5 transposon vector pBAMD1-2 integrates the operon randomly in the genome. Due to the nature of the operon, TPA is a selection marker, and no further antibiotic resistance is necessary (Brandenberg et al., 2022).
For cloning of all the constructs, the pSEVA438 plasmid vector was used with the pBBR1 ori, which is compatible with a broad range of prokaryotic organisms. The plasmid carries the XylS/Pm expression cassette, which was used as a basis for the experiments. The growth assays were done in 96-well microtiter plates incubated at 28 °C and OD600 and fluorescence (588 nm excitation, 633 nm emission) measurements were taken every 10 min over a time period of 16-24 h. The fluorescence of each well was normalized with cell count (referenced to OD600). The results were compared to the appropriate negative controls.
Sequences coding for AlkS and pAlkB were obtained by gene synthesis (IDT) and cloned via Gibson assembly into the plasmid vector. Transcription factor expression was regulated by the constitutive pEM7 promoter, replacing the XylS/Pm system. The fluorescence reporter gene mKate2 was cloned with SacI and PstI into the MCS downstream of pAlkB. To increase fluorescence intensity with clearer read-outs, a synthetic RBS from the Anderson library (BBa_J61100) was added upstream of the coding sequence via substitution PCR.
Before characterizing the transcription factor, preliminary tests were conducted to optimize the
solubility and
bioavailability of different length n-alkanes (hexane, heptane, dodecane, heptadecane). Solubility was
tested in
varying concentrations of H2O, dimethyl sulfoxide (DMSO), Tween® 80, and rhamnolipids. Long chain
alkanes could not be
brought into solution using H2O and DMSO, making them unsuitable for future experiments.
While rhamnolipids
could readily solubilize alkanes, they showed high absorption at OD600 and strong
auto-fluorescence, making
them unsuitable.
The best results were achieved by first solubilizing the alkanes in 1 % Tween80<®> in ethanol
absolute. To
allow for bioavailability this solution was suspended in H2O at 100-fold dilution.
The final PE biosensor has the AlkS-V760E transcription factor constitutively expressed by the pEM7 promoter and the AlkS-V760E/pAlkB expression strength is measured with mKate2 fluorescence as a reporter gene.
Different n-alkanes emulsified in Tween® 80 (0.1 % (v/v)) were tested as inducers of mKate 2 at different concentrations (100 mg/L, 200 mg/L) with time-resolved fluorescence measurements (figure 10). Only n-dodecane showed a change in fluorescence intensity and was used for further testing of the induction of AlkS at different concentrations.
Serial dilution experiments of n-dodecane emulsified in Tween® 80 were performed and fluorescence intensity measured over 20 h (Figure 10, A). Expression strength was calculated 6 h, 12 h and 20 h after induction with different concentrations, ranging from 2 mg/L up to 2000 mg/L. Twelve hours after induction, significant increases could be measured with inducer concentrations smaller than 200 mg/L (p<0.01). At 20 h concentrations as low as 20 mg/L were sufficient to measure a significant change in fluorescence (p<0.01). The fluorescence measurements at 20 h were used to further analyze the dose-response curve (Figure 5, B), showing inducer saturation above 2000 mg/L n-dodecane.
Since the XylS/Pm expression system is natively found on the pSEVA438 plasmid only the two point mutations, K38R and L224Q, needed to be introduced. Two primer pairs were used to add the single base pair substitutions. The sensitivity of XylS-mt towards was studied using the native Ps1/Ps2 promoter system but found to yield low expression levels in the TPA sensitive range. To mitigate this problem, the Ps1/Ps2 promoter system was substituted with pEM7 to further test the functionality in different scenarios. The fluorescence reporter gene mKate2 was cloned with SacI and PstI into the MCS downstream of Pm, add-on PCR was used to introduce the Anderson library promoter RBS BBa_J61100. (Figure 3).
The XylS-mt sensitivity towards TPA was compared to the XylS-WT sensitivity. XylS-WT showed no sensitivity towards TPA and good sensitivity towards MBA. When comparing the sensitivities of XylS-mt and XylS-WT to MBA, the introduced mutations seemed to cause a 60-70 % decrease in expression strength (figure 4).
Co-induction with varying concentrations of TPA and m-Xylene or TPA and Toluene (5 nM, 50 nM, 500 nM m-Xylene or Toluene mixed with 0 nM, 2.5 nM, 5 nM, 10 nM, 50 nM, 500 nM, or 1 mM TPA) was tested to improve the induction of XylS-mt and the expression of the GOI. Toluene and Xylene are inductors of the genomic transcription factor XylR, previously described to jointly activate expression from the Ps1 promoter with XylS in P. putida. However, co-induction showed no increase in expression strength (data not shown).
XylS-mt was first tested with the native Ps1/Ps2 promoter system, with different inducer compositions of TPA and 3-methyl-benzoate (MBA). The Ps1/Ps2 promoter was substituted with the constitutive promoter pEM7 using add-on PCR. TPA and MBA were tested separately in serial dilutions experiments (figure 11), and in combination (figure 12).
Serial dilution experiments of only TPA showed significantly increased fluorescence compared to the uninduced controls for concentrations above 1 mM at 8 h and 12 h after induction (p<0.01) (Figure 11, C). The same experiments performed with MBA as an inducer showed an overall stronger expression strength and significant changes in fluorescence after induction with 0.01 mM MBA (p<0.001) (Figure 11, A). The calculated dose response curve (Figure 11, (B)) shows inductor saturation at 0.1 mM. For induction of TPA, no inductor saturation was observed (Figure 11, D). The fluorescence intensity of the XylS-WT compared to the XylS-mt shows an overall decreased expression strength. (Figure 11, E)
To further test the influence of the Ps1/Ps2 promoter system on XylS-mt, the co-induction was tested with previously determined MBA and TPA concentrations. Three TPA concentrations were tested with one of four MBA concentrations. Fold change and normalized fluorescence were calculated (Figure 13). At an MBA concentration of 0.0025 mM, a significant TPA dependent fold change could be measured (1.29 +/- 0.056, p < 0.001). Higher MBA concentrations (0.0075 mM MBA, 0.015 mM MBA) showed an overall decreased fold change. Decrease after TPA induction is due to referencing errors caused by TPA precipitation. The expression strength shows an overall decreased fluorescence intensity at low MBA concentrations, despite co-induction with TPA (Figure 13, right)
Alternative to the Ps1/Ps2 promoter system, the constitutively active pEM7 promoter was tested, which was previously used for the expression of AlkSV760E. The new promoter showed overall higher fluorescence intensities, compared to the previously tested Ps1/Ps2 promoter system. Significant changes above induction could be measured with 0.005 mM MBA (p<0.001) or 1 mM TPA (p<0.05) (figure 14).
Three gene constructs were obtained by gene synthesis (IDT) each with a different ribosomal binding site (BBa_J61100, BBa_J61101, BBa_K4757003). The construct also contains two SapI recognition sites, a bi-directional terminator (LUZ7 T50, BBa_K4757058), mKate2 in reverse complement with degradation tag (BBa_K4757000, BBa_K4757001), and the constitutive promoter pEM7. The sRNA coding oligo sequences were cloned scarless behind the Pm promoter with SapI golden gate assembly, yielding 27 different composite parts (By combining the seed regions BBa_K4757021 - BBa_K4757027 with the scaffolds BBa_K4757031 - BBa_K4757033 ).
To find optimal expression levels of mKate2 and establish new ribosomal binding sites for P. fluorescens, different ribosomal binding sites were tested. For the experiments, the repression of the fluorescence intensity of constitutively expressed mKate2 was measured and the fold change over the auto-fluorescence of P. fluorescens was calculated (Figure 6).
Constructs with BBa_J61100 (RBS 1) showed minimal fold-change in fluorescence levels (0.73 +/- 0.316).
The second
RBS from the Anderson library (BBa_J61101, RBS 2) had a distinct increase in fold change compared to
BBa_J61100
(18.15 +/- 3.21 compared to 0.73). The synthetic RBS (RBS 3) designed by the Salis-lab calculator
(calculated for
maximal expression strength for mKate2 mRNA) showed the strongest fluorescence (48.56 +/- 3.394).
Although RBS 3 showed highest expression strength, RBS 2 was used for the final operon as the
binding was
independent from the coding sequence (CDS).
Before ordering the different sRNA constructs, in silico analysis of the free binding energy of sRNA-mRNA hybridization was calculated and compared to literature to ensure efficient repression (figure 8).
The 27 different sRNA constructs were tested using three different scaffolds, previously used for synthetic sRNA repression, and three different binding sites. The scaffolds SgrS and MicC were chosen since they have been used by previous iGEM teams (e.g. Team Peking 2011, Team Edinburgh 2018, Team UT-Tokyo 2013) and have been established in the literature (No et al., 2019). As they lack characterization in bacteria other than E. coli , we could establish sRNAs in the novel chassis P. fluorescens . Additionally, an engineered version of SgrS (SgrS-S CUUU 6 nts stem (SgrSmt)), optimized for repression in E. coli DH5 alpha, was chosen (Noh et al., 2019). Seed regions (homologous to the mRNA) were chosen with 25 bp homology, targeting either the RBS (target 1), both the RBS (12 nt) and CDS (13 nt) (target 2), or the CDS starting with AUG (target 3).
(A)
(B)
(C)
All constructs were tested with endpoint measurements in early stationary phase to see the maximum repression capability (figure 3). Seed regions with target 3 showed the weakest repression rates (0.367 +/- 0.118 mean repression compared to 0.544 +/- 0.153 and 0.569 +/- 0.102, for target 1 and 2 respectively). Seed regions with target 1 showed the strongest repression regardless of the RBS used.
The final operon construct contained the seed region targeting only the RBS (target 1, RBS2: BBa_J61101) with the SgrS and MicC scaffolds, as they showed the highest repression strength and the RBS was not designed for a specific mRNA sequence.
After conducting repression experiments with all sRNA constructs, possible correlation between the
relative
repression strength and calculated free binding energy was calculated.
Figure 8 shows repression strength against the free binding energy. For all three tested targets no
correlation between binding energy could be found. Interestingly target 3 showed an overall increased
variance
(0.0678 mean error) compared to target 1 (0.042 mean error) and target 2 (0.0422 mean error).
The scaffolds SgrS and MicC with the RBS 2 (BBa_J661101) target region were used for further characterization of the repression characteristics.
The sRNA expression was controlled by the MBA inducible XylS-WT/Pm promoter system. By targeting the
constitutively expressed mKate2, repression strength was calculated with decrease in fluorescence
intensity
(Figure 15). Both sRNA constructs showed an overall continuous repression strength over time after
induction.
(Figure 15 A, B) with the highest repression after 20 h of 0.65 +/- 0.033 and 0.61 +/- 0.034 for SgrS
and
MicC, respectively.
The inducer concentration dependent repression strength was calculated at the time points 10 h and
15 h
(Figure 15), which showed a linear increase in repression strength with a saturation above 100 mM MBA
concentration.
(A)
(B)
(C)
(D)
The XylS-K38R-L224Q on the pSEVA438 plasmid was used as a basis for assembling the final operon. The
vector was
linearized by PCR, adding homologous overhangs for pAlkB and AlkS sequences. A new sequence containing
RBS 2
(BBa_J61101), BsaI restriction sites, and the LUZ7T50 terminator (BBa_K4757058), was synthesized with
homologous
sequences (IDT). All three insert fragments (pAlkB, RBS2-BsaI-LUZ7 T50) were assembled using Gibson
assembly.
Golden Gate assembly, with BsaI restriction enzyme, was used for inserting mKate2 behind RBS 2.
Insert and
vector sequences were verified with sequencing, but after multiple attempts with different molar ratios,
they
could neither be successfully combined nor transformed into either P. fluorescens or E.
coli DH5
alpha.
Sequences coding for WT-PETase, FAST-PETase, LCC, and AlkB were synthesized by IDT and added into the pSEVA438 vector using SacI und PstI restriction digest. The enzymes (fused to His6-tag) were expressed in P. fluorescens. Proteins were from raw cell lysate or, if excreted by SPpstu secretion tag, from culture media, were purified with Ni-NTA affinity chromatographie, and competetively eluted. Time resolved, colorimetric assays with 0.25 µg enzyme and p-nitrophenyl-acetate (pNP-Ac) and pNP-butyrate (pNP-But) were done to asses enzyme activity. Reaction was incubated at28 °C, pH7 over 90 mins, with OD415 measurements every three minutes.
AlkB was successfully transformed into P. fluorescens and confirmed by Sanger sequencing,
however, as
of now the enzyme's expression and functionality could not be tested.
AlkB is a membrane bound protein with membrane specific cofactors making the isolation difficult.
The tested enzymes, WT-PETase, FAST-PETase, and LCC, all showed catalytic activity under previously
described
conditions (figure 1).
Based on these measurements Michaelis Menten kinetics could be calculated, with the FAST-PETase purified
from the
growth media showing a 4 fold, higher vmax compared to the other WT-PETase and LCC (Figure 2,
A).
The Km value was higher for the FAST-PETase from the medium compared to the
others (Figure 2, B). The initial rate of the extracellular FAST-PETase was fitted with a
Michaelis-Mentan curve
(Figure 3).
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