Introduction

Our non-model organism

In our experiments, we work with the strain Methylobacterium extorquens AM1Δcel1, a pink-pigmented facultative methylotrophic α-proteobacterium2. The Δcel mutation ensures M. extorquens does not form biofilms, which is advantageous in an experimental setting since it ensures reliable measurements of its growth curves1. From here on, this strain is referred to as M. extorquens wild type. M. extorquens can utilize methanol as the sole carbon source and convert this into polyhydroxyalkanoates (PHA). M. extorquens has been proposed as a promising methanol-based bioproduction platform3.

The overarching goal of PHAse Out was to make cost-effective and sustainable PHA with green methanol as a feedstock. For this, we genetically engineered Methylobacterium extorquens AM1 with the aim of increasing PHA production and improving PHA extraction methods. On this page, we show the most important results that substantiate our progress towards this goal.

Overview
Fig. 1 | Research lines of PHAse Out. Visual overview of the experimental lines, their interconnectivity, and connection to the project aim.

Research lines

Characterization of promoters for M. extorquens

Goal

 Characterize various inducible and constitutive promoters for genetic engineering of M. extorquens

Method

 Cloning of the promoters into the empty pTE100 backbone with the mCherry reporter gene. Transformation into M. extorquens. Measure the mCherry fluorescence signal over time under different inducer concentrations

Hypothesis

 The cells with constitutive promoters are expected to show higher fluorescence compared to the control having no promoter. The cells with inducible promoters are expected to show dose-dependent increase in fluorescence levels in relation to the inducer

Conclusion

 The IPTG- and vanillate-inducible promoters showed significantly increased fluorescence when induced, where the induction of the IPTG inducible promoter PL/O4/A1 was the strongest. Out of the constitutive promoters, PmxaF had the highest expression, followed by Ptuf. PfumC and PcoxB showed similarly low expressions

To effectively modify M. extorquens, we required genetic elements that were proven to be functional in the organism. The toolkit for genetic elements of this organism is lacking when compared to standardized model organisms4, 5. We first created a collection of parts containing different promoters from literature, all of which are described in more detail on the Parts page . We characterized multiple inducible and constitutively active promoters to use in the phaC overexpression and autolysis experiments. We tested the constitutive promoters PmxaF, PfumC, PcoxB and Ptuf4, Isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible promoter PL/O4/A15 and vanillate inducible promoterPV106. To learn how we constructed and transformed the different plasmids4, please visit the Protocols and Notebook pages.

We used a M. extorquens optimized mCherry reporter cassette to assess promoter strength. As a negative control, a strain carrying a plasmid containing the mCherry testing cassette, without a promoter, was used. As a positive control plasmid developed by dr. Schada von Borzyskowski were used4. For the promoters that were tested, the inducible promoter sequences were cloned in front of the mCherry cassette. A growth experiment was set up with biological and technical duplicates, where the OD600nm and fluorescence were tracked.

In Figure 2, the fluorescence data indicating the promoter strength is shown as the mCherry fluorescence normalized to the OD600nm. The sampling time was placed at 24 hours of growth, which fell around the middle of the exponential phase of growth. This is further elaborated on in the dropdown below Figure 2. For PL/O4/A1, the 48-hour time point was instead used since the growth curve showed a delay. Contrary to our expectation, one of the biological duplicates of PcoxB did not show fluorescence. This indicates an aberration suppressing mCherry expression in this specific biological duplicate, therefore the data from this duplicate was excluded from the analysis.

For the constitutive promoters, the expression levels were similar to the promoter activities described in literature4. PmxaF had the highest expression, followed by Ptuf. PfumC and PcoxB which showed similarly low expressions. For the inducible promoters, multiple concentrations of the respective inducers were used. In Figure 2, the uninduced condition was compared to the highest induced condition. There was a significant 190% increase for PL/O4/A1 between the uninduced and induced conditions and a significant 30% increase in expression for PV10. For results with a broader range of inducer concentrations, please visit the specific information submitted to the parts registry for each part.

Promoter overview
Fig. 2 | Normalized fluorescence indicating promoter strength of the four constitutive promoters, and of the inducible promoters both in uninduced (-) and induced conditions (+). The mean of the biological and technical duplicates is shown. The error bars represent the standard deviation. The dotted line serves as a visual aid and represents the fluorescence signal of 250 nM of the Sulforhomadine from the iGEM calibration kit, as part of the Interlab study. For PL/O4/A1 there was a significant increase of 190% between the uninduced and induced conditions (Student’s T-test, **** p < 0.0001). For PV10 there was a significant 30% increase in expression when induced (Student’s T-test, * p < 0.05 Student’s T-test).

Determination of exponential phase of growth

    Figure S1 shows the growth curves which were used to determine the optimal time point to sample the fluorescence in order to determine promoter strength, which is in the middle of the exponential phase7. In addition, the growth curves assess the viability of the strains, thereby giving an indication of the toxicity of the construct and potential inhibition by inducer presence. The bacteria carrying the different mCherry expressing plasmids were expected to have similar growth curves. However, the curves show that presence of the PmxaF, PcoxB and Ptuf constructs inhibited the maximum OD600nm reached when compared to the control and the PfumC strain. The growth curves of the PV10 strains show reduced growth. For PL/O4/A1 , a delayed growth was seen. Based on these fluorescence measurements, the sampling time was set to be 24 hours for all strains, except PL/O4/A1, which was set at 48 hours.

    Promoter overview
    Fig. S1 | Growth curves for the mCherry expressing strains, showing the mean of the biological and technical duplicates of each promoter, with the dotted lines representing the error bars. A sigmoidal 4PL curve (black) is fitted on each graph. For PL/O4/A1, the uninduced (-) and the 1000 µM IPTG induced (+) conditions are shown, and for PV10 the uninduced (-) and the 250 µM vanillate induced (+) conditions. This graph indicates that most strains were in their exponential phase of growth at 24h. An exception is the strain with the PL/O4/A1, where the growth was delayed. The middle of its exponential phase is at around 48h. PV10 shows inhibited growth, both in the uninduced (-) as induced (+) condition.

To further expand the genetic engineering toolkit for M. extorquens, follow up experiments may include characterization of a larger set of constitutive and inducible promoters.


Overexpression of genes for increased PHA production

Goal

 Increase PHA production

Method

 Overexpression of the PHA synthase gene (phaC) and the gene coding for the GroEL/ES chaperone complex through various constitutive promoters

Hypothesis

 Overexpression of the phaC and groEL/ES genes will increase PHA production in recombinant strains in comparison to the wild type

Conclusion

 Overexpression of both phaC and groEL/ES genes resulted in increased PHA production in comparison to the wild type, with overexpression of phaC under control of the PmxaF promoter resulting in a 24-fold increase of PHA production. Co-overexpression of phaC and groEL/ES under control of the Ptuf promoter indicated possible synergistic effects with a 24-fold increase of PHA production

To improve PHA production, we chose to overexpress the PHA synthase gene phaC, which codes for Poly(3-hydroxyalkanoate) polymerase and is a key enzyme in the PHA production pathway8. This pathway in M. extorquens is further elaborated on our Modeling page . As expression of this gene in the wild type M. extorquens is regulated by available nutrients in the environment4, we aimed to express phaC independent of this regulation and in high quantity. Literature suggests that overexpression of the GroES/EL chaperone system assists in folding the produced PhaC enzyme, improving the active protein levels of this enzyme9. Therefore, we created genetic constructs with the constitutive promoters of variable expression strengths that were tested in cycle A of engineering . Both the phaC and groES/EL genes were amplified from M. extorquens genomic DNA with primers designed for Gibson Assembly using the NEBuilder10. PhaC and groEL/ES were amplified for both individual cloning and a combination of the two in one plasmid. Figure 3 shows the created constructs.

phac construct
Fig. 3 | Genetic constructs that were created for overexpression and co-overexpression. With four different promoters, PmxaF, PfumC, PcoxB and Ptuf, with either phaC, groEL/ES or phaC and groEL/ES genes combined.

See the experimental design in further detail on the Protocols page .


Nile Red staining

To quantify the PHA production of our cultures, we used fluorescent Nile Red staining, which stains PHA and biomass11,12. As Nile Red does not exclusively stain PHA, the background signal from the biomass must be determined. For this, a phaC knockout strain (ΔphaC, provided by Dr. Schada von Borzyskowski from Leiden University) was used. By removal of the phaC gene, the ΔphaC strain did not produce PHA and was used to extrapolate the amount of PHA present in PHA producing strains. This was done by staining both the samples with Nile Red and subtracting the ΔphaC fluorescence signal from the fluorescent signal of the other sample. All samples were grown as explained in the optimal cultivation conditions segment, with methanol replenishment at 40h post-inoculation and sample collection at 48h post-inoculation. The collected samples were then used for biomass determination and fluorescence measurement. The wild type strain was used here as validation of this quantification technique for further experiments. The results are shown below in Figure 4.


Fluo nile red
Fig. 4 | Fluorescence signal from Nile Red staining of ΔphaC and M. extorquens wild type at different biomasses, with the biomass indicated in mg/ml. Nile Red stains biomass and PHA. As ΔphaC does not produce PHA, the signal from the ΔphaC can be subtracted from the signal of the PHA producing strain to quantify the amount of PHA produced. The divergence of the wild type sample at 0.8 mg/mL, marked with an '*' is due to a handling error and this point was subsequently excluded from the analysis.

The Nile Red staining of the wild type and ΔphaC strain show a biomass-dependent signal increase, with the wild type showing a greater fluorescent signal than the ΔphaC. This is due to PHA accumulation in the wild type. Subtracting the fluorescent signal from the ΔphaC strain allows for quantification of PHA. This can then be used to quantify PHA accumulation via Nile Red staining in milligrams per milliliter culture.


Optimizing cultivation conditions to balance nutrient availability for maximum PHA accumulation

    To determine optimal cultivation conditions for PHA production, three different cultivation methods were adapted from literature13-15. These included the addition of one of three medium components 40 hours post-inoculation, when cells were in the stationary phase . The exact execution of the experiment can be read on our Protocols page .

    Cultivation conditions 40 hours post-inoculation Hypothesis
    Methanol (0.5% (v/v)) replenishment The carbon source is the limiting factor for PHA production. Adding methanol resolves this15.
    Nitrogen-free medium replenishment Replenishment of minerals and methanol as well as a stimulus in the form of lacking nitrogen is required for PHA production13.
    Medium replenishment Addition of all medium components will lead to increased PHA production14.

    At different time points during cultivation, samples of equal amounts of biomass were taken. These samples were subsequently stained with Nile Red to determine PHA related fluorescence12 in response to different culture conditions. The results are shown in Figure S2.

    Biomass nile red
    Fig. S2 | Triplicate samples of biomass stained with Nile Red at 15, 18, 35, 40, 56, 62, and 79 hours post-inoculation. In the stationary phase at 40 hours post-inoculation either the medium, methanol, or nitrogen free medium was replenished.

    Out of the three medium replenishment conditions, the results show the highest fluorescence intensity increase for the methanol replenishment condition. The highest fluorescence was measured at 56 hours post-inoculation. Hence, the replenishment of methanol at 40 hours post-inoculation was chosen as the preferred cultivation condition in further experiments. This is also advantageous due to the speed and simplicity of this method in large scale bioreactors, as this method does not require a complete medium change to induce PHA production15. In subsequent experiments PHA production was measured at 48 hours, rather than 56 hours, due to practical considerations.


To determine the intensity of the Nile Red signal from PHA we made a calibration curve with a sample of 99% pure PHA16. Sonicated PHA was stained with nile red and the fluorescence was measured. Read more about our calibration experiments in our Protocols page . The calibration curve is shown in Figure 5.

Calibration
Fig. 5 | PHA calibration curve acquired through Nile Red staining and measurement of sonicated PHA of known concentrations. PHA samples were prepared, stained, and measured in quintuplicate. This curve, in combination with the measurements from Figure 4, was used in further experiments to quantify PHA in the different M. extorquens strains.


phaC overexpression

To test the effect of phaC overexpression on PHA production, we inoculated strains with the PmxaF, PfumC, and Ptuf promoters and a wild type strain. The bacteria were cultured following the above established culture conditions, where initial inoculation in minimal methanol-based medium is followed by methanol replenishment after 40 hours. Samples were collected at 48 hours after inoculation and were used for biomass determination as well as Nile Red staining. Measurements were done in technical quadruplicates. The results are shown below in Figure 6. All strains containing an overexpression construct showed a significant increase in PHA production compared to the wild type (p < 0.0001). In line with previously obtained results, overexpression by PmxaF yielded the highest PHA production, with a 24-fold increase. Contrary to expectation, Ptuf’s expression strength does not translate to PHA accumulation in this experiment. PfumC-driven PHA accumulation is significantly less than that of PmxaF (p = 0.0103), prompting us to use PmxaF for future experiments.

pha prod phac
Fig. 6 | PHA production of phaC overexpressing strains with the different promoters in comparison to the wild type at 48 hours after inoculation in technical quadruplicates. PmxaF is the highest producing strain, with a 24-fold increase in comparison to the wild type and a significant (Student’s T-test, * p = 0.0103) increase in comparison to the second highest producing strain, PfumC.


Following the same workflow, GroES/EL overexpressing strains with PmxaF, PfumC, PcoxB, and Ptuf constructs were cultured and measured. The results in Figure 7 show a significant increase in all overexpressing strains compared to the wild type (p ≤ 0.0001), with a ~5-fold increase in PHA production in PmxaF, PfumC and PcoxB and a 10-fold increase in PHA production in Ptuf. Ptuf also shows a significant production increase in comparison to PcoxB (p = 0.001).

pha prod groe
Fig. 7 | PHA production of the GroES/EL overexpression strains with the different promoters in comparison to the wild type at 48 hours. Ptuf shows a significant increase in PHA accumulation over all other strains (Student’s T-test, ** p = 0.001)


Lastly, strains co-overexpressing phaC and groES/EL under the control of PfumC, PcoxB, and Ptuf, respectively, were tested as described above. PmxaF was not tested, as cloning failed and could not be repeated due to time constraints. Again, all strains show a significant increase in PHA production over wild type (p < 0.0001), with a 10-fold increase for PfumC, a 11-fold increase in PcoxB and a 24-fold increase in Ptuf, as shown in Figure 8. The increase in PHA production through co-overexpression of phaC and groES/EL under Ptuf is similar to the increase through overexpression of phaC under PmxaF. Under Ptuf, a synergistic effect was observed for the co-expression constructs in comparison to the single constructs. The PHA production of the co-expression construct surpasses that of the single protein expressing strains. Especially promising is the fact that co-overexpression of the two enzymes with Ptuf results in a PHA production within 3% of the PHA production of the PmxaF driven phaC overexpression strain. This also represents a ~5-fold increase from sole phaC overexpression by Ptuf. Future experiments could investigate whether the same effect can be observed with PmxaF, yielding an even stronger PHA-producing strain, due to the inherently stronger promoter. PfumC does not indicate a similarly potential synergistic effect, but rather an antagonistic one. Under PfumC the results obtained for the co-expression construct showed lower PHA production than for sole phaC overexpression.

pha prod phacgroe
Fig. 8 | PHA production of the phaC and groES/EL overexpression combination strains with the different promoters in comparison to the wild type at 48 hours after inoculation.

In conclusion, overexpression, and co-overexpression of phaC and groES/EL show promising results for increased PHA production and possible synergistic effects. The results from our preliminary studies could be further supplemented through protein extraction and enzyme activity assays.

Negative promoter protein interactions in Methylobacterium spp. have previously been reported with fluorescent reporter proteins. Investigating whether this is the case with our constructs would be necessary for explaining differences between promoter strengths from validated mCherry-based testing to groES/EL overexpression.

Microscopy verification of PHA in overexpression strains

We visualized PHA accumulation via the staining of granules of PHA located in M. extorquens. To substantiate this design choice and verify the presence of PHA in our overexpression lines, we carried out the Nile red staining protocol on the ΔphaC, wild type, phaC overexpression, groES/EL overexpression, and phaC + groES/EL overexpression lines under the control of PmxaF (phaC + groES/EL under PfumC). We then imaged these cells under a fluorescence microscope and compared them (Figure 9) For more details, see the Fluorescence Microscopy Protocol .

pha prod phacgroe
Fig. 9 | Comparison of M. extorquens ΔphaC, wild type, phaC overexpression, groES/EL overexpression, and phaC + groES/EL overexpression lines after staining, 535nm excitation, 605nm emission12.

In these images, there are several promising indications. ΔphaC is devoid of granules, while the wild type had small bodies located at the cell poles which responded positively to staining, indicating they are lipophilic and therefore likely PHA. The +phaC stain showed similar results, except with larger, brighter bodies, not always located at the poles. groE consistently showed smaller, but more numerous vesicles spread across the length of the cell. The combination phaC + groES/EL did not show an increase over these, being mostly similar to the +phaC. From this we can conclude that there is a cellular basis for the quantification of PHA levels via Nile Red staining, and that the overexpression and chaperones had an effect, however no obviously superior overexpression was visible at the cell level.


Knockout of carotenoid genes for increased PHA production

Goal

 Knock out genes in the carotenoid biosynthesis pathway to increase flux of acetyl-coa into PHA production

Method

 Replace genes encoding for enzymes involved in the carotenoid biosynthesis pathway by a kanamycin resistance gene

Hypothesis

 Gene targets were selected based on computational simulations using a metabolic network model of M. extorquens, which were predicted to be most effective in improving PHA production

Conclusion

 A ΔcrtI strain was successfully created and validated

To improve the production of PHA by M. extorquens, we analyzed the metabolic network model to identify promising gene knockout targets that guide the metabolic flux towards the PHB cycle. Based on the computationally predicted results, three target genes encoding enzymes involved in the carotenoid pathway were selected: dxr, ispA and crtI (Figure 10).

knockout
Fig. 10 | Overview of selected gene knockout targets dxr, ispA and crtI involved in the carotenoid biosynthesis pathway, which are predicted to effectively eliminate carotenoid production and enhance PHA production in M. extorquens, as based on our modeling results.

A detailed explanation of the construct design can be found on the Engineering page .

The upstream and downstream flanking regions for homologous recombination of crtI, dxr and ispA were successfully inserted into the pREDSIX plasmid and validated by sequencing . The subsequent insertion of the kanamycin resistance gene was successful for the vectors containing the flanking regions of crtI and dxr. Unfortunately, the insertion of the kanamycin resistance cassette into the vector containing the flanking regions of ispA proved to be difficult and no verified vector could be obtained during the lab time. Transformation of pREDSIX containing the flanking regions of crtI and the kanamycin resistance gene into M. extorquens was successful and non-fluorescent colonies were identified using fluorescence microscopy. Five non-fluorescent colonies were further used to validate the knockout strain. The genomes of these five colonies were isolated and the gene replacement was validated by amplifying the target region in the genome which should have been replaced by a kanamycin resistance gene in the transformed strains. As the kanamycin resistance cassette is roughly the same size as the native crtI gene, the amplified DNA was subsequently digested using NdeI, which should only digest the target sequence containing the kanamycin resistance cassette and not the sequence containing the crtI gene. The expected bands from a correct ΔcrtI mutant are 1228, 964 and 948 bp, compared to 3433 bp for wild type M. extorquens. The results can be seen in Figure 10 and correspond to the expected band lengths.

knockout
Fig. 11 | Validation of ΔcrtI colonies (1-5) by amplifying the target region in the genome. Amplified DNA was digested using NdeI Expected bands of a correct ΔcrtI mutant are 1228, 964 and 948 bp, compared to 3433 bp for wild type M. extorquens. Correct bands are highlighted in red.

Interestingly, the obtained ΔcrtI strains seemed as pink as wild type M. extorquens, contrary to previous research that showed that deletion of crtI resulted in a colorless M. extorquens mutant17. Due to time limitations, the PHA production by these ΔcrtI mutants could not be quantified. This would have been performed similar to the PHA quantification of the phaC overexpression strain using NR staining and subsequently validated with extraction.

Furthermore, the transformation of pREDSIX containing the flanking regions of dxr and the kanamycin resistance gene into M. extorquens repeatedly failed. This may indicate that dxr is an essential gene, and that this gene deletion is lethal, which has also been suggested previously18.


Introduction of heterologous genes for autolysis

1. Determining the effectiveness and induction of exogenous Holins/Endolysins

Goal

 Introduce plasmids containing holin/endolysin genes to assess whether M. extorquens is able to express and use these proteins to auto-lyse

Method

 Introduce the five synthetic IPTG-induced holin/endolysin plasmids, and test the lethality of induced and uninduced genes by colony survival counts

Hypothesis

 Induced genes should show a reduction in colony counts compared to the uninduced, with the most lethal gene having the lowest surviving colony count

Conclusion

 PL/O4/A1 is not fully repressed in the absence of IPTG, however all genes show statistically significant lethality, especially Gp110 endolysin and EJ1 Holin/Endolysin

To improve the efficiency of PHA extraction and remove the need for hazardous chemicals to lyse the cells, we were interested in using inducible cell lysis systems to cause cell death on demand. We selected 5 cell lysis genes that had been experimentally verified in related bacteria and codon optimized them for expression in M. extorquens. We inserted native ribosome binding sites (RBS) from pTE-16B, which is an established plasmid in M. extorquens, before all protein coding sequences, ensuring effective transcription by the host's RNA polymerase.

The high GC content of M. extorquens and phage DNA (60%+) is problematic to synthesize. The standard codon table of M. extorquens also differs from E. coli. To combat this, the De Novo DNA combined synthesis success tool19 was used to strike a balance between accurate amino acid codon sequences and successful synthesis probability. The synthesized genes can be seen below (Table 1).

Table 1 | Self lysis systems, origin, brief description and literature source.

Name Origin Description Source
T4 Lysozyme Escherichia coli phage T4 Fusion protein of a membrane lysis protein with cell penetrating peptide 20
Gg Lysozyme Gallus gallus (chicken), modified for efficiency in P. putida Gallus gallus (chicken) lysozyme sequence 21
T7 hydrolase Agrobacterium Tumefaciens phage T7 Modified A. tumefaciens phage lytic system, peptidoglycan hydrolase 22
Gp110 endolysin Salmonella Bacteriophage Salmonella Bacteriophage peptidoglycan hydrolase 23
EJ1 Holin/Endolysin Pneumococcus phage EJ1 Two part Holin-Endolysin system derived from Pneumococcus phage EJ-1 24

To transform the genes into M. extorquens and selectively induce them, we used the inducible plasmid pTE100-PL/O4/A1. We used Gibson assembly cloning to create the constructs. We verified the functionality of the genes by sequencing the inserted regions. Experiments were continued with sequencing-verified plasmids. See the Protocols page for detailed information about the construct cloning and transformation into M. extorquens.

Since we expected a lethal phenotype for induced holin/endolysin systems, we used a survival study setup to assess the effect of the inserts in M. extorquens. We used pTE100-PL/O4/A1 without insert as our negative control, as it is expected to be non-lethal, and a plasmid free competent M. extorquens culture as our positive control, as this should be totally lethal due to lacking antibiotic resistances. The details for this can be found on the Protocols page .

survival
Fig. 13 | Survival study for assessing autolysis systems under inducing and non-inducing conditions in M. extorquens, compared to positive (non-lethal) and negative (lethal) controls. Biological duplicates employed, with confidence intervals calculated from mean and variance of the duplicates.

These results were statistically analyzed (two tailed T-test, p < 0.05) by comparing the uninduced average colony counts with the average colony count for the controls, then comparing the induced and uninduced populations of the same gene. This showed that all the autolysis systems have statistically decreased populations in comparison to the negative control (non-lethal genotype). The Gp110 endolysin and EJ1 Holin/Endolysin showed the most significant decrease in population, going as low as single digit colony counts. Between the IPTG negative and IPTG positive plates, there was no significant difference in colony counts. This combination indicates that the autolysis cassettes influenced colony survival, however, autolysis cassettes showed similar effects on the population under uninduced and induced conditions. From this it can be concluded that the inducer was “leaky” and was not sufficiently repressed under non-induced conditions, allowing for expression of lysis genes even when no inducer was present.


2. Determining the effects of Holins/Endolysins on solvent sensitivity

Goal

 Test the effect of the heterologous Holins/Endolysins on the solvent resistance of M. extorquens

Method

 Using the transformed M. extorquens from the survival experiment, spot plating on a butanol-containing plate and compare colony growth and opacity

Hypothesis

 Colonies carrying genes with lower colony forming unit averages will have lower opacity colonies compared to wild type

Conclusion

 High lethality genes show reduced growth on Butanol plates

The previous results indicate a correlation between reduction in cell viability and the autolytic systems. However, this correlation cannot be fully substantiated to determine causation. As such, we performed further analysis of the effect of the autolysis systems on cell wall integrity. Solvent tolerant bacteria, such as M. extorquens, are reliant on the integrity of their cell walls, efflux pumps, and various other mechanisms for their resistance to solvents25. M. extorquens is generally solvent tolerant due to its methylotrophic nature. Given that the lysis genes should negatively affect cell well integrity, we examined the difference in solvent tolerance between our engineered strains and a wild type control by exposing them to butanol. See Protocols page for more information on the experimental set-up.

survival rates
Fig. 12 | Survival rates of M. extorquens cells carrying autolysis genes on varying butanol concentrations, IPTG induced and uninduced states. Survival rates quantified via image derived grayscale values. Experiments were performed in technical triplicates.

Statistical analysis of the rate of change in growth in response to butanol shows a significant difference (p < 0.05) between Gp110 endolysin and EJ1 Holin/Endolysin as compared to the other strains, with Gp110 and EJ1 showing less cell viability than the other strains. Additionally, there is a significant difference (p < 0.05) between the Gp110 endolysin and EJ1 Holin/Endolysin and the control, with these displaying less cell viability than the control. The Gp110 endolysin and EJ1 Holin/Endolysin strains show a near zero cell viability under solvent stress compared to the control and other cell lines. This supports our previous results, which indicate the Gp110 endolysin and EJ1 Holin/Endolysin inserts have the largest effect on cell viability. As the inserted genes lyse cells by either hydrolyzing the cell wall, by inserting holin proteins into the cell membrane, or through a combination of these mechanisms, the solvent experiment results support our hypothesis that the insertion of these genes into M. extorquens reduced cell wall integrity. Taken together, the experiments performed for the development of an autolysis system in M. extorquens show the functional incorporation of the inserts, a significant negative effect on cell viability, and a significant effect of Gp110 endolysin and EJ1 Holin/Endolysin on solvent tolerance.

In conclusion, we were able to successfully insert and verify the presence of our autolysis genes. After this, we indicated all autolysis genes reduced the number of colony forming units after transformation and growth on plates, and two of these genes were causing significantly reduced cell viability under solvent stress. We were unable to detect significant increases in cell death after induction with IPTG. The combination of both a reduced colony forming unit rate and a reduced tolerance to solvents in our most lethal genes supports the cause of cell death being related to the integrity of the cell membrane of the M. extorquens cells. The target of our autolysis genes were the cell wall and membrane, therefore this supports that we were able to successfully induce autolysis in M. extorquens. However, as we were not able to control the induction of the autolysis system, we were unable to test the viability of this process for increased PHA extraction efficiency.

PHA extraction

    Solvent extraction using chloroform is the most well established extraction method for PHA26. Extraction expert Jonathan Salt recommended using solvent-based extraction with chloroform and acetone, since it is cheapest for large scale extraction methods. However, due to concerns raised by safety experts , we focussed on DMC-based solvent extraction27. Read more about the safety concerns of using chloroform on our Safety page . The complete protocol can be found on our Protocols page . No discernible product quantities were extracted, and no increase in extracted product compared to the ΔPhaC strain was found .

    To test whether the fault laid with the extraction or if the strains did not produce PHA, a solvent extraction using chloroform was performed. 572 milligram of extracted product was obtained from 150mL of wild type M. extorquens (Figure S3). Due to time constraints, optimizing the DMC-based extraction was not possible. It was likewise not possible to analyze the product from the chloroform extraction. To improve DMC-based extraction in future experiments we recommended using a dry-pellet for extraction, as the adhesiveness of the wet cell pellet to the falcon tubes was difficult to work with when transferring the wet-pellet with DMC to the extraction set-up.

    PHA
    Fig. S3 | Obtained product from a chloroform-based extraction, extracted from 150mL of wild type M. extorquens


Conclusion

Through our experimental workflow, PHAse Out aimed to establish a cost-effective and sustainable method for producing PHA with M. extorquens using green methanol. Our main achievements and findings include the successful characterization of various promoters, notably demonstrating significant increases in fluorescence for IPTG- and vanillate-inducible promoters, with the IPTG-inducible promoter PL/O4/A1 standing out. Among the constitutive promoters, PmxaF exhibited the highest expression. Furthermore, overexpression of the PHA synthase gene (phaC) and the GroEL/ES chaperone complex resulted in a remarkable 24-fold increase in PHA production, especially when controlled by the PmxaF promoter. Co-overexpression of phaC and groEL/ES under the Ptuf promoter also indicated possibly promising synergistic effects. The development and integration of autolysis systems with exogenous holin/endolysin genes showed a significant reduction in cell viability, with Gp110 endolysin and EJ1 Holin/Endolysin displaying the most pronounced effects on cell integrity. These systems have potential applications in cell lysis processes. In addition, the assessment of solvent tolerance revealed that specific autolysis genes significantly reduced cell viability under solvent stress, confirming their influence on cell wall integrity. With these findings, we further and support the scientific developments made toward creating sustainable PHA production methods, with the ultimate goal of creating a more sustainable future.

References
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