Team:Wageningen UR/Wetlab/Auxotrophy

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Co-dependency

Co-dependency

Due to its proximity to cattle, the safety of the Cattlelyst biofilter is extremely important. Complementing the two safety circuits designed to ensure the containment of the genetically modified bacteria in the biofilter, this study offered promising preliminary results regarding the establishment of a cross-feeding community of Escherichia coli and Pseudomonas putida, based on amino acids exchange and carbon-source dependency. Specifically, auxotrophic and overproducing strains for tryptophane and tyrosine were characterized, and carbon-source dependency was shown to play an important role in the maintenance of the ratio between the bacterial species during co-culture.

Introduction

Cattlelyst aims at reducing the emission of both ammonia and methane from cattle stalls and is based on genetically modified bacteria able to efficiently metabolize ammonia and methane. Inside the biofilter, two synthetically engineered bacterial strains, a methanotroph and an ammoniatroph (bacterium able to metabolize ammonia) cooperate to purify the air. Due to the physical characteristics of the biofilter and its proximity to the surrounding natural environment, it is of utmost importance to guarantee the containment of the genetically modified bacterial strains used inside the biofilter. To answer this need, three complementary projects were carried out within the 2021 iGEM project. Specifically, two safety circuits – one based on the presence of methane and the other on a quorum sensing mechanism - were implemented inside the synthetically engineered bacterial strains. Nevertheless, in stressful conditions, the bacteria will be prone to mutate sequences essential for these circuits to operate and escape [1]. To complement the aforementioned measures, this project aimed at establishing a synthetic cross-feeding community of E. coli and P. putida based on amino acids exchange and on carbon-source dependency. This choice was based on the easiness to obtain auxotrophic strains for amino acids in laboratory conditions [2] and the fitness benefits that derive from the establishment of cross feeding communities based on amino acid auxotrophies [3], [4]. Codependency of E. coli and P. putida was designed to rely on the knock-out and overproduction of different amino acids in both bacteria. In this scenario, E. coli was knocked-out for amino acid 1 (AA1) and engineered to overproduce amino acid 2 (AA2), while P. putida was knocked-out for amino acid 2 (AA2) and modified to overproduce amino acid 1 (AA1)(i.e. cross-feeders) (Figure 1).

Carbon-source dependency

Carbon-source dependency was included to confer an additional layer of safety to the system. Here, P. putida was engineered to rely solely on E. coli for the carbon-source necessary to accumulate biomass (Figure 1). E. coli was shown to excrete acetate both during anaerobic and aerobic growth [5], and P. putida was proven to grow on acetate as sole carbon-source [6]. Thus, a P. putida strain not capable of using glucose as a carbon-source was used in this project. This strain presents a double knock-out for the operon gtsABCD (encoding the ATP-dependent ABC glucose transporter) and for the gene gcd (encoding the glucose dehydrogenase) [7]. The two knock-outs are reported to impede the uptake of glucose by preventing its transport through the inner membrane, mediated by the ABC transporter, and by blocking the peripheral oxidative route, which, through the action of Gcd, converts glucose to the intermediate D-gluconate. Altogether, the goal of this minor thesis project was to make E. coli and P. putida dependent on each other. The double auxotrophy and carbon-source dependency, together with the safety circuits, should ensure the containment of the genetically engineered bacteria inside the biofilter.

Figure 1: Cross-feeding community of E. coli and P. putida based on amino acid auxotrophy and carbon-source dependency. In this graphical representation, E. coli and P. putida are knocked-out respectively for amino acid 1 (AA1) and amino acid 2 (AA2) while overproducing the other amino acid. Auxotrophic and overproducing characteristics of the two bacteria make them cross-feeders. Co-culturing of the cross-feeders results in the reciprocal exchange of amino acids. In addition, E. coli is shown to secrete acetate, carbon-source on which P. putida can rely entirely for growth.

Approach

Literature study

A literature study was initially performed to determine which amino acids represent the best candidates to establish co-dependency between E. coli and P. putida. The criteria that were taken into consideration are presented below. Based on these criteria a comprehensive table was compiled (Results section - Table 1) and the most promising amino acids were selected.

• Criteria for the amino acids selection

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  In the literature study, several criteria were taken into consideration:

  • Amino acid composition of the bacteria, based on which priority was given to amino acids with a lower abundance in the bacteria;
  • Amino acids abundance in nature, so that priority was given to rare amino acids not easily found in the surrounding environment;
  • Amino acid ratio between yield/composition. Priority in this case was given to amino acids energetically cheap to produce or amino acids that, even though energetically costly, are not prevalent in the cell;
  • Hydrophobicity of the amino acid. Priority was given to amino acids with high hydrophobicity, which secretion could take place via passive diffusion without the need of specialized transport-ers;
  • Number of knock-outs needed to obtain the auxotrophy, with priority given to amino acid auxotrophies that can be obtained with a single knock-out;
  • Prevalence in literature. Priority was given to amino acid auxotrophies frequently described in literature as stable and easy to establish.

Titration experiments

Following the preliminary literature study, the six most promising amino acids (leucine, lysine, arginine, tryptophan, tyrosine and histidine) were subjected to experimental analysis to determine which amino acids are needed in the lowest concentrations to re-establish wild type (WT) growth of the respective E. coli auxotrophic strain. Six E. coli strains belonging to the KEIO collection [8], auxotrophic for the most promising amino acids were incubated in a microplate reader with increasing concentrations of the required amino acids (ranging from 0.5 μM to 100 μM). Growth curves and doubling times of each strain were generated and analyzed.

• List of E. coli strains used during this experiment

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  Genotype	Reference
  ΔargH	[8]
  ΔhisB	[8]
  ΔleuA	[8]
  ΔlysA	[8]
  ΔtyrA	[8]
  ΔtrpD	[8]

• Plate reader experiments

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  • Day 1

  Each auxotrophic and WT strain was inoculated in LB and grown overnight.

  • Day 2

  Overnight grown cultures of each auxotrophic strain were centrifuged at 6000g for 5 minutes and washed 3X in M9 medium. The auxotrophic cells were added either to M9 medium supplemented with glucose and increasing concentrations of the required amino acids (ranging from 0.5 μM to 100 μM) or to conditioned medium (CM). The cells were then added to a microplate with an initial OD600 of 0.005 and were grown for 24 hours at 37 °C (for E. coli) or 30 °C (for P. putida) with shaking. On each microplate, the same amino acids concentrations were added to E. coli WT strains as control.

  • Day 3

  Growth curves were generated and analyzed using GraphPad Prism. Doubling time was determined via the manual identification of the tangent at the inflection point of each growth curve. The angular coefficient μ of the tangent was then calculated and the doubling time was determined as: LN(2)/μ. The doubling times of each auxotrophic strain were normalized for the respective WT and graphically represented using GraphPad Prism.

Overproduction study

Following growth curve analysis, Trp, Tyr, and His were selected as the most promising amino acids to establish a cross-feeding community of E. coli and P. putida. The cross-feeding behavior requires each strain to be auxotrophic for one amino acid while simultaneously overproducing the other amino acid. In this context, two approaches were tested to select the best overproduction strategy: general overproduction and targeted overproduction.

• General
• Targeted

In this strategy, the knock-out of genes encoding for enzymes belonging to the TCA cycle - malate dehydrogenase (mdh) and phosphoenolpyruvate carboxylase (ppc) – was hypothesized to lead to the interruption of the cycle and the accumulation of intermediates of this pathway. This, in turn, was assumed to result in an increased production of a mixture of different amino acids deriving from the TCA cycle [3], that could sustain the growth of the partner auxotrophic strain.

In this strategy, the overproduction of the complementary amino acid was assumed to result from the elimination of the native feedback inhibition of the biosynthetic pathway of Trp, Tyr, and His.

E. coli overproducing strains belonging to the KEIO collection were tested for their ability to secrete amounts of amino acids high enough to sustain the growth of a biosensor strain auxo-trophic for the corresponding amino acid.

• List of E. coli strains used during this experiment

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  Genotype	Reference
  ΔhisL	[8]
  ΔtyrR	[8]
  ΔtrpR	[8]
  Δmdh	[8]
  Δppc	[8]

• Plate reader experiments

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  • Day 1

  The overproducing strains were separately grown overnight in 5 mL of M9 supplemented with glucose (0.4% m/vol).

  • Day 2

  The samples were then centrifuged at 3000g for 10 minutes and the supernatants were filter-sterilized with a 0.2 μm filter. The conditioned medium (CM) was then supplemented with 5X concentrated M9 and glucose (ex. 400 μL CM : 100 μL 5X M9). The relative biosensor strains were inoculated in each medium.

  • Day 3

  Growth of the biosensor strains was assessed.

Creation of cross-feeder strains

Following the overproduction study, Trp and Tyr were selected to establish the cross-feeding community of E. coli and P. putida. The overproduction of Tyr in Pseudomonas species was described to be complex in literature [9], while the overproduction of Trp was shown to be possible via the expression of an overproduction plasmid harboring a feedback resistant version of trpE (trpES40F) [10]. Therefore, P. putida was chosen to be auxotrophic for Tyr and overproducing Trp, while E. coli was designated to be auxotrophic for Trp and overproducing Tyr. In practice, E. coli and P. putida auxotrophic strains were firstly obtained and subsequently, overproduction was engineered on the auxotrophic strains genomic background.

1. Creation of auxotrophic strains

• E. coli auxotrophic strain

E. coli auxotrophic for Trp was obtained from the KEIO collection. Specifically, E. coli ΔtrpD was used.

• P. putida auxotrophic strain

The creation of an auxotrophic P. putida strain for Tyr requires the knock-out of two genes: tyrA and phhAB [11]. The knock-outs in P. putida EM42 were performed via a two-step deletion method relying on the integrative vector pGNW and on homologous recombination.

• Knock-outs of tyrA and phhAB

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  Figure 2: Knock-outs of tyrA and phhAB in P. putida EM42. Schematic representation of the biosynthetic pathways of Trp, Tyr, and Phe. TyrA and PhhA contribute to the biosynthesis of Tyr. Figure was adapted from [11].

• Plate reader experiments

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  • Preliminary

  Knock-outs in P. putida EM42 were performed via a two-step deletion method relying on homologous recombination, assisted by the integrative vector pGNW. pGNW plasmids harbor the ORI R6K, which needs the $pi; protein for plasmid replication. The $pi; protein is encoded by pirR6K, present in pir+ bacteria, such as DH5α λpir. 500bp were selected upstream (homology region 1 - H1) and downstream (homology region 2 - H2) of the genes to be deleted and amplified with primers bearing Golden Gate-compatible BsaI sites.

  • Day 1

  Golden Gate assembly was performed to insert the upstream and downstream homology regions (H1 and H2) in pGNW. After Golden Gate, competent E. coli DH5α λpir were transformed via heat-shock and plated on LB agar/kana50.

  • Day 2

  Following transformation, the colonies were tested via colony PCR. In case of successful transformation, the pGNW – H1H2 plasmid was transferred from E. coli DH5α λpir to P. putida EM42 via triparental conjugation.

  • Day 3

  After conjugation, P. putida EM42 colonies were grown overnight in LB to enable the chromosomal integration of pGNW – H1H2 via homologous recombination.

  • Day 4

  The cells were then transformed via electroporation with the pQURE6-H plasmid, bearing the I-SceI endonuclease under the XylS/Pm expression system. After electroporation, cells were recovered in LB medium supplemented with 3-Methylbenzoate (3-MB) for 1 hour at 30 °C and 300 rpm. The addition of 3-MB resulted in the expression of the I-SceI endonuclease, which cuts the integrated pGNW in the genome introducing a potentially lethal double strand break in the recombinants. This resulted in two possible outcomes: restoration of the wild type genotype or deletion of the gene of interest.

  • Day 5

  To screen for the correct mutation event the colonies were tested via PCR using sequence specific primers.

2. Creation and characterization of overproducing strains

• E. coli overproducing strain

The knock-out of tyrR was performed in the mutant genomic background of E. coli ΔtrpD following the λ-red protocol.

• Protocol λ-red for knock-outs in E. coli

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  • Preliminary

  The plasmid pSC020, harboring the λ-red operon under the control of an arabinose promoter (pAra) was isolated from an E. coli bearing strain.

  • Day 1

  pSC020 was transformed into E. coli BW25113 ΔtrpD via electroporation. Recovery was performed in LB medium at 30 °C, 500 rpm for 1 h. After recovery, the cells were plated in LB agar/Amp100 plates.

  • Day 2

  The linear fragment used to perform gene knock-out was composed of a cassette containing a gene encoding gentamycin resistance (genR), flanked by two 50 bp regions. The linear fragment was obtained by amplifying the genR cassette using primers harboring the specific 50 bp of homology with the target sequence.

  • Day 3

  The fragment was transformed into E. coli BW25113 ΔtrpD electrocompetent cells. The cell culture was supplemented with 0.01 M L-arabinose to induce the expression of the λ-red operon and allow homologous recombination to take place. After transformation, the cells were plated on LB agar/gen10 plates.

  • Day 4

  Resulting colonies were screened via colony PCR using sequence specific primers.

• P. putida overproducing strain

The overproduction of Trp in P. putida EM42 was designed to rely on the construction of an overexpression vector harboring trpES40F. The S40F mutation was chosen to remove the feed-back inhibition that high concentrations of Trp would cause on the TrpE enzyme.

• Protocol pSEVA64-trpES40F plasmid construction

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  • Preliminary

  The cloning procedure aimed at inserting trpE in pSEVA64 was initially designed in silico using Benchling and subsequently performed in the laboratory via Golden Gate assembly. The coding sequence was designed to be preceded by a constitutive promoter (BBa_J23100), a Ribosome Binding Site (RBS BBa_B0034) and to be terminated by the ribosomal RNA gene T1 terminator (rrnB).

  • Day 1

  The trpEG gene was amplified from WT E. coli MG1655 with sequence specific primers bearing BsaI sites on the overhangs. The amplification results were loaded on an electrophoresis agarose gel. The bands of interest were subsequently purified from gel.

  • Day 2

  Golden Gate assembly was performed to ligate the trpE gene within a pSEVA64 vector, bearing a gentamycin resistance cassette. The plasmid was then transformed in chemically competent E. coli DH5α cells via heat shock. Recovery was performed in LB medium at 37 °C, 500 rpm for 1 h. After recovery, the cells were plated on LB agar/gen10 plates.

  • Day 3

  Following overnight incubation at 37°C, single colonies were tested via colony PCR. The correct colonies were inoculated in 5 mL of LB/gen10 for overnight incubation.

  • Day 4

  Subsequently, plasmid isolation was performed. The S40F mutation was introduced in trpE via PCR. The linear PCR product, containing a 5’P-terminal residue was subjected to DpnI digestion and incubated for 1 hour at 37 °C. The digested product was then loaded on agarose gel, run for 30 mins at 100 V, and purified. Following gel extraction, the linear product was ligated overnight at 16 °C using the T4 ligase to obtain a circular vector.

  • Day 5

  The ligation mix was transformed into chemically competent E. coli DH5α cells via heat shock. The cells were then plated on LB agar/gen10 plates.

  • Day 6

  Single colonies were picked, DNA was isolated via MiniPrep and the plasmid was sequenced.

Growth curves to assess amino acids overproduction

The overproduction of Tyr was tested in the single mutant strains E. coli BW25113 ΔtyrR and the overproduction of Trp was assessed in P. putida EM42 ΔtyrA and P. putida EM42 ΔphhAB.

• Protocol amino acids overproduction assessment

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  • Day 1

  Each auxotrophic/biosensor, overproducing, and WT strain was inoculated in LB and grown overnight.

  • Day 2

  Two E. coli strains belonging to the KEIO collection, auxotrophic for Tyr and Trp were used as biosensors. Specifically, E. coli ΔtyrA was inoculated in each of the CM derived from the overnight growth of three biological replicates of E. coli ΔtyrR supplemented with 5X concentrated M9 and glucose. Similarly, E. coli ΔtrpD was inoculated in each of the CM derived from the overnight growth of three biological replicates of P. putida EM42 ΔtyrA and P. putida EM42 ΔphhAB supplemented with 5X concentrated M9 and glucose. In parallel, E. coli ΔtyrA and E. coli ΔtrpD were incubated with increasing concentrations of the required amino acids (ranging from 2 μM to 50 μM) to create a calibration curve.

  • Day 3

  Following incubation, the growth curves of each strain were generated and analyzed. To create the calibration curve, the final OD600 of each strain was plotted against the respective amino acid concentration. Because the amino acids overproduction was expected to fall within the 10-15 μM range, only the 2-25 μM range was considered to generate the calibration curves. In addition to the newly generated final OD600 values of the biosensor strains, the previously obtained values were plotted as well.

Carbon-source dependency characterization

In parallel to the establishment of an amino acids codependency between E. coli and P. putida, a carbon-source dependency was engineered to confer an ulterior layer of safety to the system. In this context, E. coli - shown to natively excrete acetate during aerobic growth due to overflowing metabolism - was used as carbon donor, while P. putida – shown to be able to grow on acetate as sole carbon-source – was the carbon receiver. Specifically, P. putida EM42 Δgts Δgcd, a strain solely reliant on acetate for biomass generation was used in this project. In preparation for the co-culture experiment, the method for determining the relative abundance of the two strains in co-culture was validated. Following validation, co-cultures of the two strains were set up.

• Validation of selective plating method

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  • Day 1

  E. coli and P. putida were individually grown overnight in LB.

  • Day 2

  The strains were washed 3X times in M9 and diluted to an OD600 of 1. P. putida EM42 Δgts Δgcd and E. coli ΔtrpD were combined in Eppendorf tubes in different ratios (1:100, 10:1, 1:1, 10:1, 100:1) and subsequently spread on selective plates. LB agar/cm25 plates were used to select for P. putida EM42 Δgts Δgcd due to the natural resistance of the bacterium to cm, while LB agar/kana50 plates were used to select for E. coli ΔtrpD due to the strain resistance to kana conferred by the replacement of trpD with a kanamycin resistance cassette. The plates were incubated overnight at 30°C.

  • Day 3

  The colonies on each plate were counted and a Pearson correlation test was performed to determine the presence of linear correlation between the initial ratios (t0) in tubes and the number of colonies found on the selective plates (t24).

• Protocol co-culture experiment

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  • Day 1

  E. coli and P. putida were individually grown overnight in LB.

  • Day 2

  The strains were washed 3X times in M9 and diluted to an OD600 of 1. E. coli ΔtrpD and P. putida EM42 Δgts Δgcd, and E. coli ΔtrpD and P. putida EM42 were co-inoculated in a 1:1 ratio at an initial OD600 of 0.01 in M9 with glucose (0.4%) and Tyr (100 μM). 100 μL of the co-culture was immediately plated on selective plates before overnight growth (t0). The remaining co-culture volume was left to grow overnight at 30 °C shaking.

  • Day 3

  The colonies on each t0 plate were counted, while the overnight grown co-culture was plated on selective plates (t24).

  • Day 4

  The colonies on each t24 plate were counted. Statistical difference between P. putida/E. coli ratios at t0 and t24 was calculated using a t-test.

Results

Literature study

Based on the criteria described in the Approach section, a comprehensive table was compiled and the most promising amino acids were selected. Ultimately, the preliminary literature study indicated that Trp and Arg are the most promising amino acids to establish a cross-feeding community of E. coli and P. putida.

Titration experiments

Overall, the amino acid needed in the lowest concentration to restore WT growth was Trp. The growth curves indicate that the addition of Trp – in the 25 μM to 50 μM range – to E. coli ΔtrpD would restore the final OD600 of the strain to values similar to the WT. Similarly, the addition of 50 μM of His almost reached the WT OD600 of E. coli ΔhisB, while the addition of 50 μM of Tyr to E. coli ΔtyrA restored more than half of the final OD600 reached by the WT. Despite exhibiting higher needed concentrations, Tyr was required in lower amounts than Leu, Lys, and Arg to restore the WT growth of the respective auxotrophic E. coli strains. Altogether, these results suggested that the amino acids that are needed in the lowest concentrations to re-establish WT growth in auxotrophic E. coli strains are Trp, His, and Tyr (Figure 3). These results are in accordance with the predictions obtained by PIPE. Specifically, PIPE was able to successfully predict that Arg is needed in higher concentrations than Trp to re-establish WT growth in E. coli auxotrophic strains.

• Growth curves

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  Figure 3: Growth curves of (A) E. coli ΔlysA, (B) E. coli ΔleuA, (C) E. coli ΔargH, (D) E. coli ΔhisB, (E) E. coli ΔtyrA, and (F) E. coli ΔtrpD. The X axis represents the time in hours while the Y axis depicts the OD600. Vertical bars represent the standard deviation of the three replicates.

In addition to the comparison of the final OD600 values, the doubling times of all the strains were calculated. Two amino acids concentrations - 25 μM and 50 μM - were considered when comparing the doubling times of all the auxotrophic strains (Figure 4). The doubling times were normalized for the WT and graphically represented to allow easier comparison. At 25 μM and 50 μM all the auxotrophic strains had a longer doubling time than the WT. Nevertheless, among the six amino acids, the addition of Trp, His, and Tyr conferred the lowest doubling times to the respective auxotrophic strains both at 25 μM and 50 μM (Figure 4). Therefore, the choice of Trp, His, and Tyr for the creation of E. coli and P. putida cross-feeding strains was further corroborated.

Figure 4:  Graphical representation of the doubling times of each auxotrophic strain normalized for the WT. Two amino acids concentrations are represented 25 uM (A) and 50 uM (B). Each bar represents the normalized doubling time. The horizontal line indicates the WT value.

Following the choice of Trp, His, and Tyr, further analysis were carried out on the growth curves of E. coli ΔtrpD, E. coli ΔhisB¸ and E. coli ΔtyrA. Specifically, the OD600 of the auxotrophic strains was shown to increase proportionally to the increase in concentration of the respective amino acids added to the medium (Figure 5). The statistical significance of the positive linear correlation was tested by means of a Pearson correlation test (Trp r = 0.977, p value < 0.001; Tyr r = 0.983 p value < 0.001; His r = 0.986, p value < 0.001).

• correlation between OD and respective amino acids

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  Figure 5: Positive linear correlation between the final OD600 and respective amino acids concentration. On the X axis is depicted the concentration while on the Y axis is represented the OD600. Vertical bars represent the standard deviation of the three replicates.

Overproduction strategy: general or targeted?

Overall, only the targeted overproducers E. coli ΔtyrR and E. coli ΔtrpR were shown to support the growth of the respective biosensors. The biosensors strains E. coli ΔtyrA and E. coli ΔtrpD growing on the CM of their overproducing strains showed an OD600 as high as 0.2 after 24 hours and higher than 1 after 48 hours of growth. Conversely, no growth could be detected for the biosensors inoculated on the CM of E. coli Δmdh and E. coli ΔhisL. Ultimately, based on these results, the targeted overproduction strategy was chosen to establish the cross-feeding community of E. coli and P. putida.

Creation of auxotrophic strains

The creation of an auxotrophic P. putida EM42 strain for Tyr necessitates the knock-out of two genes: tyrA and phhAB. After following the knock-out protocol, colony PCR was performed. The band size of the mutated genotype was expected to be ~1290 bp for ΔphhAB and ~1360 bp for ΔtyrA, while the WT – used as a negative control - was expected to be ~3000 bp in both cases. Electrophoresis of the PCR amplification revealed the presence of two P. putida EM42 colonies knocked-out for phhAB and seven colonies knocked-out for tyrA (Figure 6).

• Knock-outs

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  Figure 6: Knock-outs of tyrA and phhAB in P. putida EM42. Colony PCR performed on P. putida EM42 colonies obtained via homologous recombination.

  The knock-out of tyrR in E. coli ΔtrpD and the construction of the overproduction plasmid pSEVA64-trpES40F were unsuccessful
  In parallel to the creation of the P. putida EM42 mutant strains, the creation of an E. coli strain double mutant for trpD and tyrR was attempted. The knock-out of tyrR was performed in the mutant genomic background of E. coli ΔtrpD following the λ-red protocol. Nevertheless, PCR analysis of the obtained colonies did not show the expected results but bands with size comparable to the negative control. Unfortunately, due to time constraints, the creation of an E. coli ΔtrpD ΔtyrR strain could not be attempted again. Alongside the creation of E. coli and P. putida strains, the overproduction of Trp in P. putida EM42 was designed to rely on the construction of an overexpression vector harboring trpES40F. The S40F mutation has been extensively characterized and was chosen to remove the feedback inhibition that high concentrations of Trp would cause on the trpE enzyme. The trpE gene was therefore isolated from E. coli and cloned inside the pSEVA64 vector. trpE was successfully integrated via Golden Gate assembly in the pSEVA64 vector. Subsequently, the creation of the S40F mutation was carried out via PCR. Nevertheless, sequencing results showed the introduction of a deletion instead of the expected codon substitution. Similarly to the creation of double knock-outs in E. coli and P. putida, due to time constraints, the creation of the overexpression vector pSEVA64-trpES40F could not be fully carried out.

Overproduction

Despite the unsuccessful creation of the double mutant strains E. coli ΔtrpD ΔtyrR and P. putida EM42 ΔtyrA ΔphhAB , the overproduction of Tyr was tested in the single mutant strains E. coli ΔtyrR and the overproduction of Trp was assessed in P. putida EM42 ΔtyrA and P. putida EM42 ΔphhAB. Two E. coli strains belonging to the KEIO collection auxotrophic for Tyr and Trp were used as biosensors. To create the calibration curve, the final OD600 of each strain was plotted against the respective amino acid concentration. Because the amino acids overproduction was expected to fall within the 10-15 μM range, only the 2-25 μM range was considered to generate the calibration curves. In addition to the newly generated final OD600 values of the biosensor strains, the previously obtained values (Figure 5) were plotted as well. Previous and recent data were shown to fall within the same standard line (Figure 7), proving the consistency between the first and the present microplate experiment. To determine the concentration of Trp secreted by P. putida EM42 ΔtyrA and P. putida EM42 ΔphhAB , the final OD600 value of E. coli ΔtrpD - incubated in P. putida EM42 ΔtyrA and P. putida EM42 ΔphhAB CM – were interpolated in the Trp standard curve (Figure 7A – respectively pink and orange dots). Likewise, to determine the concentration of Tyr secreted by E. coli ΔtyrR, the final OD600 value of E. coli ΔtyrA - incubated in E. coli ΔtyrR CM - was interpolated in the Tyr standard curve (Figure 7B – yellow dot). Ultimately, E. coli ΔtyrR was shown to secrete 3.12 μM of Tyr, P. putida EM42 ΔtyrA was shown to secrete 1.8 μM of Trp, while P. putida EM42 ΔphhAB was reported not to secrete Trp.

Figure 7: Calibration curves used to determine the concentration of Trp (A) and Tyr (B) respectively secreted by E. coli ΔtyrR and P. putida ΔtyrA. Black dots represent the original values while blue colored dots represent the newly obtained values. The X axis represents the amino acid concentration, while the Y axis indicates the OD600 of the cultures. Pink and orange dots respectively indicate P. putida EM42 ΔtyrA and P. putida EM42 ΔphhAB CM, while the yellow dot indicates E. coli ΔtyrR CM.

Carbon-source dependency between E. coli and P. putida

In parallel to the establishment of an amino acids codependency between E. coli and P. putida, a carbon-source dependency was engineered to confer an ulterior layer of safety to the system.

Firstly, the selective plating method was found to be a reliable representation of the ratio of P. putida and E. coli found in tube. Specifically, a Pearson correlation test was performed to determine the presence of linear correlation between the initial ratios (t0) in tubes and the number of colonies found on the selective plates (Figure 8). Correlation was found (r = 0.99, p value < 0.01) and the selective plating method was subsequently used to assess the ratio of E. coli and P. putida in co-culture. P. putida EM42 and P. putida EM42 Δgts Δgcd exhibited different final ratios when co-cultured with E. coli ΔtrpD. No significant difference was found between the P. putida EM42 Δgts Δgcd or E. coli ΔtrpD ratio at t0 and t24, indicating that the co-culture maintained the same 1:1 ratio of the initial culture after 24 hours of growth. Conversely, the co-culture of P. putida EM42 and E. coli ΔtrpD showed a significant increase in P. putida abundance after 24 hours of growth, leading to a 4:1 ratio in favor of P. putida EM42 at the end of the co-culture experiment.

Figure 8: Graphical representation of the ratios of P. putida and E. coli in co-culture at t0 and t24. Each dark circle indicates a single replicate, while the bars indicate the mean of replicates at the time points indicated below the graph. The letter “P” indicates P. putida, while the letter “E” indicates E. coli. t0 = 0 hours, t24 = 24 hours. Only significant differences are plotted and are indicated with a ** (**p value < 0.01).

• Selective plating and growth curves

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  Figure 9:Correlation between tubes and selective plates. The X axis represents the log10 of the P. putida/E. coli ratio found in tube, while the Y axis represents the log10 of the P. putida/E. coli ration found after plating on selective plates.

  P. putida EM42 Δgts Δgcd presented minimal growth on glucose
  Alongside the co-culture experiments, P. putida EM42 Δgts Δgcd was subjected to ulterior analysis. P. putida EM42 and P. putida EM42 Δgts Δgcd growth on acetate followed a similar trend. Both strains were shown to grow in M9 supplemented with 0.2% and 0.5% w/v acetate but were unable to grow at 1% w/v and higher concentrations (Figure 10). Of particular interest is the growth of P. putida EM42 Δgts Δgcd on glucose. The OD600 of the strain was shown to increase proportionally to the increase in glucose concentrations, indicating the capability of P. putida EM42 Δgts Δgcd to grow on glucose. Nevertheless, the growth of P. putida EM42 Δgts Δgcd on glucose is remarkably lower than P. putida EM42.

  

  Figure 10:Growth curves of P. putida EM42 and P. putida EM42 Δgts Δgcd on acetate and glucose. The X axis represents the time in hours ranging from 0 to 30 hours. The Y axis represents the OD600 of each culture at each time point ranging from 0 to 1.5 (A-C). In graph D, the represented OD600 values range from 0 to 0.10, due to overall lower values.