Increasing cellular fitness and product yields in Pseudomonas putida through an engineered phosphoketolase shunt

doi:10.21203/rs.3.rs-1722450/v1

PDF

Research Article

Increasing cellular fitness and product yields in Pseudomonas putida through an engineered phosphoketolase shunt

https://doi.org/10.21203/rs.3.rs-1722450/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Pseudomonas putida as a cell factory is increasingly being used due to its remarkable features such as fast growth, versatile and robust metabolism, extensive genetic toolbox and high tolerance to oxidative stress and toxic compounds. This interest is driven by the need to improve microbial performance to a level that enables biologically possible processes to become economically feasible and thereby foster the transition from an oil-based economy to a more sustainable bio-based one. To this end, one of the current strategies is to maximize the product-substrate yield of an aerobic biocatalyst such as P. putida during growth on glycolytic carbon sources, such as glycerol and xylose. We demonstrate that this can be achieved by implementing the phosphoketolase shunt, through which pyruvate decarboxylation is prevented, and thus carbon loss is minimized.

Results

In this study, we introduced the phosphoketolase shunt in the metabolism of P. putida KT2440. To maximize the effect of this pathway, we first tested and selected a phosphoketolase (Xfpk) enzyme with high activity in P. putida. Results of the enzymatic assays revealed that the most efficient Xfpk was the one isolated from Bifidobacterium breve. Using this enzyme, we improved the P. putida growth rate on glycerol and xylose by 44 and 167 %, respectively, as well as the biomass yield quantified by OD_600nm by 50 and 30 %, respectively. Finally, we achieved 38.5 % and 25.9 % more mevalonate-glycerol and flaviolin-glycerol yield, respectively. A similar effect was observed on the mevalonate-xylose and flaviolin-xylose yields, which increased 48.7 and 49.4 %, respectively.

Conclusions

P. putida with the Xfpk shunt grew faster, reached higher final OD_600nm and provided better product-substrate yields. By reducing the pyruvate decarboxylation flux, we significantly improved the performance of this important workhorse for industrial applications. This work encompasses the first steps towards full implementation of the non-oxidative glycolysis (NOG) or the glycolysis alternative high carbon yield cycle (GATCHYC), in which a substrate is converted into products without CO₂ loss These enhanced properties of P. putida will be crucial for its subsequent use in a range of industrial processes.

There is a pressing need to transition to a sustainable and biobased economy (Staffas et al., 2013). The deployment of micro-organisms to produce chemicals currently derived from fossil fuels is a promising green alternative to this end (Paula & Birrer.,2006). One of the key challenges when using micro-organisms as cell factories is the optimization of the titer, rate, and yield (TRY) to make the production process competitive with the petrochemical industry (Nelsen and Kiesling, 2016).

Pseudomonas putida is an attractive host that is increasingly being developed as a cell factory for a wide range of biotechnological applications (Poblete-Castro et al., 2012 ; Martin-Pascual et al., 2021). Its high metabolic versatility, ability to tolerate environmental stresses and wide use of a variety of carbon sources make this bacterium one of the laboratory workhorses that can fulfil the demands of industrial biotechnology (Nikel et al., 2014; Ankenbauer et al., 2020).

In current industrial cultivation processes, feedstock costs remain one of the major bottlenecks (Caspeta and Nielsen, 2013). Therefore, the substrate to product ratio must be as close to the theoretical maximum as possible. Many high-value products, such as isoprenoids, butanol, and polyketides, are derived from the intermediate acetyl–CoA, a key compound connecting the glycolysis and the tricarboxylic acid (TCA) cycle (Cordova & Alper., 2016). P. putida, like many other bacteria grown on glycolytic carbon sources, produces acetyl-CoA through pyruvate decarboxylation. However, in this process, carbon is lost in the form of CO₂, lowering the theoretical carbon yield. Nature has cunning ways to resolve this problem by itself, through the usage of phosphoketolases (Xfpks). These enzymes are widely distributed among Bifidobacteria. These bacteria lack the aldolase and glucose-6-phosphate NADP⁺ oxidoreductase enzymes and use an alternative route to metabolize carbohydrates, in which phosphoketolases are key. These enzymes irreversibly cleave the sugar phosphates xylulose-5-phosphate (X5P) and fructose-6-phosphate (F6P) to glyceraldehyde-3-phosphate (G3P) and erythrose-4-phosphate (E4P) respectively, releasing an acetyl-phosphate (AcP) in the process. This AcP is then converted by the phosphotransacetylase (Pta) to acetyl-CoA, circumventing pyruvate carboxylation (Henard et al., 2017). One of the most famous examples of using this enzyme is the non-oxidative glycolysis (NOG) (Bogorad et al., 2013; Lin et al. 2018). The NOG pathway generates acetyl CoA from sugars or sugar phosphates without carbon loss. To such an end, the Xfpk catalyzes the two aforementioned reactions, in which E4P and G3P are formed. To achieve complete carbon conservation, three molecules of F6P are needed. They are broken down into three AcP and two E4P molecules. The E4P molecules need to undergo carbon rearrangement by the transaldolase and transketolase reactions to regenerate two molecules of F6P.

Recent metabolic engineering efforts have shown that the implementation of a phosphoketolase can improve carbon yields and the production of acetyl-CoA–derived products. For instance, the introduction of a phosphoketolase from B. adolescentis into E. coli showed an improved poly-β-hydroxybutyrate (PHB) yield (63.7%), fatty acid yield (14.36%) and mevalonate yield (64.3%) (Wang et al., 2019). Overexpression of the phosphoketolase gene from B. animalis in a C. glutamicum strain resulted in a 14% increase in the glutamic acid yield from glucose as well as suppression of CO₂ emission (Chinen et al., 2007). Similarly, Meadows et al., (2016) combined a xylulose-5-phosphate-specific phosphoketolase and three other heterologous enzymes to rewire the central carbon metabolism for more acetyl-CoA supply in S. cerevisiae, resulting in engineered strains that produce more farnesene and require less oxygen.

In this paper, we first characterized several Xfpk enzymes from Bifidobacteria species through in vitro assays. Secondly, we demonstrate that the introduction of the phosphoketolase shunt in P. putida KT2440 can improve biomass formation on glycerol and xylose, consuming less substrate in the process. At last, we prove that the carbon conserving pathway can significantly increase the yield of the acetyl-CoA derived compounds, such as malonyl-CoA and mevalonate.

Selecting the best phosphoketolase candidate

To maximize carbon conservation in P. putida, we introduced the phosphoketolase shunt. The introduction of the Xfpk will cleave the respective sugar phosphates F6P and X5P, and release AcP in the process, which can be directly converted to acetyl-CoA. We hypothesized that through this introduction, pyruvate decarboxylation would be circumvented, and the carbon loss minimized.

(Fig. 1).

To select a Xfpk candidate with high enzymatic activity, an in vitro assay was performed. We cloned all candidate genes (Xfpk from B. adolescentis, B. animalis and B. breve) under the control of a strong constitutive promoter (BBa_J23100) and RBS (0034) in the medium copy number pSEVAb83 vector. Functional expression of all enzymes was assessed by detection of AcP generated from F6P in cell-free extracts (Fig. 2). The Xfpk enzymes from B. adolescentis and B. animalis produced 1.26 and 1.19 mM AcP/OD₆₀₀, respectively. Both only produced amounts slightly higher compared to the empty vector control (0.80 mM). This suggests that both enzymes are active, yet do not cleave F6P with high efficiency. However, the Xfpk from B. breve displayed high reactivity towards F6P. An AcP concentration of 36.25 mM/OD₆₀₀ was measured, roughly 30-fold higher than the other two candidates. Therefore, the Xfpk from B. breve was selected for further experiments to assess growth and production.

Effect of the phosphoketolase on the glycerol metabolism of P putida KT2440

To determine whether expression of the Xfpk from B. breve impacts the fitness of P. putida, we assessed its effect on cells grown on glycerol. Glycerol is a major waste product of the biodiesel industry and has a higher degree of reduction than glucose, making it an excellent substrate for microbial fermentation (Nikel et al., 2014). To facilitate growth on glycerol, we constructed a glpR knockout mutant. This gene encodes a glycerol repressor and causes inconsistent lag-phases when cultured on glycerol (Nikel et al., 2015). Subsequently, P. putida ΔglpR strains, harbouring either an empty pSEVAb83 plasmid or the pSEVAb83 encoding the xfpk from B. breve, were grown in M9 minimal medium supplemented with 200 mM glycerol. The growth experiments revealed that Xfpk overexpression resulted in a 44.3% increase in specific growth rate from 0.12 to 0.18 h^− 1. Moreover, the introduction of the Xfpk increased the maximum OD_600nm from 4.4 to 6.6, an increase of 50% (Fig. 3B). We assumed that this increase could be an effect of enhanced glycerol consumption. However, both strains consumed glycerol to a similar extent, implying that most formed biomass is a direct result of carbon conservation (Fig. 3b). To use the phosphoketolase shunt to a further extent, we overexpressed the fructose-1,6-biphosphatase (Fbp) to increase the flux towards F6P. However, this did not have any added benefit over overexpressing the Xfpk alone (Data not shown).

Exploiting the phosphoketolase shunt for production of acetyl-CoA derived compounds from glycerol

After demonstrating the beneficial effect of the Xfpk on growth, we shifted to exploit this advantageous strain to produce acetyl-CoA derived compounds from glycerol. A wide variety of interesting valuable compounds can be derived from the intermediate acetyl-CoA e.g., isoprenoids, 1-butanol, and polyketides (Cordova & Alper., 2016). As described previously, in a normal glycolytic regime, acetyl-CoA is produced by pyruvate decarboxylation. In this conversion, carbon is lost in the form of CO₂, negatively impacting the yield of acetyl-CoA derived products. By rewiring the metabolic flux through the phosphoketolase shunt, this loss can be prevented, improving yields in the process. To assess this beneficial effect, we took two acetyl-CoA derived compounds, malonyl-CoA, and mevalonate, as a proof of concept. Malonyl-CoA is a malonic acid that can be used to produce fatty alcohols and is derived directly from acetyl-CoA by the enzyme acetyl-CoA carboxylase consisting of four subunits (AccABCD).

For easy detection of increased malonyl-CoA levels, we used the previously repurposed type III polyketide synthase RppA (Yang et al., 2018). This enzyme converts five molecules of malonyl-CoA to 1,3,6,8-tetrahydroxynapthaene, which is subsequently nonenzymatically oxidized to flaviolin. The produced flaviolin is red coloured by itself, allowing spectrophotometrically quantification. Within our experiments, we overexpressed both rppA as well as all the several native subunits in P. putida

The second acetyl-CoA derived compound, mevalonate, is a key compound in industrial biochemistry. It is a metabolic precursor for terpenoids, which can be used in production of cosmetics and biofuels (Yoon et al., 2009). Mevalonate is produced from acetyl-CoA in a three-step process. First one acetyl-CoA is converted to acetoacetyl-CoA. Secondly, a second acetyl-CoA will condense with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Finally, this HMG-CoA is converted to mevalonate. For our experiments, we used the mvaE and mvaS genes from Enterococcus faecalis. The mvaE gene encodes a bifunctional protein that catalyzes both the first and last reaction in the mevalonate production pathway (Yoon et al., 2009).

For the production experiments, we relocated the Xfpk to a pSEVA62b vector and all product expression genes were cloned under a constitutive J23100 promoter into a pSEVA23b vector.

The production experiments for malonyl-CoA revealed a yield of 0.02 and 0.03 g/g for P. putida ΔglpR with an empty plasmid and with the Xfpk shunt, respectively (Fig. 3C). Therefore, P. putida ΔglpR with the Xfpk shunt produced 38.5% more than the control. Similar results were obtained for mevalonate production, in which P. putida ΔglpR with an empty plasmid had a yield of 0.011 and with the Xfpk shunt of 0.015 g/g: 25.9% higher (Fig. 3D).

Effect of the phosphoketolase on engineered xylose metabolism in P putida KT2440

The Xfpk is a promiscuous enzyme, which besides cleaving the sugar-phosphate F6P, also cleaves X5P. As X5P is the breakdown product of xylose degradation, we hypothesized that introducing the phosphoketolase shunt could enhance growth on xylose. Xylose is a major constituent of hemicellulose which has been proposed as an alternative microbial feedstock (Scapini et al., 2021). Xylose utilization requires a combination of two genes, xylA encoding xylose isomerase and xylB encoding xylulokinase. Additional overexpression of xylE which encodes a xylose/H⁺ symporter has been described to improve growth on xylose even further (Dvorak & de Lorenzo, 2018; Elmore et al., 2020). The xylose utilization genes, derived from E. coli, were codon-optimized for P. putida using the Jcat tool. The xylABE genes alone and together with the xfpk were cloned in the low copy number vector pSEVAb62 under the expression of the strong constitutive BBa_J23100 promoter. Both plasmids were transformed in a P. putida strain with a Δgcd background. The gcd gene encodes a glucose dehydrogenase, which has been reported to break down xylose to xylonate, a dead-end product (Dvorak & de Lorenzo., 2018).

Plasmid born expression of the xylose utilization genes resulted in very long lag phases (> 312 h) and deviated growth patterns (Data not shown). Therefore, we decided to chromosomally express the operons. The xylose operons, both with and without the xfpk, and under the control of the constitutive Ptac promoter were chromosomally integrated into KT2440 Δgcd downstream of the PP_5322 gene, resulting in strains KT2440Δgcd: XylABE and KT2440Δgcd: XylABE-Xfpk. This locus has been described for its high basal expression, yet low impact on cellular fitness (Chaves et al., 2020). These new plasmid-free strains showed a reduced lag phase of 12 hours. Corresponding with the results of the glycerol experiment, strains expressing the Xfpk, showed a faster growth rate and higher cell density compared to their non-expressing counterpart. KT2440Δgcd: XylABE grew with a specific growth rate of 0.02 h^− 1 and reached the stationary phase after 216 hours at an OD_600nm of 5.73. On the contrary, KT2440Δgcd: Xyl-Xfpk had a specific growth rate of 0.05 h^− 1 and a final OD_600nm of 7.4; an increase of 167% and 30.2%, respectively. Moreover, KT2440Δgcd: XylABE-Xfpk reached a higher OD while using less substrate than its non-expressing counterpart, highlighting the major impact carbon conservation has (Fig. 4B). As with glycerol, we equipped the xylose strains with the plasmids to produce malonyl and mevalonate. The production experiments for malonyl-CoA revealed a yield of 0.08 and 0.12 g/g for KT2440Δgcd: XylABE and KT2440 Δgcd: XylABE-Xfpk, respectively (Fig. 4C). Therefore, KT2440 Δgcd: XylABE-Xfpk produced 49,4% more than the control. Similar results were obtained for mevalonate production, in which KT2440Δgcd: XylABE had a yield of 0.022 and KT2440 Δgcd: XylABE-Xfpk 0.042 g/g: 48,7% higher (Fig. 4D).

In this paper, we show that the introduction of the phosphoketolase shunt increased the cellular fitness of P. putida, enabling it to reach higher cell densities and growth rates. Additionally, yields were enhanced due to carbon conservation. Although we only showcase its beneficial effect on glycerol and xylose, we believe its catabolic repertoire can be further extended. Hemicellulose, of which xylose is a major constituent, contains other promising substrates that would profit from the phosphoketolase shunt, such as arabinose, mannose, and galactose (Scapini et al., 2021). For arabinose, Elmore et al., (2020) engineered recently both the oxidative and isomerase pathway in P. putida. The oxidative pathway breaks down arabinose to the TCA intermediate 2-ketoglutarate, whilst the isomerase pathway is analogous to the xylose pathway. Therefore, for the Xfpk shunt to be used to its maximum efficiency, only the isomerase pathway, which has X5P as product, would suffice. The other two substrates, mannose, and galactose, both break down to F6P, allowing full utilization of the Xfpk shunt as well.

The Xfpk shunt, as described in this paper, could be further enhanced towards a full NOG or the recently engineered GATCHYC (glycolysis alternative high carbon yield cycle) (Bogorad et al., 2013; Hellgren et al., 2020). Both cycles display full carbon conservation with a 100% acetyl-CoA yield from its designated substrate. However, the NOG pathway contains two bidirectional steps (transaldolase and transketolase), which kinetically limits the pathway (Anderson et al., 2017). On the other hand, the GATCHYC relies mostly on unidirectional steps and the promiscuous activity of the Xfpk, which would cleave the sugar-phosphate sedoheptulose-7-phosphate (S7P) as well. The Xfpk from B. breve has been reported to use X5P and F6P in a 3:2 ratio (Bergman et al., 2016). It is likely that it also possesses a side activity towards S7P, making implementation of the GATCHYC noteworthy. However, whereas full conversion of substrate to acetyl-CoA is a major benefit in both pathways, their major downfall is the insufficient formation of redox cofactors. To resolve this problem, alternative electron donors could potentially be added. One of the most promising candidates is formate, which can be directly derived by electrochemical reduction of CO₂ (Claassens et al., 2018). Moreover, it has already been showcased that supplementation of formate to P. putida KT2440 drastically increased NADH formation (Zobel et al., 2015). Therefore, a combination of this carbon conservation pathway with formate might yield a superior platform strain.

Another recently engineered route to overcome the redox limitation of the NOG is the EP-bifido pathway (Wang et al., 2019). This route was demonstrated with glucose as a carbon source and pushes the flux towards the oxidative pentose phosphate pathway. In this pathway, NADPH is generated at the expense of 1 CO₂. However, due to the introduction of the Xfpk, more carbon loss was eventually prevented, and product yield was enhanced. The metabolism of P. putida consists of a cyclic architecture, termed EDEMP, merged from the Enter-Doudoroff (ED), the Embden-Meyerhof-Parnas (EMP) and the Pentose Phosphate (PP) pathway (Nikel et al., 2015). This cycle recycles hexose phosphates, generating reducing equivalents in the form of NADPH. Replacing the EDEMP with the EP- bifido pathway could have great implications for the metabolism of P. putida as it would produce reducing equivalents whilst conserving carbon. This synergistic effect could further enhance product formation. Moreover, as NADPH is a well-known combater of oxidative stress, it could even further increase P. putida as an industrial workhorse (Nikel et al., 2021).

Native glycolytic routes are limited by low carbon efficiencies, largely due to carbon losses associated with pyruvate decarboxylation to acetyl-CoA. The introduction of the Xfpk shunt from Bifidobacteria offers a strategic bypass to redirect the main glycolytic flux to enhance carbon yields. In this paper, we screened the activities of three Xfpk enzymes in P. putida. Furthermore, we showcased the exemplary effect carbon conservation can have on the cellular fitness and production in P. putida when grown on glycerol or xylose. Altogether, this work provides a framework to further build upon this carbon conserving mechanism to strengthen P. putida as an industriophile.

Bacterial strains and growth conditions

P. putida and E. coli cultures were incubated at 30 ^oC and 37 ^oC respectively. For genetic modification and plasmid contraction, strains were cultured in Lysogeny broth (LB) medium containing 10 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract, unless otherwise indicated. For the preparation of solid media, 1.5% (w/v) agar was added. Antibiotics were used whenever required at the following concentrations: kanamycin (Km) 50 µg/ml, gentamycin (Gm) 10 µg/ml, chloramphenicol (Cm) 50 µg/ml and apramycin (Apra) 50 µg/ml. Flasks experiments were performed in 250 mL Erlenmeyer flasks filled with 25 mL of M9 media (Hartmans et al., 1989) or MOPS media and incubated in a rotary shaker at 200 rpm at 30 ^oC. Flaks were inoculated at a starting OD₆₀₀ of 0.1 from an overnight LB culture. Samples were taken at various time points for quantification of cell density and HPLC analysis.

Plasmid construction

DNA fragments were amplified using Q5 Hot start High Fidelity DNA Polymerase (New England Biolabs) and separated by electrophoresis using a 1% (w/v) agarose gel. DNA was purified by the NucleoSpin Gel and PCR clean up kit (Macharey-Nagel, Germany). Plasmids were constructed using Golden Gate using the SEVA assembly protocol or through Gibson Assembly (Damalas et al., 2019; Gibson et al., 2010). The phosphoketolase genes were obtained from the genomic DNA of the Bifidobacterium strains: B. animalis, B. adolescentis, B. breve. The rppA gene and all accA-D genes were taken from an in-house plasmid (Batianis et al., unpublished). The mvaE and mvaS genes were derived from pSMART-MEV1 ordered from Addgene. All plasmids were transformed via heat shock in chemically competent E. coli Dh5α λpyr and transformants were selected on LB plates with corresponding antibiotics. Colonies were screened by colony PCR using Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific Inc. Waltheim, MA, USA). The plasmids from successful screenings were extracted and verified by Sanger DNA sequencing (MACROGEN Inc, the Netherlands). Correct plasmids were transformed into P. putida using electroporation.

Strain construction

Gene knockouts were constructed using the protocol previously described in Wirth et al., 2020. First, regions of approximately 500 bp upstream and downstream of the target genes were amplified from the genomic DNA of P. putida KT2440. The TS1 and TS2 fragments were cloned into the non-replicative pGNW plasmid and propagated into E. coli Dh5α λpir. Positive colonies were transformed into P. putida via electroporation and successful cointegrations were screened by PCR. The second plasmid pQURE6-H (Volke et al., 2020) was introduced to the selected co-integrate. The transformants were grown on an LB agar plate with Gm and induced with 2 mM 3-methylbenzoic acid (3-mBz). The 3-mBz was used to induce the XylS-dependent Pm promoter, driving the expression of the I-SceI homing nuclease. Deletions were confirmed by colony PCR. Successful recombinants were cured of pQURE6-H by omitting 3-mBz from the medium and selected for antibiotic sensitivity.

Crude cell extraction

Cell-free extracts were obtained from 25 ml of culture broth in 50 ml Greiner Tubes and cultivated at 30°C, 200 rpm orbital shaking until an OD600 of ca. 0.5–0.6. The cultures were centrifugated at 4,000 rpm for 10 min at 4ºC. Cell pellets were washed once with 50 ml of precooled 1X PBS buffer (pH = 7.0) with 10 mM 2-mercaptoethanol at 4ºC. The pellets were resuspended in 750 uL of pre-cooled 1X PBS buffer (pH = 7.0) with 10 mM 2-mercaptoethanol. After that, the cell suspension was transferred to a pre-chilled tube with 0.5 mm silica beads and homogenized using a FastPrep®-24 (MP Biomedicals, SantaAna, CA, USA) (2 cycles of 30 s, 5 min resting on ice in between runs). The homogenized mixture was centrifuged at 7500 x g for 30 min at 4°C to remove insoluble cell debris. The cell-free extracts were stored at − 20 ºC for further use.

Phosphoketolase activity assay

Phosphoketolase activity was measured using the ferric hydroxamate method, based on the chemical conversion of enzymatically produced acetyl-phosphate into ferric acetyl hydroxamate, according to the protocol from Wang et al., 2019. The standard reactions were carried out in 1.5 mL of Eppendorf tube in a total assay volume of 100 µL consisting of 50 mM Tris (pH 7.5), 5 mM MgCl₂, 5 mM potassium phosphate, 1 mM thiamine pyrophosphate and 10 mM F6P as a substrate. Crude cell-free extract was added to start the reaction and incubated at 30°C for 1 hour. To stop the enzymatic reaction, 60 µL hydroxylamine hydrochloride (2 M, pH 6.5) was added to 40 uL of assay solution. After incubation for 10 min at room temperature, 120 µL colouring reagent consisting of 15% (w/v) trichloroacetic acid, 4 M HCl, and FeCl₃•6H₂O (5% [w/v] in 0.1 M HCl) were added to generate ferric hydroxamate, which was then spectrophotometrically quantified at 505 nm by comparing to a series of lithium potassium acetyl-phosphate standards (Sigma).

Analytical Methods

Extracellular metabolite concentrations were determined by high-performance liquid chromatography (HPLC). Glycerol, xylose and mevalonate concentrations were detected and quantified using an ICS5000 HPLC (Thermo Scientific) with a refractive index detector (Shodex RI-101, sample frequency 5 Hz) and a Thermo UV/VIS detector (λ = 210nm) coupled to an Animex HPX-87H column (BioRad) at 60 ̊C. Separations were performed using 0.016 N H₂SO₄ as an eluate at a flow rate of 0.6 mL/min. Culture samples were centrifuged at 16,000 x g for 15 min. Supernatant and standards were mixed with 6 mM propionic acid as an internal standard at a ratio of 4:1.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors approved the manuscript.

Availability of data and materials

All data generated and analysed in this study are included in this article and supplementary file.

Competing interests

The authors declare no conflict of interest.

Funding

This work was financed by the European Union’s Horizon2020 Research and Innovation Programme under grant agreement Nos. 635536 (EmPowerPutida) and 730976 (IBISBA) to V.A.P.M.d.S. Additionally, this work was also part of the research program Putida for plastics supported by the Dutch Research Council (NWO) [GSGT.2019.028]. Funding for open access charge: Dutch Research Council.

Authors contributions

L.B. conceptualization; L.B., M.M.P., K.K., M.T and S.H. methodology; L.B. and M.M.P. writing-initial draft; L.B., M.M.P., K.K., M.T, S.H., R.v.K and V.A.P.M.d.S. writing-review and editing; R.v.K. and V.A.P.M.d.S. supervision.

Andersen, J. L., Flamm, C., Merkle, D., & Stadler, P. F. (2017). Chemical transformation motifs—Modelling pathways as integer hyperflows. IEEE/ACM transactions on computational biology and bioinformatics, 16(2), 510–523.
Ankenbauer, A., Schäfer, R. A., Viegas, S. C., Pobre, V., Voß, B., Arraiano, C. M., & Takors, R. (2020). Pseudomonas putida KT2440 is naturally endowed to withstand industrial-scale stress conditions. Microbial biotechnology, 13(4), 1145–1161.
Bagdasarian, M. A., Lurz, R., Rückert, B., Franklin, F. C. H., Bagdasarian, M. M., Frey, J., & Timmis, K. N. (1981). Specific-purpose plasmid cloning vectors II. Broad host range, high copy number, RSF 1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene, 16(1–3), 237–247.
Bergman, A., Siewers, V., Nielsen, J., & Chen, Y. (2016). Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae. Amb Express, 6(1), 115.
Bogorad, I.W., Lin, T.-S., Liao, J.C., 2013. Synthetic non-oxidative glycolysis enables complete carbon conservation. Nature 502, 693–697. https://doi.org/10.1038/ nature12575.
Caspeta, L., & Nielsen, J. (2013). Economic and environmental impacts of microbial biodiesel. Nature biotechnology, 31(9), 789–793.
Chaves, J. E., Wilton, R., Gao, Y., Munoz, N. M., Burnet, M. C., Schmitz, Z., … Michener, J. K. (2020). Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design. Metabolic engineering communications, 11, e00139.
Chinen, A., Kozlov, Y. I., Hara, Y., Izui, H., & Yasueda, H. (2007). Innovative metabolic pathway design for efficient l-glutamate production by suppressing CO2 emission. Journal of bioscience and bioengineering, 103(3), 262–269.
Claassens, N. J., Sánchez-Andrea, I., Sousa, D. Z., & Bar-Even, A. (2018). Towards sustainable feedstocks: A guide to electron donors for microbial carbon fixation. Current opinion in biotechnology, 50, 195–205.
Cordova, L. T., & Alper, H. S. (2016). Central metabolic nodes for diverse biochemical production. Current opinion in chemical biology, 35, 37–42.
Damalas, S. G., Batianis, C., Martin-Pascual, M., Lorenzo, V., & Martins dos Santos, V. A. P. (2020). SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial Biotechnology, 13(6), 1793–1806. https://doi.org/10.1111/1751-7915.13609
Dvořák, P., & de Lorenzo, V. (2018). Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose. Metabolic engineering, 48, 94–108.
Elmore, J. R., Dexter, G. N., Salvachúa, D., O’Brien, M., Klingeman, D. M., Gorday, K., Michener, J. K., Peterson, D. J., Beckham, G. T., & Guss, A. M. (2020). Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose, xylose, arabinose, p-coumaric acid, and acetic acid. Metabolic Engineering, 62, 62–71. https://doi.org/10.1016/j.ymben.2020.08.001
Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R. Y., Algire, M. A., … Venter, J. C. (2010). Creation of a bacterial cell controlled by a chemically synthesized genome. science, 329(5987), 52–56.
Grant, S. G., Jessee, J., Bloom, F. R., & Hanahan, D. (1990). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proceedings of the National Academy of Sciences, 87(12), 4645–4649.
Hartmans, S., Smits, J. P., van der Werf, M. J., Volkering, F. et al., Metabolism of styrene oxide and 2-phenylethanol in the styrene-degrading Xanthobacter strain 124X. Appl. Environ. Microbiol. 1989, 55, 2850–2855.
Hellgren, J., Godina, A., Nielsen, J., & Siewers, V. (2020). Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products. Metabolic Engineering, 62, 150–160.
Henard, C. A., Smith, H. K., & Guarnieri, M. T. (2017). Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst. Metabolic engineering, 41, 152–158.
Herrero, M., De Lorenzo, V., & Timmis, K. N. (1990). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. Journal of bacteriology, 172(11), 6557–6567.
Lin, P.P., Jaeger, A.J., Wu, T.-Y., Xu, S.C., Lee, A.S., Gao, F., Chen, P.-W., Liao, J.C., 2018. Construction and evolution of an Escherichia coli strain relying on nonoxidative glycolysis for sugar catabolism. Proc. Natl. Acad. Sci. U.S.A. 115, 3538–3546. https://doi.org/10.1073/pnas.1802191115.
Martin-Pascual, M., Batianis, C., Bruinsma, L., Asin-Garcia, E., Garcia-Morales, L., Weusthuis, R. A., … Dos Santos, V. A. M. (2021). A navigation guide of synthetic biology tools for Pseudomonas putida. Biotechnology Advances, 107732.
Meadows, A. L., Hawkins, K. M., Tsegaye, Y., Antipov, E., Kim, Y., Raetz, L., … Zhao, L. (2016). Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature, 537(7622), 694–697.
Nielsen, J., & Keasling, J. D. (2016). Engineering cellular metabolism. Cell, 164(6), 1185–1197.
Nikel, P. I., Chavarría, M., Fuhrer, T., Sauer, U., & De Lorenzo, V. (2015). Pseudomonas putida KT2440 strain metabolizes glucose through a cycle formed by enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways. Journal of Biological Chemistry, 290(43), 25920–25932.
Nikel, P. I., Fuhrer, T., Chavarría, M., Sánchez-Pascuala, A., Sauer, U., & de Lorenzo, V. (2021). Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress. The ISME journal, 15(6), 1751–1766.
Nikel, P. I., Kim, J. & de Lorenzo, V. Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440. Environ. Microbiol. 16, 239–254 (2014).
Nikel, P. I., Martínez-García, E., & De Lorenzo, V. (2014). Biotechnological domestication of pseudomonads using synthetic biology. Nature Reviews Microbiology, 12(5), 368–379.
Nikel, P. I., Romero-Campero, F. J., Zeidman, J. A., Goñi-Moreno, Á., & de Lorenzo, V. (2015). The glycerol-dependent metabolic persistence of Pseudomonas putida KT2440 reflects the regulatory logic of the GlpR repressor. MBio, 6(2), e00340-15.
Paula, L., & Birrer, F. (2006). Including public perspectives in industrial biotechnology and the biobased economy. Journal of Agricultural and Environmental Ethics, 19(3), 253–267.
Poblete-Castro, I., Becker, J., Dohnt, K., Dos Santos, V. M., & Wittmann, C. (2012). Industrial biotechnology of Pseudomonas putida and related species. Applied microbiology and biotechnology, 93(6), 2279–2290.
Scapini, T., Dos Santos, M. S., Bonatto, C., Wancura, J. H., Mulinari, J., Camargo, A. F., … Treichel, H. (2021). Hydrothermal pretreatment of lignocellulosic biomass for hemicellulose recovery. Bioresource Technology, 126033.
Staffas, L., Gustavsson, M., & McCormick, K. (2013). Strategies and policies for the bioeconomy and bio-based economy: An analysis of official national approaches. Sustainability, 5(6), 2751–2769.
Volke, D. C., Friis, L., Wirth, N. T., Turlin, J., & Nikel, P. I. (2020). Synthetic control of plasmid replication enables target-and self-curing of vectors and expedites genome engineering of Pseudomonas putida. Metabolic engineering communications, 10, e00126.
Wang, Q., Xu, J., Sun, Z., Luan, Y., Li, Y., Wang, J.,.. . Qi, Q. (2019). Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO2 emission. Metabolic engineering, 51, 79–87.
Wirth, N. T., Kozaeva, E., & Nikel, P. I. (2020). Accelerated genome engineering of Pseudomonas putida by I- SceI―mediated recombination and CRISPR-Cas9 counterselection. Microbial Biotechnology, 13(1), 233–249. https://doi.org/10.1111/1751-7915.13396
Yang, D., Kim, W. J., Yoo, S. M., Choi, J. H., Ha, S. H., Lee, M. H., & Lee, S. Y. (2018). Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proceedings of the National Academy of Sciences, 115(40), 9835–9844.
Yoon, S.-H., Lee, S.-H., Das, A., Ryu, H.-K., Jang, H.-J., Kim, J.-Y.,.. . Kim, S.-W. (2009). Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. Journal of biotechnology, 140(3–4), 218–226.
Zobel, S., Kuepper, J., Ebert, B., Wierckx, N., & Blank, L. M. (2017). Metabolic response of Pseudomonas putida to increased NADH regeneration rates. Engineering in Life Sciences, 17(1), 47–57.

No competing interests reported.

Supplementarydata.docx

PDF

Editorial decision: Major revision
19 Jun, 2022
Reviews received at journal
08 Jun, 2022
Reviewers agreed at journal
06 Jun, 2022
Reviewers invited by journal
05 Jun, 2022
Editor assigned by journal
04 Jun, 2022
Submission checks completed at journal
04 Jun, 2022
First submitted to journal
03 Jun, 2022

You are reading this latest preprint version

Increasing cellular fitness and product yields in Pseudomonas putida through an engineered phosphoketolase shunt

Status:

Version 1

Abstract

Figures

Background

Results

Selecting the best phosphoketolase candidate

Exploiting the phosphoketolase shunt for production of acetyl-CoA derived compounds from glycerol

Discussion

Conclusion

Material & Methods

Bacterial strains and growth conditions

Plasmid construction

Strain construction

Crude cell extraction

Phosphoketolase activity assay

Analytical Methods

Declarations

References

Competing Interests

Supplementary Files

Status:

Version 1