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 OD600nm of 5.73. On the contrary, KT2440Δgcd: Xyl-Xfpk had a specific growth rate of 0.05 h− 1 and a final OD600nm 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).