Engineered Passive Glucose Uptake in Pseudomonas taiwanensis VLB120 Increases Resource Efficiency for Bioproduction

Abstract

Glucose is the most abundant monosaccharide and a principal substrate in biotechnological production processes. In Pseudomonas, this sugar is either imported directly into the cytosol or first oxidised to gluconate in the periplasm. While gluconate is taken up via a proton-driven symporter, the import of glucose is mediated by an ABC-type transporter, and hence both require energy. In this study, we heterologously expressed the energy-independent glucose facilitator protein (Glf) from Zymomonas mobilis to replace the native energy-demanding glucose transport systems, thereby increasing the metabolic energy efficiency. The implementation of passive glucose uptake in engineered production strains significantly increased product titres and yields of the two different aromatic products, cinnamic acid (+10%-15%) and resveratrol (+26%; 18.1 mg/g) in batch cultures.

Keywords: Pseudomonas; ATP consumption; glucose transport; metabolic engineering; strain optimization.

FIGURE 1

Engineered glucose uptake in P. taiwanensis . (A) Overview of native and (B) engineered glucose transporters in P. taiwanensis VLB120. Energetic drivers are illustrated according to the mechanism of transport indicated by asterisks. (C) Genetic organisation of glucose uptake‐encoding genes in wild‐type and engineered P. taiwanensis VLB120. 'gtsD, remaining 32 bp of truncated coding sequence of native GtsD; EDEMP cycle, Entner–Doudoroff‐Embden‐Meyerhof‐Parnas‐pentose phosphate cycle; Gcd, PQQ‐dependent glucose dehydrogenase; Glf_Zm, glucose facilitator protein from Zymomonas mobilis ; Glk, glucokinase; Gnl, gluconolactonase; GnuK, d‐gluconate kinase; GtsABCD, glucose ABC transporter; GntP, d‐gluconate transporter; OMP, outer membrane porins; oprB‐I, coding sequence of a carbohydrate‐selective porin; Pgl, 6‐phosphogluconolactonase; P _gts , presumed promoter region (200 bp upstream of the start codon of gtsA PVLB_20095–80); PQQ, pyrroloquinoline quinone; Zwf, Glucose‐6‐phosphate dehydrogenase (Nikel et al. 2015).

FIGURE 2

Biomass yields and kinetics of different Glf_Zm strains. (A) Final biomass yields (g_biomass/g_glucose) resulting from the determined cell dry weight of P. taiwanensis VLB120 GRC3, GRC3Δgcd and GRC3Δ6MC‐III with either replaced glucose transporter gene gtsABCD by Zm_glf indicated by Δ for GtsABCD or with Zm_glf expression from landing pad PVLB_06360/65. Grown in shaken‐flask cultures with 30 mM (5.4 g L⁻¹) glucose and 3‐fold buffered MSM for 24 h with inoculation of 1% (v/v) from the adaption culture. (B) Growth rate of the same strains determined in Growth Profiler experiment from OD₆₀₀ equivalent values obtained in 3‐fold buffered MSM with 20 mM glucose (Figure S5). Error bars represent the standard deviation (n = 3 in A or n = 4 in B). Statistical analysis was made by 1‐way ANOVA (ns, p > 0.05; *, p < 0.05; **, p < 0.01; ****, p < 0.0001). Ns, not significant; Y_X/S, biomass yield; μ, growth rate.

FIGURE 3

(A) Schematic overview of cinnamate biosynthesis and (B) cinnamate production by different Glf_Zm strains. Cinnamate titre and OD₆₀₀ of GRC3 PHE and GRC3 PHE Δgcd with markerless cinnamate production module (attTn7::FRT‐P _14f ‐AtPAL2) with either replaced glucose transporter genes gtsABCD by Zm_glf or with Zm_glf expression from landing pad PVLB_06360/65. Strains were grown in 24‐square deep well plate with 30 mM (5.4 g L⁻¹) glucose, 3‐fold buffered MSM, sampled after 115 h in stationary phase. Error bars represent the standard deviation (n = 4). Statistical analysis was made by 2‐way ANOVA (ns, p > 0.05; **, p < 0.01; ****, p < 0.0001). E4P, erythrose‐4‐phoasphate; EDEMP cycle, Entner‐Doudoroff‐Embden‐Meyerhof‐Parnas‐pentose phosphate cycle; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway.

FIGURE 4

(A) Schematic overview of resveratrol biosynthesis and (B) resveratrol production by different Glf_Zm strains. Resveratrol and p‐coumarate titre of GRC3Δ6MC‐III with stilbene module (attTn7::FRT‐P _14f ‐his.AhSTS‐Sc4CL^A294G ) with either replaced glucose transporter gene gtsABCD by Zm_glf or with additional Zm_glf expression from landing pad PVLB_06360/65. Grown in 24‐square deep well plate with 30 mM (5.4 g L⁻¹) glucose, 3‐fold buffered MSM and 1 mM p‐coumarate for 24 h. Error bars represent the standard deviation (n = 3). Statistical analysis was made by 2‐way ANOVA (****, p < 0.0001). EDEMP cycle, Entner‐Doudoroff‐Embden‐Meyerhof‐Parnas‐pentose phosphate cycle.