Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

Abstract

Background: Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.

Results: Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C(10):C(10)). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/g(glucose) corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.

Conclusions: A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

Figure 1

Rhamnolipid biosynthesis pathway in P. putida. All enzymatic steps required for the synthesis of essential precursors including host-intrinsic enzymes and exogenous RhlA and RhlB are shown. *) Annotated in P. putida as XanA [31].

Figure 2

Toxicity studies with di-rhamnolipids using different bacteria. (a) Inhibiting effect of di-rhamnolipids on E. coli. Di-rhamnolipid concentrations tested were 0 g/L (■), 5 g/L (▲), 10 g/L (♦), 25 g/L (□), 50 g/L (○), and 90 g/L (◊). (b) Inhibiting effect of di-rhamnolipids on B. subtilis. Di-rhamnolipid concentrations tested were 0 mg/L (■), 2.5 mg/L (●), 5 mg/L (▲), 10 mg/L (♦), 25 mg/L (□), 50 mg/L (○), 75 mg/L (Δ), and 90 mg/L (◊). (c) Inhibiting effect of di-rhamnolipids on C. glutamicum. Di-rhamnolipid concentrations tested were 0 mg/L (■), 2.5 mg/L (●), 5 mg/L (▲), 10 mg/L (♦), 25 mg/L (□), 50 mg/L (○), 75 mg/L (Δ), and 90 mg/L (◊). (d) Growth rates resulting from toxicity experiments with B. subtilis (■) and C. glutamicum (●). (e) Growth rates resulting from toxicity experiments with E. coli.

Figure 3

Theoretical yields in rhamnolipid production with P. putida under different conditions. (a) Rhamnolipid yields on alternative carbon substrates. The yields were calculated for zero growth (black bars), zero growth and 30 mmol ATP/(g_CDW h) maintenance metabolism (white bars), growth at a rate of 0.4 1/h with 30 mmol ATP/(g_CDW h) maintenance metabolism (gray bars), and a growth rate of 0.8 1/h with 50 mmol/(g_CDW h) maintenance metabolism (shaded bars). The substrate uptake rates were constrained to 120 mCmol/(g_CDW h). (b) Rhamnolipid production capacity depending on biomass formation and maintenance metabolism for alternative carbon substrates. The course of rhamnolipid production for uptake of glucose (■), glycerol (●), sucrose (▲) and octanoate (♦) are displayed. The black curves display rhamnolipid production in dependence on the rate of growth; grey dashed curves display rhamnolipid production in dependence of maintenance metabolism.

Figure 4

Scheme of utilized pathways during rhamnolipid production by P. putida. ED, Entner-Doudoroff pathway; PP, pentose phosphate pathway; PDC, pyruvate decarboxylation; G1P, glucose-1-phosphate; HAA, β-D-(β-D-hydroxyalkanoyloxy)alkanoic acid; RhlA, 3-hydroxyacyl-ACP:3-hydroxyacyl-ACP O-3-hydroxyacyltransferase; RhlB, rhamnosyltransferase I.

Figure 5

Thin layer chromatography of rhamnolipids. The sample of P. aeruginosa PAO1 (lane 2), grown in PPGAS-medium at 37°C, contains mono- and di-rhamnolipid as does the commercial rhamnolipid (JBR425, Jeneil Biosurfactant Co.) (lane 1). P. putida, expressing the rhlAB operon of P. aeruginosa from the plasmid pVLT33_rhlAB, cultivated in glucose containing LB-medium produced mono-rhamnolipid (lane 4). Empty vector control pVLT33 (lane 3). The band located above the di-rhamnolipids is IPTG (i.e., lanes 3 and 4).

Figure 6

Molecular structure of mono-rhamnolipids from recombinant P. putida. The major compound (C₁₀:C₁₀) is indicated by thick black lines, while minor compounds are indicated by thin lines.

Figure 7

Percentage distribution of compounds gained by hydrophobic adsorption from the supernatant of a fermentation of P. putida KT42C1 pVLT31_rhlAB.

Figure 8

Uncoupling of rhamnolipid production and cell growth of P. putida in a 50 mL baffled flask. (a) Fermentation characteristics including cell growth (■) and course of rhamnolipid (▲) and glucose (♦) concentrations and their respective fitted courses. CDW, cell dry weight. (b) Specific rates resulting from the fitted experimental data. The black line represents the course of the growth rate, while the dashed line and the dotted line show the specific glucose uptake rate and the specific rhamnolipid production rate respectively.

Figure 9

Kinetics of rhamnolipid-production in P. putida in a stirred 300 mL reactor. (a) Development of biomass (■) and glucose (▲) concentrations. The experimental data is depicted by symbols, while the lines present the fits using Equations (1) to (3). CDW, cell dry weight. (b) Specific rates characterizing rhamnolipid production in P. putida. The solid line presents the measured ¹³CO₂ production rate originating from ¹³C-labeled glucose. The dashed line represents the glucose uptake rate and was calculated from the fitted glucose concentration curve. The dotted line shows the rate of CO₂ evolution, produced in the rhamnolipid synthesis pathway. The curve was calculated from the experimentally determined yield of rhamnolipid on glucose and the estimated rate of glucose consumption.