Driven By Demand Metabolic Engineering - Recombinant Rhamnolipid Synthesis in Pseudomonas Putida As an Example

Metabolic engineering of secondary metabolite producers implicitly relies on high flux through central carbon metabolism. This high flux caused by the demand for carbon and energy for the synthesis of the molecule of interest, however is rarely matched, requiring substantial improvements of central carbon metabolism operation.

It was recently shown that central carbon metabolism of Pseudomonas putida is metabolically regulated, while for example the carbon substrate degradation pathways beta-ketoadipate and Entner-Doudoroff are transcriptionally regulated [1]. From this finding we deduce a metabolic engineering strategy that relies on metabolic demand:

“High transcriptional activity in a peripheral pathway that is translated into high enzymatic activity (high metabolic demand) allows significant rerouting of metabolic resources to the product of choice”.

Indeed, we have evidence from extreme growth conditions (e.g., growth in the presence of a second phase of octanol) that P. putida can match an increased metabolic demand by tripling the glucose uptake rate without producing any side products, thus only biomass and CO₂ [2]. We tested this metabolic engineering strategy using rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways: Fatty acid de novo synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-) rhamnose, respectively. Hence, opposing to single pathway molecules, rhamnolipid synthesis causes demand of two central carbon metabolism intermediates; glucose-6-phosphate is required for rhamnose synthesis, while the lipid moiety is derived from lipid de novo synthesis, thus consuming acetyl-CoA.

We previously presented a P. putida KT2440 able to produce up to 0.2 g/L rhamnolipids by recombinant expression of the two responsible genes (rhlAB) from Pseudomonas aeruginosa [3]. Here we show that we could raise rhamnolipid concentration to greater than 3 g/L. Following the above sketched strategy of driven by demand, a synthetic promoter library was developed using an approach reported in literature based on degenerated primers [4]. This technique yields an array of transcriptional activity, potentially allowing the identification of optimal enzyme activity for high flux towards the rhamnolipid synthesis. The best producing strain was able to reach a titer of 3 g/L with a yield of 40% [Cmol_RL/Cmol_Glc], which is around 60% of the theoretical yield. While native producer P. aeruginosa synthesizes 40 g/L [5], the carbon yield is significantly lower. With around 10% [Cmol_RL/Cmol_Glc] it ranges below 10% of the theoretical yield. Notably, also the specific rate of rhamnolipid production is significantly higher using this novel recombinant P. putida (43 mg_RL/(g_CDW h) opposing to 27 mg_RL/(g_CDW h)).

This high rhamnolipid synthesis rate is possible, because the activated rhamnose pathway triples its flux, while the flux through de novo fatty acid synthesis increases by at least 40%. We here show that P. putida´s central carbon metabolism is capable of meeting metabolic demand generated by engineering transcription in peripheral pathways, thereby enabling significant rerouting of carbon fluxes towards the target compound, here, industrially interesting rhamnolipids. Hence, the engineering strategy of driven by demand is highly applicable to P. putida, arguing for this intriguing organism as a host in industrial biotechnology.

References

1.         Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR: The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J Bacteriol 2002, 184:3909-3916.

2.         Blank LM, Ionidis G, Ebert BE, Bühler B, Schmid A: Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase. FEBS J 2008, 275:5173-5190.

3.         Wittgens A, Tiso T, Arndt TT, Wenk P, Hemmerich J, Muller C, Wichmann R, Kupper B, Zwick M, Wilhelm S, et al: Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440. Microb Cell Fact 2011, 10.

4.         Jensen PR, Hammer K: The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 1998, 61:82-87.

5.         Müller MM, Hörmann B, Syldatk C, Hausmann R: Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems. Appl Microbiol Biotechnol 2010, 87:167-174.