Creating metabolic demand as an engineering strategy in Pseudomonas putida – Rhamnolipid synthesis as an example

Abstract

Metabolic engineering of microbial cell factories for the production of heterologous secondary metabolites implicitly relies on the intensification of intracellular flux directed toward the product of choice. Apart from reactions following peripheral pathways, enzymes of the central carbon metabolism are usually targeted for the enhancement of precursor supply. In Pseudomonas putida, a Gram-negative soil bacterium, central carbon metabolism, i.e., the reactions required for the synthesis of all 12 biomass precursors, was shown to be regulated at the metabolic level and not at the transcriptional level. The bacterium's central carbon metabolism appears to be driven by demand to react rapidly to ever-changing environmental conditions. In contrast, peripheral pathways that are only required for growth under certain conditions are regulated transcriptionally. In this work, we show that this regulation regime can be exploited for metabolic engineering.We tested this driven-by-demand metabolic engineering strategy using rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways, i.e., fatty acid de novo synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-)rhamnose, respectively. In contrast to single-pathway molecules, rhamnolipid synthesis causes demand for two central carbon metabolism intermediates, i.e., acetyl-CoA for HAA and glucose-6-phosphate for rhamnose synthesis.Following the above-outlined strategy of driven by demand, a synthetic promoter library was developed to identify the optimal expression of the two essential genes (rhlAB) for rhamnolipid synthesis. The best rhamnolipid-synthesizing strain had a yield of 40% rhamnolipids on sugar [CmolRL/CmolGlc], which is approximately 55% of the theoretical yield. The rate of rhamnolipid synthesis of this strain was also high. Compared to an exponentially growing wild type, the rhamnose pathway increased its flux by 300%, whereas the flux through de novo fatty acid synthesis increased by 50%.We show that the central carbon metabolism of P. putida is capable of meeting the metabolic demand generated by engineering transcription in peripheral pathways, thereby enabling a significant rerouting of carbon flux toward the product of interest, in this case, rhamnolipids of industrial interest.

High titer heterologous rhamnolipid production

“…It is noticeable that rhamnolipid production started when glucose started to be limiting corresponding to the onset of the glucose feeding. The recorded value of 18 mg/(g h) is comparable to the maximum value for the P. putida KT42C1 pVLT31_ rhlAB reported by Wittgens et al (2011) and about 38% for the very similar construct P. putida KT2440 pSynPro8_ rhlAB grown on a complex rich medium containing additional glucose (Tiso et al 2016). However, the comparison to experimental data reported on complex rich medium must be considered with caution.…”

Bio-upcycling of polyethylene terephthalate

“…Accessing non-biodegradable plastics of petrochemical origin (and in the future of biological origin) as carbon source for fermentations enables biotechnology to valorize enormous waste streams for the sustainable production of many valuable products by exploiting the metabolic versatility of microorganisms. Products such as aromatics, organic acids, glycolipids and lipid derivatives as well as biopolymers and fuel molecules are just some examples 46,47,63,64 . More importantly, the established biosynthetic pathways for plastic monomer metabolism can also be adapted to function in different organisms, widening the applicability of the described approach even further.…”

Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida—a step forward to tailor-made rhamnolipids

“…The non-pathogenic P. putida KT2440 is a well-established host organism for the heterologous production of rhamnolipids, which allows to circumvent the complex regulatory networks and pathogenicity of native rhamnolipid producers (Wittgens et al 2011;Tiso et al 2016;Beuker et al 2016aBeuker et al , 2016b, and was used for the first time in this study for the biosynthesis of longchain rhamnolipids by expression of rhl-genes from B. glumae PG1. All rhamnolipid species produced by P. putida after heterologous expression of rhl-genes from B. glumae contained the same fatty acids (mainly C14 or C14-C14) as those produced by B. glumae wild-type which are also known from B.…”

“…The successful heterologous production of rhamnolipids in the non-pathogenic P. putida KT2440 expressing rhl-genes from P. aeruginosa is well-established and qualifies this strain as a suitable host organism to circumvent the complex regulatory system of native rhamnolipid producers (Wittgens et al 2011;Tiso et al 2016;Beuker et al 2016b). It further offers the possibility for the production of both major individual species (mono-and/or di-rhamnolipids) after expression of respective rhl-genes or operons (Wittgens et al 2017) respectively.…”