Designer rhamnolipid production

Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of applications, e.g., in detergents, food, bioremediation, medicine/pharmacology, and crop science. These properties include their emulsification and foaming capacities and their ability to lower the surface tension. Further, aspects like biocompatibility and environmental friendliness, both features of rhamnolipids [1] are becoming increasingly important. Rhamnolipids thus constitute suitable substitutes for synthetic surfactants produced from fossil resources. Native producers of rhamnolipids are mainly pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth [2]. As most biosurfactants, rhamnolipids are synthesized in mixtures. We here show our approach to alter the native mixture of surfactant molecules to produce specific new-to-nature combinations. The molecular structure (Figure 1) can on the one hand be altered in the hydrophilic moiety by changing the number of rhamnose molecules. We achieved this by using only distinct genes from the native rhamnolipid synthesis pathway. On the other hand, we were also able to change the length of the fatty acids in the hydrophobic part. This chain length is determined by the acyl-transferase (RhlA). Using rhlA genes from different organisms enables our microbial cell factory to synthesize molecules with different chain lengths [3]. The different molecular structures have further been shown to feature diverse physico-chemical properties [4]. Exploiting the natural structural diversity will thus allow for the synthesis of designer rhamnolipids tailormade for specific applications. We thus present a novel approach to use biochemical engineering to create tailormade products for a more sustainable future. Please click Additional Files below to see the full abstract

A scalable bubble-free membrane aerator for biosurfactant production

The bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production

Metabolic Engineering of Pseudomonas putida KT2440 for enhanced rhamnolipid production

The production of chemicals and fuels is mainly based on fossil resources. The reduced availability of these resources and thus the increasing prices for crude oil as well as the resulting pollution of the environment require alternative strategies to be developed. One approach is the employment of microorganisms for the production of platform molecules using renewable resources as substrate. Biosurfactants, such as rhamnolipids, are an example for such products as they can be naturally produced by microorganisms and are biodegradable in contrast to chemical surfactants. The bio-based production of chemicals has to be efficient and sustainable to become competitive on the market. Several strategies can be applied to increase the efficiency of a microbial cell factory, e.g., streamlining the chassis. Here, we show the heterologous production of rhamnolipids with the non-pathogenic Pseudomonas putida KT2440 with the aim of increasing the yield. P. putida KT2440 is a well-characterized microorganism and its genome is sequenced and well annotated. Thus, the targeted removal of genes is possible and can lead to a reduction of the metabolic burden and by-product formation, which can result in a higher yield. Furthermore, the efficient supply of precursors is an important factor for optimized production processes. Rhamnolipids are amphiphilic molecules containing rhamnose and ß-hydroxy fatty acids. These precursors are synthesized by two pathways, the fatty acid de novo synthesis and the rhamnose pathway. We performed gene deletions to avoid the synthesis of by-products, like pyoverdine, exopolysaccharides, and large surface proteins and energy consuming devices as the flagellum. Most of the genome-reduced mutants reached a higher yield compared to the strain with wildtype background. With the best chassis, the yield could be increased by 35%. Furthermore, we conducted the overexpression of genes for precursor supply, either plasmid-based or genomically integrated. In this regard, the genes for the phosphoglucomutase, the complete rhamnose-synthesis pathway operon, and different enzymes in the pathway for acetyl-CoA synthesis were targeted. Various combinations were tested, and the highest yield reached was 51% higher compared to the initial rhamnolipid producer. Finally, a genome-reduced mutant was equipped with the overexpression modules and the rhamnolipid titer was increased from approximately 590 mg/L for the wildtype background to 960 mg/L, which represents a 63% increase. In conclusion, we were able to enhance the yield of rhamnolipids per glucose using metabolic engineering

Coupling an Electroactive Pseudomonas putida KT2440 with Bioelectrochemical Rhamnolipid Production

Sufficient supply of oxygen is amajor bottleneck in industrial biotechnological synthesis. One example is the heterologous production of rhamnolipids using Pseudomonas putida KT2440. Typically, the synthesis is accompanied by strong foam formation in the reactor vessel hampering the process. It is caused by the extensive bubbling needed to sustain the high respirative oxygen demand in the presence of the produced surfactants. One way to reduce the oxygen requirement is to enable the cells to use the anode of a bioelectrochemical system (BES) as an alternative sink for their metabolically derived electrons. We here used a P. putida KT2440 strain that interacts with the anode using mediated extracellular electron transfer via intrinsically produced phenazines, to performheterologous rhamnolipid production under oxygen limitation. The strain P. putida RL-PCA successfully produced 30.4 � 4.7mg/Lmono-rhamnolipids togetherwith 11.2 � 0.8mg/L of phenazine-1-carboxylic acid (PCA) in 500-mL benchtop BES reactors and 30.5 � 0.5 mg/L rhamnolipids accompanied by 25.7 � 8.0 mg/L PCA in electrode containing standard 1-L bioreactors. Hence, this study marks a first proof of concept to produce glycolipid surfactants in oxygen-limited BES with an industrially relevant strain

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

The human pathogenic bacterium Pseudomonasaeruginosa produces rhamnolipids, glycolipids with functionsfor bacterial motility, biofilm formation, and uptake of hydrophobicsubstrates. Rhamnolipids represent a chemically heterogeneousgroup of secondary metabolites composed of one ortwo rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosyntheticpathway involves rhamnosyltransferase I encoded by the rhlABoperon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoicacids (HAAs) followed by their coupling to one rhamnose moiety.The resulting mono-rhamnolipids are converted to dirhamnolipidsin a third reaction catalyzed by therhamnosyltransferase II RhlC. However, the mechanism behindthe biosynthesis of rhamnolipids containing only a singlefatty acid is still unknown. To understand the role of proteinsinvolved in rhamnolipid biosynthesis the heterologous expressionof rhl-genes in non-pathogenic Pseudomonas putidaKT2440 strains was used in this study to circumvent the complexquorum sensing regulation in P. aeruginosa. Our resultsreveal that RhlA and RhlB are independently involved inrhamnolipid biosynthesis and not in the form of a RhlAB heterodimercomplex as it has been previously postulated.Furthermore, we demonstrate that mono-rhamnolipids providedextracellularly as well as HAAs as their precursors are generallytaken up into the cell and are subsequently converted todi-rhamnolipids by P. putida and the native host P. aeruginosa.Finally, our results throw light on the biosynthesis ofrhamnolipids containing one fatty acid,which occurs by hydrolyzationof typical rhamnolipids containing two fatty acids,valuable for the production of designer rhamnolipids with desiredphysicochemical properties

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

<p>Abstract</p> <p>Background</p> <p>Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen <it>Pseudomonas aeruginosa </it>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 <it>P. aeruginosa</it>. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.</p> <p>Results</p> <p>Here, the metabolic engineering of a rhamnolipid producing <it>Pseudomonas putida </it>KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, <it>P. putida </it>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 <it>Bacillus subtilis, Corynebacterium glutamicum</it>, and <it>Escherichia coli. P. putida </it>KT2440 expressing the <it>rhlAB</it>-genes from <it>P. aeruginosa </it>PAO1 produces mono-rhamnolipids of <it>P. aeruginosa </it>PAO1 type (mainly C₁₀:C₁₀). 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 <it>P. aeruginosa </it>PAO1 at yields of about 0.15 g/g_glucosecorresponding 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.</p> <p>Conclusions</p> <p>A functional alternative to the pathogenic rhamnolipid producer <it>P. aeruginosa </it>was constructed and characterized. <it>P. putida </it>KT24C1 pVLT31_<it>rhlAB </it>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 <it>P. aeruginosa</it>.</p

Accessing natural diversity of rhamnolipids by metabolic engineering of Pseudomonas putida

The petrochemical industry is essential for many consumer goods currently in use. Implementing innovative biobased processes, renewable resources are used as substitutes for crude oil in goods such as household detergents, producing bioderived cleaning agents. However, biosurfactants are still expensive compared to their chemically synthesized counterparts. In this work, the exploitation of Pseudomonas putida for the heterologous synthesis of rhamnolipids was the main focus. Rhamnolipids are glycolipids that have a high potential for industrial applications. However, the production of rhamnolipids using the wild-type producer Pseudomonas aeruginosa entails a number of significant limitations, as P. aeruginosa is a human pathogen, and purification is expensive because rhamnolipid production by P. aeruginosa requires plant oils as a carbon and energy source. Introducing rhamnolipid synthesis genes into P. putida enabled the first microbial cell factory for the production of rhamnolipids from glucose. The synthesis of these secondary metabolites was uncoupled from growth and circumvented sophisticated fermentation procedures. A closer look at the underlying metabolic network during rhamnolipid synthesis revealed that P. putida upregulated its central carbon metabolism in order to meet the created demand. This finding was exploited as a metabolic engineering strategy and led to the development of a high-producing strain featuring a 3 g/L titer and a 40% carbon yield. Subsequently, we focused on the enhancement of transcriptional activity by introducing synthetic promoter libraries. This approach was successful, and a wide range of rhamnolipid productivity was achieved; however, the high flux towards the product was unstable in the long run. After establishing rhamnolipid production, we focused on the diversification of the product spectrum. Initially, only mono-rhamnolipids were synthesized. By introducing and eliminating specific genes in the rhamnolipid biosynthesis pathway, we were also able to produce di-rhamnolipids and the rhamnolipid precursor, hydroxyalkanoyloxy alkanoic acid. Lastly, we streamlined the host metabolism and optimized the fermentation conditions, including medium composition. The highest titer was achieved in a fed-batch flask experiment in which 7 g/L of surfactants were produced. The results are discussed in the context of rational strain engineering for the production of designer surfactants