Interdisciplinary PhD projects 2019

Nowadays, new biobased ingredients are discussed for many industrial products as an alternative for petrochemical substrates. Biosurfactants and bioactive alkaloids are examples for such biobased products e.g. for application in agrochemicals. Recombinant production of such high value compounds in established microbial production platforms like Pseudomonas putida KT2440, as applied by the BioSC FocusLabs Bio² and CombiCom, represents one strategy to get access to the wealth of those natural products. Key factors for a successful recombinant production strategy are, besides establishing a robust expression of the biosynthetic genes, an optimal process control and the understanding of the physiological effects of the produced compounds on the host cells.  ResPuTra aimed at the elucidation of the yet unexplored response of a genetically streamlined P. putida chassis to the production of stressing compounds by whole-transcriptome sequencing (RNA-Seq) to deduce further traits utilizable for the optimization of production strains and processes. In particular, we investigated the response toward the production of potentially membrane damaging rhamnolipid biosurfactants (one target molecule of Bio2) using a salicylate inducible system for biosynthetic gene expression. We observed a striking response of P. putida KT2440 GR18 already towards salicylate exposure. Especially genes that are involved in iron homeostasis were upregulated, putatively due to a strong demand of iron for the detoxification of aromatics in the siderophore deficient strain background. However, the massive transcriptome change including the upregulation of many transcription factors and transport proteins also indicate a severe stress response during salicylate exposure. 
During the production of rhamnolipids, especially putative genes for unspecific export proteins were found to be highly upregulated. Those represent interesting targets for studies on the secretion of rhamnolipids and the tolerance of P. putida with regard to that, aiming to construct a strain with increased rhamnolipid secretion. Promoters that respond to membrane-active components such as rhamnolipids may also be used to build-up a biosensor for non-invasive and real-time monitoring of product formation. The remarkably strong stress response to salicylate exposure hampered the unambiguous identification of rhamnolipid responses, however, a small set of candidate genes could be identified.  
In conclusion, ResPuTra revealed potential targets to optimize the heterologous expression of genes within the salicylate-inducible systems in general. For optimization, the production of rhamnolipids for further strain engineering towards more efficient secretion was suggested. In addition, the existing vectors may be optimized towards a biosensor for membrane-active substances or other potential candidates for the construction can be examined to that end in the future. 

Publication

Gauthier, C, Lavoie, S, Kubicki, S, Piochon, M, Cloutier, M, Dagenais-Roy, M, Groleau, M-C, Pichette, A, Thies, S and Déziel, E (2024). Structural characterization of a nonionic rhamnolipid from Burkholderia lata. Carbohydrate Research 535: 108991.   

The co-cultivation of bacteria with different physiological properties can offer several benefits for industrial biotechnology. However, cell growth and gene expression within synthetic microbial consortia need to be tightly controlled and, as a prerequisite, precisely monitored. The project SynCom therefore aimed to develop a model co-cultivation platform for synthetic microbial consortia in which growth performance of individual Pseudomonas putida KT2440 strains and selective target gene expression of different fluorescence reporters can be online monitored during co-cultivation. For this purpose, three fluorescent reporter proteins with distinguishable emission spectra were selected, the respective genes cloned and expressed thereby creating three P. putida strains producing one of these reporter proteins each. To this end, we constructed integrative vectors with different fluorescent protein-encoding reporter genes via ligase-independent cloning allowing to achieve stable integration of the reporter genes at different chromosomal positions. Afterwards, the strain libraries were analyzed and sorted regarding their respective growth behavior and fluorescence signal intensity and strains providing a constant and stable expression level were selected.  Measurement of the fluorescence intensity and cell density during cultivation showed a correlation of the two signals, and thus suggested that the fluorescence may be used as a biomass-indicator. To ensure maximal sensitivity and specificity of the measurements, an in-house built BioLector device with freely adjustable wavelength combinations was used to determine optimum excitation and emission wavelengths for each strain (Figure 1). Subsequently, all three strains were grown in co-culture. It was shown that the growth of each strain could be followed individually within the co-culture via its specific fluorescence signal.  
This project therefore provides a robust setup for gaining insights in the growth behavior of the biotechnologically relevant bacterium P. putida KT2440 during synthetic co-cultivation settings. 

The ProdAnchor mini-project, a joint effort of BioSC FocusLabs greenRelease and CombiCom, aimed at establishing an innovative purification method for prodigiosin synthetase PigC from bacterial expression cultures using small antimicrobial peptides as “anchors” to immobilize PigC on polymer surfaces. Prodigiosin synthetase PigC, a key enzyme for production of natural compounds in CombiCom, catalyzes the final step of the biosynthesis pathway of prodigiosin. Prodigiosin is a red hydrophobic pigment that shows promising bioactivities, such as anticancer and antibiotic effects. Since PigC activity is dependent on cell membrane attachment, isolation of pure and active PigC fractions in aqueous buffers remains challenging. In ProdAnchor, PigC has been genetically combined with three anchor peptides from different organisms (Bacillus subtilis, long-tailed macaques, and Japanese horseshoe crabs), which all show polymer-binding properties. All three C-terminal PigC-anchor peptide fusion constructs (PAPs) maintained full PigC catalytic activity in expression cultures. After immobilization of PAPs on polypropylene reaction tubes, residual PigC activity was observed upon addition of substrate solution, resulting in characteristic red/pink coloration by formation of prodiginines. To further fine-tune immobilization, washing and elution of PAPs, a column chromatography approach was tested with several polymer materials. However, in the elution fraction no sufficiently pure and active PigC was detected so far. 
In summary, the mini-project ProdAnchor has successfully shown that small peptides like hydrophobic anchor peptides can be fused to PigC without impairing PigC catalytic activity. Detergent screening lead the way to optimal composition of possible elution buffers that maintain PigC activity. Unfortunately, the surface to protein ratio was not sufficient for PigC purification through binding to polymer beads in a column approach, so future work is required to fine-tune purification conditions. 

MELOBEE aimed on the production of mannosylerythritol lipids (MEL) from microbial oil (single cell oil, SCO) obtained by fermentation of sugar beet pulp (SBP). MEL are valuable glycolipids with potential medical and cosmetic applications. Until now production is economically unfavorable, one reason is the high cost for plant oils, which are used as substrate in conventional production of MEL. In contrast, SCO has the advantage that it can be produced sustainably from renewable resources and waste streams. This was demonstrated for the example of SBP, which is a side stream from sugar industry and available at low cost.

The oleaginous yeast Cryptococcus curvatus was successfully cultivated in a 2 L laboratory fermenterusing 100 g/L SBP as substrate. After 7 days of cultivation, cells were harvested and subsequently lyophilized, whereupon about 60 g of a dry powder consisting of the lipid containing cell bodies were obtained.

For extraction of SCO, supercritical CO₂ extraction was applied₂ to improve the economy of the otherwise costly isolation process. After extraction, pure SCO with a yield of about 10% (w/w) was obtained. The consistency and smell resembled rapeseed or sunflower oil.

The purified SCO was afterwards used as a substrate for fermentation with the yeast-type fungus Pseudozyma aphidis, which is well established for MEL production. In shake flask experiments, suitability of SCO as a substrate for MEL synthesis was investigated and compared with sunflower oil, soybean oil and glucose. MEL production was verified by thin layer chromatography (TLC).

It could be shown that SCO is principally a suitable substrate for MEL production, but resulted in less yields than sunflower or soybean oil. While the dry weights of ethyl acetate extracts

showed a high amount of 27 g/L dissolved contents, TLC analysis showed only a faint band for MEL, indicating that the major part of extracted material consisted of other species. Sunflower and soybean oil gave 8 and 6 g/L extractable contents, respectively, mainly consisting of MEL according to TLC. Glucose, supporting reports from literature, resulted in no MEL formation at all, indicating the significance of using oils for efficient production of glycolipids with P. aphidis.

All in all, the approach to use SCO instead of plant oils for production of MEL appears to be promising, even though achieved titers and yields were lower than for sunflower and soybean oil. Since conventional MEL production is hampered by economic concerns which particularly address substrate cost, the substitution of classically used oils by SCO made from sustainable resources could aid in the development of an economically viable MEL production process.