“…A second approach used again a combination of strain and process engineering. Bator et al (2020a) adapted P. putida to the usage of ethanol as carbon source and developed a lab-scale process in which ethanol was added to a foam collection bottle to collapse the foam. The formed solution containing the collapsed foam and ethanol was recycled back into the fermenter.…”
E. coli HB101 pRK2013 Sm R , hsdR-M + , proA2, leuB6, thi-1, recA; harboring plasmid pRK2013 Ditta et al., 1980 E. coli DH5α pYTSK01K_0G7 DH5α harboring plasmid pYTSK01K_0G7 This study E. coli DH5α pVLT33-PA_rhlABC DH5α harboring plasmid pVLT33-PA_rhlABC Wittgens et al., 2017 E. coli DH5α pBNT Km DH5α harboring plasmid pBNTmcs(t)Km This study S. cerevisiae VL6-48 pYTSK10K_0G7_rhlAB VL6-48 harboring plasmid pYTSK10K_0G7_rhlAB This study E. coli DH5α pYTSK10K_1G7_rhlAB DH5α harboring plasmid pYTSK10K_1G7_rhlAB This study E. coli DH5α pRhon5Hi-2-eyfp DH5α harboring plasmid pRhon5Hi-2-eyfp Troost et al., 2019 S. cerevisiae VL6-48 pYTSK40K_1G7_rhlAB VL6-48 harboring plasmid pYTSK40K_1G7_rhlAB This study E. coli DH5α pYTSK40K_0G7_rhlAB DH5α harboring plasmid pYTSK40K_0G7_rhlAB This study E. coli S17-1 pYTSK40K_1G7_rhlAB S17-1 harboring plasmid pYTSK40K_1G7_rhlAB This study E. coli DH5αλpir pSK02 DH5α λpir harboring Tn7 delivery vector pSK02 for chromosomal integration Bator et al., 2020b E. coli DH5α pSW-2 DH5α harboring plasmid pSW-2 encoding I-SceI nuclease Martinez-Garcia and de Lorenzo, 2011 E. coli DH5αλpir pTNS-1 DH5α λpir harboring plasmid pTNS-1 Choi et al., 2005 E. coli DH5αλpir p pha DH5α λpir harboring plasmid p pha Mato Aguirre, 2019 E. coli PIR2 pEMG-flag1 PIR2 harboring plasmid pEMG-flag1 Blesken et al., 2020 E. coli PIR2 pEMG-flag2 PIR2 harboring plasmid pEMG-flag2 Blesken et al., 2020 E. coli DH5αλpir pMaW03 DH5α λpir harboring plasmid pMaW03 This study P. putida chassis strains P. putida KT2440 Wild type Bagdasarian et al., 1981 P. putida KT2440 flag PP_4328-PP_4344 and PP_4351-PP_4397 Blesken et al., 2020 P. putida KT2440 phaG PP_1408 This study P. putida KT2440 pha PP_5003-PP_5008 Blesken et al., 2020 P. putida KT2440 phaG pha PP_1408 and PP_5003-PP_5008 (phaC1ZC2DFI) This study P. putida KT2440 flag pha PP_4328-PP_4344, PP_4351-PP_4397 and PP_5003-PP_5008 This study P. putida strains used for biosurfactant production P. putida KT2440 pPS05 KT2440 harboring plasmid pPS05 Tiso et al., 2016 P. putida KT2440 SK4 rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 sal mRL E SK40 rhlAB, eYFP, attTn7 integrated, P nagAa /nagR, Gm R , tnsABCD This study P. putida KT2440 flag SK4 PP_4328-PP_4344 and PP_4351-PP_4397, rhlAB, attTn7 integrated, P 14ffg Blesken et al., 2020 P. putida KT2440 phaG SK4 PP_1408, rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 pha SK4 PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg Blesken et al., 2020 P. putida KT2440 phaG pha SK4 PP_1408 and PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 flag pha SK4 PP_4328-PP_4344 and PP_4351-PP_4397 and PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg
“…A second approach used again a combination of strain and process engineering. Bator et al (2020a) adapted P. putida to the usage of ethanol as carbon source and developed a lab-scale process in which ethanol was added to a foam collection bottle to collapse the foam. The formed solution containing the collapsed foam and ethanol was recycled back into the fermenter.…”
E. coli HB101 pRK2013 Sm R , hsdR-M + , proA2, leuB6, thi-1, recA; harboring plasmid pRK2013 Ditta et al., 1980 E. coli DH5α pYTSK01K_0G7 DH5α harboring plasmid pYTSK01K_0G7 This study E. coli DH5α pVLT33-PA_rhlABC DH5α harboring plasmid pVLT33-PA_rhlABC Wittgens et al., 2017 E. coli DH5α pBNT Km DH5α harboring plasmid pBNTmcs(t)Km This study S. cerevisiae VL6-48 pYTSK10K_0G7_rhlAB VL6-48 harboring plasmid pYTSK10K_0G7_rhlAB This study E. coli DH5α pYTSK10K_1G7_rhlAB DH5α harboring plasmid pYTSK10K_1G7_rhlAB This study E. coli DH5α pRhon5Hi-2-eyfp DH5α harboring plasmid pRhon5Hi-2-eyfp Troost et al., 2019 S. cerevisiae VL6-48 pYTSK40K_1G7_rhlAB VL6-48 harboring plasmid pYTSK40K_1G7_rhlAB This study E. coli DH5α pYTSK40K_0G7_rhlAB DH5α harboring plasmid pYTSK40K_0G7_rhlAB This study E. coli S17-1 pYTSK40K_1G7_rhlAB S17-1 harboring plasmid pYTSK40K_1G7_rhlAB This study E. coli DH5αλpir pSK02 DH5α λpir harboring Tn7 delivery vector pSK02 for chromosomal integration Bator et al., 2020b E. coli DH5α pSW-2 DH5α harboring plasmid pSW-2 encoding I-SceI nuclease Martinez-Garcia and de Lorenzo, 2011 E. coli DH5αλpir pTNS-1 DH5α λpir harboring plasmid pTNS-1 Choi et al., 2005 E. coli DH5αλpir p pha DH5α λpir harboring plasmid p pha Mato Aguirre, 2019 E. coli PIR2 pEMG-flag1 PIR2 harboring plasmid pEMG-flag1 Blesken et al., 2020 E. coli PIR2 pEMG-flag2 PIR2 harboring plasmid pEMG-flag2 Blesken et al., 2020 E. coli DH5αλpir pMaW03 DH5α λpir harboring plasmid pMaW03 This study P. putida chassis strains P. putida KT2440 Wild type Bagdasarian et al., 1981 P. putida KT2440 flag PP_4328-PP_4344 and PP_4351-PP_4397 Blesken et al., 2020 P. putida KT2440 phaG PP_1408 This study P. putida KT2440 pha PP_5003-PP_5008 Blesken et al., 2020 P. putida KT2440 phaG pha PP_1408 and PP_5003-PP_5008 (phaC1ZC2DFI) This study P. putida KT2440 flag pha PP_4328-PP_4344, PP_4351-PP_4397 and PP_5003-PP_5008 This study P. putida strains used for biosurfactant production P. putida KT2440 pPS05 KT2440 harboring plasmid pPS05 Tiso et al., 2016 P. putida KT2440 SK4 rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 sal mRL E SK40 rhlAB, eYFP, attTn7 integrated, P nagAa /nagR, Gm R , tnsABCD This study P. putida KT2440 flag SK4 PP_4328-PP_4344 and PP_4351-PP_4397, rhlAB, attTn7 integrated, P 14ffg Blesken et al., 2020 P. putida KT2440 phaG SK4 PP_1408, rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 pha SK4 PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg Blesken et al., 2020 P. putida KT2440 phaG pha SK4 PP_1408 and PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 flag pha SK4 PP_4328-PP_4344 and PP_4351-PP_4397 and PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg
A key challenge to advance the efficiency of bioprocesses is the uncoupling of biomass from product formation, as biomass represents a by-product that is in most cases difficult to recycle efficiently. Using the example of rhamnolipid biosurfactants, a temperature-sensitive heterologous production system under translation control of a fourU RNA thermometer from Salmonella was established to allow separating phases of preferred growth from product formation. Rhamnolipids as bulk chemicals represent a model system for future processes of industrial biotechnology and are therefore tied to the efficiency requirements in competition with the chemical industry. Experimental data confirms function of the RNA thermometer and suggests a major effect of temperature on specific rhamnolipid production rates with an increase of the average production rate by a factor of 11 between 25 and 38 °C, while the major part of this increase is attributable to the regulatory effect of the RNA thermometer rather than an unspecific overall increase in bacterial metabolism. The production capacity of the developed temperature sensitive-system was evaluated in a simple batch process driven by a temperature switch. Product formation was evaluated by efficiency parameters and yields, confirming increased product formation rates and product-per-biomass yields compared to a high titer heterologous rhamnolipid production process from literature.
“…Moreover, the proteomics studies revealed a downregulated synthesis of several proteins encoded within the ped cluster. Recently, proteins of the ped cluster have been shown to be involved in the oxidation of alcohols by P. putida KT2440 (Bator, Karmainski, et al, 2020; Li et al, 2020; Thompson et al, 2020). Subsequently, using cytochrome c , electrons are transferred to the cytochrome c oxidase (PP_2675, PedF), which was also less abundant here, in turn reducing O 2 as part of the respiratory electron transfer chain (García‐Horsman et al, 1994; Wehrmann et al, 2020).…”
The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen.Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrialscale bioprocesses.
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