Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

Abstract

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

Heterologous Rhamnolipid Biosynthesis: Advantages, Challenges, and the Opportunity to Produce Tailor-Made Rhamnolipids

“…This strategy will also allow to uncouple additional genes from their native regulation, e.g., responsible for the educt synthesis, which are usually strongly regulated in the wild types (Aguirre-Ramírez et al, 2012), to further improve the rhamnolipid biosynthesis. Metabolic engineering was also successfully used to lower the intrinsic metabolic burden by deleting high energy or resource-demanding side activities, e.g., the biosynthesis of polyhydroxyalkanoate (PHA) and the formation of flagella, which strongly increased the amounts of rhamnolipids (Wittgens et al, 2011;Tiso et al, 2016Tiso et al, , 2020. This strain engineering in conjunction with strategies for bioprocess development (feeding strategies, media compositions, downstream processing, etc.)…”

“…Especially for applications in the (microbial) enhanced oil recovery, rhamnolipids were produced in E. coli using the common T7 expression system (Wang et al, 2007;Han et al, 2014;Jafari et al, 2014;Du et al, 2017), while others established a constitutive expression of rhlAB in E. coli (Kryachko et al, 2013). More frequently, P. putida KT2440 wild type or engineered strains were used as heterologous host for rhamnolipid biosynthesis using an inducible tac-promoter or constitutive expressed and partly synthetic promoters (Wittgens et al, 2011(Wittgens et al, , 2017Wittgens, 2013;Behrens et al, 2016;Beuker et al, 2016a,b;Tiso et al, 2016Tiso et al, , 2017Tiso et al, , 2020Anic et al, 2017Anic et al, , 2018Noll et al, 2019; Table 1). Besides well-known short-chain rhamnolipids from P. aeruginosa also the heterologous production of long chain rhamnolipids was established in this organism by expressing rhlAB and rhlC from B. glumae .…”

Coupling an Electroactive Pseudomonas putida KT2440 with Bioelectrochemical Rhamnolipid Production

“…Phenazine production plasmids have been generated previously [ 19 ]. The genes rhlA and rhlB for rhamnolipid production originated from P. aeruginosa PAO1 and were previously cloned into the pSK02 plasmid [ 16 ], which served as the template for this study. Plasmids were assembled via Gibson assembly (New England Biolabs-Gibson Assembly) [ 22 ].…”

“…However, the costly aeration and the subsequent problems with strong reactor foaming still impose severe technical challenges [ 12 ]. This also hinders the scale-up of the process towards the industrial production of rhamnolipids and it requires substantial technical effort to overcome this challenge [ 13 , 14 , 15 , 16 ]. To reduce this bottleneck, which comes with the need for sufficient oxygen supply for aerobic growth and product synthesis, we here explore an alternative approach for rhamnolipid production using P. putida as a biocatalyst under oxygen-limitation in bioelectrochemical systems (BESs).…”

Uncoupling Foam Fractionation and Foam Adsorption for Enhanced Biosurfactant Synthesis and Recovery

“…To reduce production costs and prevent health concerns, efforts to develop a competitive non-pathogenic RL and HAA production host have increased significantly over the last two decades [ 12 , 20 , 21 , 22 ]. In this study, we use Pseudomonas putida KT2440 strains with a corresponding integration of the rhlA and rhlB genes as constructed previously [ 23 , 24 , 25 ].…”