Pseudomonas putida KT2440 is a well-established chassis in industrial biotechnology. To increase the substrate spectrum, we implemented three alternative xylose utilization pathways, namely the Isomerase, Weimberg, and Dahms pathways. The synthetic operons contain genes from Escherichia coli and Pseudomonas taiwanensis. For isolating the Dahms pathway in P. putida KT2440 two genes (PP_2836 and PP_4283), encoding an endogenous enzyme of the Weimberg pathway and a regulator for glycolaldehyde degradation, were deleted. Before and after adaptive laboratory evolution, these strains were characterized in terms of growth and synthesis of mono-rhamnolipids and pyocyanin. The engineered strain using the Weimberg pathway reached the highest maximal growth rate of 0.30 h −1. After adaptive laboratory evolution the lag phase was reduced significantly. The highest titers of 720 mg L −1 mono-rhamnolipids and 30 mg L −1 pyocyanin were reached by the evolved strain using the Weimberg or an engineered strain using the Isomerase pathway, respectively. The different stoichiometries of the three xylose utilization pathways may allow engineering of tailored chassis for valuable bioproduct synthesis.

BackgroundItaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L−1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production.ResultsIn this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO3. This integrated approach resulted in a maximum itaconate titer of 220 g L−1, with a total acid titer of 248 g L−1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus.ConclusionIn this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism.

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

DH5αλpir pTNS1DH5αλpir harboring plasmid pTNS1 Choi et al., 2005 DH5αλpir pSK02 DH5 αλpir harboring Tn7 delivery vector pSK02 for chromosomal integration; containing rhlAB genes from P. aeruginosa PA01 Bator et al., 2020 P. taiwanensis VLB120 wild type Panke et al., 1998 P. putida DOT-T1E wild type Ramos et al., 1998 S12 wild type Hartmans et al., 1990 KT2440 wild type Bagdasarian et al., 1981