Akkumulation von L-Malat und D-Lactat in Arabidopsis thaliana und Laccase/HBT-vermittelte Delignifizierung von Spartina alterniflora und Phragmites australis

  • Accumulation of L-malate and D-lactate in Arabidopsis thaliana and laccase/HBT mediated delignification of Spartina alterniflora and Phragmites australis

Heil, Alexander; Kreuzaler, Fritz (Thesis advisor); Conrath, Uwe (Thesis advisor)

Aachen (2016)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2016

Abstract

The current work contains two projects "Accumulation of L-malate and D-lactate in Arabidopsis thaliana" (A) "Laccase/HBT mediated delignification of Spartina alterniflora and Phragmites australis" (B). In project A, L-malate and D-lactate accumulated in A. thaliana plants. The accumulation of L-malate is carried out by modification of the plant metabolism with the enzymes PEPC, MDH and the tonoplast dicarboxylate transporter (TDT). Gene pepci2 (Hydrilla verticillata), mdh5 (Zea mays) and tdt (Arabidopsis thaliana) were cloned in a plant expression vector pTRA-K-PEPCi2-TDT-$\lambda$F-MDH5. These genes were combined and stable transformed into A. thaliana plants. The Enzyme PEPC catalyzes the addition of bicarbonate to phosphoenolpyruvate (PEP) to form the oxaloacetate and inorganic phosphate. Further the oxaloacetate is visibly reduced to the L-malate by the MDH. L-malate was transported by TDT from the cytosol in to the vacuole, and accumulated there. For the accumulation of D-lactate in A. thaliana plants was used the vector pTRA-K-RbcS-1A-D-LDHA-pA3'g7 and stable transformed in to A. thaliana plants. The vector contained the previously isolated D-ldhA gene from E. coli, coding for a largely one-way direction working, pyruvate-reducing D-LDH. The expression of the transgenes pepci2, mdh5, tdt and ldhA the increased activity of enzymes MDH and PEPC was shown using qRT-PCR and the enzyme activity also measured in A. thaliana plants in the T1 generation. An increased L-malate content of the leaf lysate of transformants of T1 generation has been demonstrated using the enzymatic test of L-malate. The detection of D-LDH activity in the cell lysate of transformants failed. Also D-lactate was enzymatically undetectable. The next generation (T2) of both types of transformants was further analyzed by GC/MS. "D-lactate transformants" contains the average of 2.32 times more D/L-lactate than of "L-malate transformants". Vice versa, L-malate transformants contained average 2.08 times more L-malate. Using statistical analysis, a significant difference between two types of transformants of lactate and malate concentrations could be detected. In project B, "Laccase/HBT mediated delignification of Spartina alterniflora and Phragmites australis" (B), shows the groundwork of a chemical enzymatic method for the delignification of non-wood plants. The main aim of this project is to develop sustainable and environmentally friendly lignin degradation. The enzymatic method contains "laccase/mediator system" and the chemical method includes the H2O2 and NaOH. Model plants were the non-wood plants such as Spartina alterniflora (smooth mud grass) and Phragmites australis (reed). In total three enzymes were selected: the manganese peroxidase (MnP), the lignin peroxidase (LiP) and the laccase (Lacc). Each of the corresponding genes could be cloned and expressed in the yeast expression vector pPIC9K in P. pastoris. But only the laccase was synthesized in a sufficient quantity by P. pastoris, therefore only laccase in combination with mediator HBT and H2O2 were used for the delignification of plants.By photometric measurements of the absorbance and the determination of integral values of spectral ranges, of the samples with laccase, HBT and H2O2, have been achieved the reduction of lignin content in the plant material by 25 %.