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RNA Silencing of Lactate Dehydrogenase Gene in Rhizopus Oryzae

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RNA Silencing of Lactate Dehydrogenase Gene in Rhizopus Oryzae RNA Silencing of Lactate Dehydrogenase Gene in Rhizopus oryzae Neda Haghayegh Jahromi Ali Hashemi Gheinani This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Science with a Major in Resource Recovery – Industrial Biotechnology, 120 ECTS credits No. 9/2010 Neda Haghayegh Jahromi, [email protected] Ali Hashemi Gheinani, [email protected] Master thesis Subject Category: Technology University College of Borås School of Engineering SE-501 90 BORÅS Telephone +46 033 435 4640 Examiner: Dr.Ilona Sarvari Horvath Supervisor, name: Dr.Elisabeth Feuk-Lagerstedt Supervisor, address: University of Borås ,School of Engineering SE-501 90 BORÅS Date: 10.12.03 Keywords: RNA interference, Knockdown, Rhizopus oryzae, siRNA, Lactate dehydrogenase, Lactic acid, Ethanol iii Abstract RNA silencing with direct delivery of siRNA has been used to suppress ldhA gene expression in filamentous fungus Rhizopus oryzae. Here, for the first time we show that, introducing small interfering RNA which consequently forms silencing complexes can alter the gene expression and we report a significant reduction of lactic acid production for isolates containing short (25 nt) synthetic siRNA. In all samples lactic acid production was reduced comparing with wild types. The average concentration of lactic acid production by Rhizopus oryzae during batch fermentation process where glucose has been used as a sole carbon source, diminished from 2.06 g/l in wild types to 0.36 g/l in knockdown samples which signify 5.7 times decrease. Interestingly, the average concentration of ethanol production was increased from 0.38 g/l in wild types to 0.45 g/l in knockdown samples. In some samples we were able to report even a 10 fold decrease in lactic acid production. Since R.oryzae is capable to assimilate a wide range of carbohydrates hydrolysed from lignocellulosic material in order to produce many economically valuable bulk material such as ethanol, these results suggest that RNA silencing is a useful method for industrial biotechnology to be applied in fungus Rhizopus oryzae in order to trigger the metabolism and gene expression toward a desired product. Keywords: RNA interference, Knockdown, Rhizopus oryzae, siRNA, Lactate dehydrogenase, Lactic acid, Ethanol iv List of Figures FIGURE 1.RHIZOPUS ORYZAE .......................................................................................................................................... 1 FIGURE 2. A SIMPLIFIED PATHWAY FROM CARBON SOURCE TO PRODUCTS ............................................................................... 2 FIGURE 3.VARIOUS GROWTH FORMS OF FUNGI. (A) ASEPTATE HYPHA OF MUCORMUCEDO (ZYGOMYCOTA).THE HYPHA BRANCHES TO FORM A MYCELIUM. (B) SEPTATE BRANCHED HYPHA OF TRICHODERMA VIRIDE (ASCOMYCOTA). SEPTA ARE INDICATED BY ARROWS. (C) YEAST CELLS OF SCHIZOSACCHAROMYCES POMBE (ASCOMYCOTA) DIVIDING BY BINARY FISSION. (D) YEAST CELLS OF DIOSZEGIA TAKASHIMAE (BASIDIOMYCOTA) DIVIDING BY BUDDING. (E) PSEUDOHYPHA OF CANDIDA PARAPSILOSIS (ASCOMYCOTA), WHICH IS REGARDED AS AN INTERMEDIATE STAGE BETWEEN YEAST CELLS AND TRUE HYPHAE. (F) THALLUS OF RHIZOPHLYCTIS ROSEA (CHYTRIDIOMYCOTA) FROM WHICH A SYSTEM OF BRANCHING RHIZOIDS EXTENDS INTO THE SUBSTRATE. (G) PLASMODIA OF PLASMODIOPHORA BRASSICAE (PLASMODIOPHOROMYCOTA) INSIDE CABBAGE ROOT CELLS. SCALE BAR ¼ 20 MM (A,B,F,G) OR 10 MM (C_E).(WEBSTER AND WEBER, 2007) ........................................................ 6 FIGURE 4.R.ORYZAE ON AGAR ........................................................................................................................................ 6 FIGURE 5.R.ORYZAE HYPHAE .......................................................................................................................................... 6 FIGURE 6.R.ORYZAE MYCELIA ........................................................................................................................................ 6 FIGURE 7.THE HIERARCHY OF BIOLOGICAL CLASSIFICATION'S EIGHT MAJOR TAXONOMIC RANKS FOR R.ORYZAE (INTERMEDIATE MINOR RANKINGS ARE NOT SHOWN.) ................................................................................................................................ 7 FIGURE 8.A REPRESENTATION OF THE UNIVERSAL PHYLOGENETIC TREE, BASED ON COMPARISONS OF THE GENES ENCODING SMALL SUBUNIT (16S OR 18S) RIBOSOMAL RNA. THE LENGTHS OF THE LINES LINKING ORGANISMS TO THEIR NEAREST BRANCH POINT REPRESENT INFERRED EVOLUTIONARY DISTANCES (RRNA GENE SEQUENCE DIVERGENCE)(DEACON, 2006). .............. 7 FIGURE 9.NJ TREE OF RHIZOPUS STRAINS INFERRED FROM 622 NUCLEOTIDES FROM LSU RDNA D1 AND D2 REGION. NUMBERS GIVEN ON BRANCHES INDICATE THE CONFIDENCE LEVEL FROM 1K REPLICATES BS SAMPLING [44] ....................................... 8 FIGURE 10.MICROGRAPH SHOWING MUCORALES(KENDRICK, 2000) ..................................................................................... 9 FIGURE 11.MATURE SPORANGIUM OF MUCOR SP. FUNGUS(KENDRICK, 2000) ....................................................................... 9 FIGURE 12.SCHEMATIC DIAGRAM OF RHIZOPUS SP. (KENDRICK, 2000) ................................................................................ 10 FIGURE 13.DIAGRAMMATIC REPRESENTATION OF A FUNGAL HYPHA(DEACON, 2006) ............................................................. 11 FIGURE 14.R.ORYZAE AFTER 48 HOURS BATCH FERMENTATION ........................................................................................... 12 FIGURE 15.R.ORYZAE AFTER 48 HOURS BATCH FERMENTATION ........................................................................................... 12 FIGURE 16.DIAGRAM OF A HYPHAL MODULAR UNIT(MAHESHWARI, 2005) .......................................................................... 12 FIGURE 17.ERGOSTEROL(HANSON, 2008) ..................................................................................................................... 13 FIGURE 18.N-ACETYLGLUCOSAMINE(HANSON, 2008) ...................................................................................................... 14 FIGURE 19.TRANSMISSION ELECTRON MICROSCOPY OF ULTRATHIN SECTIONS OF FUNGAL CELL SHOWING DIFFERENT LAYERS OF CELL WALL AND PLASMA MEMBRANE (MICHAEL J. CARLILE, 2001). .................................................................................. 15 FIGURE 20.STRUCTURAL FORMULAE OF THE PRINCIPAL POLYMERS IN R.ORYZAE ..................................................................... 15 FIGURE 21.FLUORESCENCE MICROSCOPIC IMAGES (100×) OF RHODAMINE B BY (A) R. ORYZAE MYCELIA, (B) INTACT CELL WALL, (C) CHITIN, (S) CHITOSAN, (E) PHOSPHOMANNAN AND (F) GLUCAN(DAS ET AL., 2008) ....................................................... 17 FIGURE 22.THE CYTOSKELETON IN FUNGI(WEBSTER AND WEBER, 2007) ............................................................................. 19 FIGURE 23.THE ROLE OF CONVENTIONAL KINA AND KIP2 FAMILY A COMPARISON OF A WILD TYPE WITH A CONVENTIONAL KINESIN DELETION MUTANT (TAKEN FROM REQUENA ET AL. 2001). B SCHEME OF AMTWITH THEMTPLUS END COMPLEX. THIS PROTEIN COMPLEX CONSISTS OF SEVERAL PROTEINS, E.G. KIPA OR LIS1, CONVENTIONAL KINESIN TRANSPORTS VESICLES AND COMPONENTS OF THE PLUS END COMPLEX, FOR INSTANCE DYNEIN (ZHANG ET AL. 2003). A DIRECT INTERACTION BETWEEN KINA AND DYNEIN OR DYNACTIN HAS NOT YET BEEN VERIFIED. MODIFIED AFTER HESTERMANN ET AL. (2002) C, D WHEN KIPA, WHICH IS SUGGESTED TO BE INVOLVED IN THE DELIVERY OF CELL END MARKERS, IS MISSING, HYPHAE LOSE DIRECTIONALITY. IMAGES TAKEN FROM (FISCHER, 2007). ....................................................................................... 19 FIGURE 24.THE ROLE OF CLAMP CONNECTIONS IN MAINTAINING A REGULAR DIKARYON(DEACON, 2006). .................................. 21 FIGURE 25.ELECTRON MICROGRAPHS OF YEAST NUCLEUS SHOWING NUCLEOLAR SUBCOMPARTMENTALIZATION. A–B—FROM S. CEREVISIAE SHOWING FC, GC, AND DFC (A) AND RDNA LOCALIZATION ONLY IN THE FC (B); C–D—THE NUCLEOLUS OF S. POMBE SHOWING NDB FROM GAR-1 MUTANT CELLS, THE TRUNCATED GAR2P IS ACCUMULATED IN THE DB (C). THE BAR ¼ 1MM REPRODUCED FROM CHROMOSOMA 1997, 105:542–552 AND CHROMOSOMA 1999, 108:103–113, BY COPYRIGHT PERMISSION OF SPRINGER VERLAG ...................................................................................................... 21 FIGURE 26.IMAGES OF GFP-TUBA (A TUBULIN) IN A. NIDULANS SHOWING CYTOPLASMIC MICROTUBULES (A) SPINDLE MICROTUBULES (B) AND ASTRAL MICROTUBULES EMANATED FROM THE POLES OF AN ELONGATING SPINDLE (B, C)(XIANG AND FISCHER, 2004). ..................................................................................................................................................................... 23 v FIGURE 27.RIBBON-LIKE AGGREGATES OF CHITIN MICROFIBRILS (R) PRODUCED IN VITRO FROM CHITOSOMES (C) ISOLATED FROM MUCOR ROUXII, WHEN INCUBATED WITH SUBSTRATE (N-ACETYLGLUCOSAMINE) AND A PROTEOLYTIC ACTIVATOR. (COURTESY OF C.E. BRACKER; FROM BARTNICKI-GARCIA ET AL(DEACON, 2006). ......................................................................... 25 FIGURE 28.TWO POSSIBLE PATHWAYS TO CONVERT C:G TO A:T (IRELAN AND SELKER, 1996) .................................................. 28 FIGURE 29.SCHEMATIC ILLUSTRATION OF RIP AND MIP(IRELAN AND SELKER, 1996).A:BOTH COPIES OF DUPLICATION ARE RETAINED THROUGH THE SEXUAL CYCLE. MOST OFTEN BOTH ARE FOUND TO HAVE SUFFERED FROM RIP OR MIP (FILLED BOXES).B: DUPLICATION HAPPED BUT RIP AND MIP WERE NOT HAPPENED. C: DUPLICATED SEQUENCE IS DELETED. D: FIRST RIP HAPPED AND THEN DUPLICATION WAS DELETED. ................................................................................................................
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