Sakunda Anggarini

English

博士(生命科学)山口大学Yamaguchi Universit

Yamaguchi University Navigator for Open access Collection and Archives

 ïî  íï øÚ±®³¿¬ Ò±òé÷ ø  îôððð  ñ  èðð ÷ ¿¾±«¬  èðð ©±®¼­(Summary of the Doctoral Dissertation) (Dissertation Title)Improvement of The Heat Resistance Ability of Thermotolerant TISTR 548ø Ì×ÍÌÎ ëìè ÷(Name) Sakunda AnggariniFundamentally, microorganisms have an upper-temperature limit of survival thatis called a critical high temperature (CHT) (Matshushita ., 2015; Kosaka ., 2019),which may be a combined results of various cellular elements including general andparticular metabolisms. Mesophilic microbes that have a CHT close to the range of globalwarming temperatures would be damaged by an increase in temperature.Thermotolerant microorganisms are able to grow and/or perform fermentation at highertemperatures than those for mesophilic microorganisms. In this study, two experimentson the enhancement of thermotolerance of  were carried out. One experimentwas on improvement of thermotolerance of by genes for reactiveoxygen species (ROS)-scavenging enzymes (RSEs) and heat shock proteins (HSPs), andthe other experiment was on improvement of the thermotolerance of by metalions.A temperature close to the critical high temperature (CHT) influences membranestability and generates ROS, resulting in macromolecule damage such as proteinunfolding or denaturation. However, in mesophiles, including  only a few genesfor ROS-scavenging enzymes (RSEs) and heat shock proteins (HSPs) have been identifiedas thermotolerant genes that are essential at the CHT. We attempted to increase theexpression of genes for RSEs ( and 0186) andgenes for HSPs (0844). The influence of these genes at the CHT of TISTR 548 wasevaluated by the 2-step cultivation method, determination of ROS and cell length, and ïî  íï øÚ±®³¿¬ Ò±òé÷ ø  îôððð  ñ  èðð ÷ ¿¾±«¬  èðð ©±®¼­examination of resistance to other stresses. The results showed that increased expressionof genes for RSEs and HSPs led to an up-shift of the CHT of TISTR 548, thoughlevel of ROS. However, among the genes for HSPs that were tested, the genesand were unable to raise the CHT when their expression was increased, eventhough they were able to decrease the accumulation level of ROS to some extent.Examination of the effect of oxidative stress on transformants by addition of H2O2 to theculture medium showed that the conclusion was essentially consistent with the results ofmeasurement of levels of ROS. The change of cell morphology was also relativelyconsistent with the effects of the genes on cell growth at the CHT. Moreover, whenglucose or ethanol was added to the culture medium, none of the transformants that hadany gene for RSEs and HSPs showed that was different growth from that of the control.However, some of the transformants showed enhanced tolerance to acetic acid. Thesefindings suggest that molecules damaged by ROS or unfolded proteins that prevent cellgrowth accumulate in cells at the CHT. Therefore, prevention of the accumulation of ROSmight be effective way for increasing the CHT. The expression of genes for RSEs isthought to play a direct role in prevention of the accumulation of ROS, whereas theexpression of genes for HSPs is assumed to play an indirect role by maintaining thequality of proteins including RSEs, which in turn helps to prevent accumulation of ROSand increase heat resistance capability.Furthermore, with the aim of improving the ability for thermotolerance, weinvestigated the effects of various metal ions on growth of  TISTR 548at the CHT. Addition of Mg2+ and K+ clearly improved growth by 1°C, but Mn2+, Ni2+, Co2+,Al3+, Fe3+, and Zn2+ showed negligible effects. These results indicate that only specificmetals are required for the enhancement of growth of specific microorganisms. Tounderstand the physiological functions associated with the addition of Mg2+ or K+, cellmorphology, intracellular level of ROS, and ethanol productivity were investigated at ïî  íï øÚ±®³¿¬ Ò±òé÷ ø  îôððð  ñ  èðð ÷ ¿¾±«¬  èðð ©±®¼­39°C. Cell elongation was repressed by Mg2+but not by K+. The greatest reduction inintracellular levels of ROS was achieved by addition of only K+, followed by both metalsand addition of only Mg2+. Additionally, ethanol productivity was recovered by theaddition of both metals. Moreover, the addition of Mg2+ or K+ at a nonpermissivetemperature to cultures of 26 thermosensitive, single gene-disrupted mutantsof .  TISTR 548 resulted in improvement of metal ion-specific growth of severalmutants. Remarkably, K+ repressed the growth of two mutants. These results suggestthat K+ and Mg2+ enhance cell growth at the CHT via different mechanisms, which areinvolved in the maintenance of low intracellular levels of ROS. For more details, under aCHT condition, the addition of Mg2+ resulted in stabilization of both the outer and innermembranes, and leakage electrons from respiratory chain was suppressed. Membranestabilization also helps to maintain intracellular ions, cytoplasmic sensors, proteins, andRNAs. On the other hand, when the K+ was added, the repression of K+ leakage capable tomaintain the homeostasis for cellular metabolism whereby intracellular ROS is reduced.However, the observation of combinative usage of Mg2+ and K+ exhibits a distinct resultcompares to the single effect.In conclusion, the results of both of the studies described above suggest that thereare two ways to address the problem of accumulation of ROS at a CHT: (1) suppression ofthe generation of ROS and (2) elimination of accumulated ROS. In fact, increasedexpression of genes for RSEs suppressed the generation of ROS and improved growth athigh temperatures, thus increasing the CHT. On the other hand, Mg2+ may have an effecton accumulation of ROS by mainly blocking the generation of ROS preventing electronleakage through strengthening the membrane structure. The actions of the increasedexpression of genes for HSPs and addition of K+ appear to improve thermostability ineither or both of these two ways. It is possible that the combination of these genes andmetals greatly improves the thermostability of . Therefore, further study onstable high temperature fermentation is needed.

Improvement of heat resistant ability of thermotolerant zymomonas mobilis TISTR 548

http://petit.lib.yamaguchi-u.ac.jp/G0000006y2j2/file/28406/20201209161052/DT15100044_Abstract.pdf

Improvement of heat resistant ability of thermotolerant zymomonas mobilis TISTR 548

Abstract

Similar works

Full text

Available Versions

Yamaguchi University Navigator for Open access Collection and Archives