세포질 내 발린 생합성 경로 최적화를 통한 재조합 효모의 아이소부탄올 생산성 향상에 관한 연구

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부 농생명공학전공, 2016. 2. 서진호.Global environmental problems due to fossil fuel and its depletion have promoted the development of microorganisms for synthesizing alternative liquid biofuels. Compared to ethanol, a traditional biofuel, biobutanol has significant advantages such as higher energy density, lower hygroscopicity and compatibility with existing transportation infrastructures. Especially, isobutanol has a higher octane number, a standard measure of the performance of gasoline. Also, it has been applied in various industrial fields and used as an important platform chemical. In this study, Saccharomyces cerevisiae which has been traditionally used for industrial ethanol production was engineered to produce isobutanol. Naturally S. cerevisiae produces isobutanol at low concentrations by the valine biosynthesis pathway and the Ehrlich pathway. However, there are some problems because of different location of the valine biosynthesis pathway and the Ehrlich pathway. Therefore, this thesis was focused on optimizing the cytosolic valine biosynthesis pathway for improving isobutanol production. First, investigations of various genes from bacteria involved in the valine biosynthesis pathway were conducted. The alsS gene from Bacillus subtilis which has high specificity to pyruvate was selected for expression of acetolactate synthase (ALS). The genes coding for ketolacid reductoisomerase (KARI) and dihydroxyacid dehydratase (DHAD) from Escherichia coli and Corynebacterium glutamicum were cloned and introduced to the D452-2 strain with keto acid decarboxylase (KDC) of Lactococcus lactis. All of genes which have the mitochondria targeting sequences were modified. The yeast strain containing the endogenous genes of ALS, KARI and DHAD was used as the control strain. Flask fermentations with 40 g/L glucose was carried out under micro-aerobic conditions. As a result, the isobutanol titer and yield of the strain expressing alsS from B. subtilis, ilvC and ilvD from C. glutamicum and kivD from L. lactis were the highest among engineered strains used in this study. Second, to improve the activity of DHAD, engineering of the Fe-S cluster was conducted. The Grx3 protein controls iron sensing. Cfd1 is an important factor of the cytosolic Fe-S cluster assembly. The CRISPR/Cas9 system was used to replace the GRX3 gene in the chromosome of S. cerevisiae with the CFD1 gene to construct the D_FeS strain. The D_FeS-SK_CCMDC strain was constructed by introducing alsS from B. subtilis, ilvC and ilvD from C. glutamicum and kivD from L. lactis. Compared to the D-SK_CCMDC, the strain D_FeS-SK_CCMDC produced isobutanol titer more than 60%. Finally, the D_FeS-SK_CCMDC was cultivated in a reactor with gas trapping and the fermentation condition was controlled by altering agitation speed and aeration. The final concentration of isobutanol of the D_FeS-SK_CCMDC was 246 mg/L with isobutanol yield (6.15 mg isobutanol/g glucose), corresponding to a 25% increase in titer and a 32% increase in yield than those obtained in flask fermentation. These results suggested that modulation of the cytosolic valine biosynthetic pathway in combination with optimization of a fermentation process can be a successful strategy for producing isobutanol in S. cerevisiae.I. INTRODUCTION 1 1. Isobutanol 1 1.1. Advanced biofuel 1 1.2. Building block chemical 4 2. Microbial production of isobutanol 6 3. Isobutanol biosynthesis pathway 9 3.1. The valine biosynthesis pathway 9 3.2. The Ehrlich pathway 11 4. Cytosolic localization of the valine biosynthesis of S. cerevisiae 13 5. Cytosolic Fe-S cluster related to activity of dihydroxyacid dehydratase 19 6. Gas trapping based in situ removal system 25 7. Research objectives 26 II. MATERIALS AND METHODS 27 1. Reagents 27 2. Strains and plasmids 28 2.1. Strains 28 2.2. Plasmids 32 3. DNA manipulation and transformation 37 3.1. Enzymes 37 3.2. Polymerase chain reaction (PCR) 37 3.3. Isolation of DNA fragments and DNA sequencing 37 3.4. Transformation of E. coli 38 3.5. Preparation of plasmid DNA and bacteria genomic DNA 39 3.6. Yeast transformation 39 4. Media and culture conditions 40 4.1. Media 40 4.2. Cultivations in flasks 40 4.3. Cultivations in bioreactor with gas trapping 41 5. Analysis 43 5.1 Dry cell weight 43 5.2 Metabolite detection 43 5.3 Isobutanol detection 44 III. RESULTS AND DISCUSSIONS 46 1. Construction of the efficient isobutanol biosynthesis system 46 1.1. Comparison of acetolactate synthases from S. cerevisiae and B. subtilis 46 1.2. Evaluation of ketolacid reductoisomerase and dihydroxyacid dehydratase from various microorganisms 50 2. Engineering Fe homeostasis and Fe-S cluster synthesis 54 2.1. Construction of engineered strain by using CRISPR/Cas9 system 54 2.2. Production of isobutanol from the strain engineered for Fe-S cluster 57 3. Isobutanol production in bioreactor with gas trapping 60 3.1. Confirmation of effect of gas trapping in different conditions 60 3.2. Optimization of fermentation condition 63 IV. CONCLUSIONS 67 V. REFERENCES 68 국 문 초 록 79Maste

Changes in the Diagnostic Efficiency of Thyroid Fine-Needle Aspiration Biopsy during the Era of Increased Thyroid Cancer Screening in Korea

Purpose The volume of thyroid cancer screening and subsequent thyroid fine-needle aspiration (FNA) have rapidly increased in South Korea. We analyzed the thyroid cancer diagnoses/thyroid FNA ratio according to the annual number of FNA to evaluate changes in the diagnostic efficiency of FNA. Materials and Methods This was a nationwide population-based retrospective cohort study. The overall thyroid cancer diagnoses/thyroid FNA ratio and annual incremental thyroid cancer diagnoses/incremental thyroid FNA ratio were indirectly calculated using data obtained from the Korea Central Cancer Registry database and the Korean National Health Insurance Service claims database from 2004 to 2012. Pearson correlation analyses were performed to evaluate the strength of linear associations between variables. Results The number of thyroid FNA increased from 28,596 to 177,805 (6.2-fold increase) from 2004 to 2012. The overall thyroid cancer diagnoses/thyroid FNA ratio decreased from 36.5% in 2004 to 25.1% in 2012 and was negatively correlated to the number of FNA (R= -0.977, p < 0.001). The annual incremental thyroid cancer diagnoses/incremental thyroid FNA ratios (range, 15.3% to 30.7%) were always lower than the overall thyroid cancer diagnoses/thyroid FNA ratio in each year and also worsened according to the increase in the number of FNA (R=-0.853, p=0.007). Conclusion The diagnostic performance of both overall and annual incremental thyroid FNA worsened, whereas the number of thyroid FNA procedures increased. More sophisticated indications for FNA are required to improve its diagnostic efficiency, considering the increased burden of screening-detected thyroid nodules.ope

Hepatocellular carcinoma in Budd-Chiari syndrome: a single center experience with long-term follow-up in South Korea

AIM: To evaluate long-term clinical course of Budd-Chiari syndrome (BCS) and predictive factors associated with the development of hepatocellular carcinoma (HCC) and survival. METHODS: We analyzed 67 patients with BCS between June 1988 and May 2008. The diagnosis of BCS was confirmed by hepatic venous outflow obstruction shown on abdominal ultrasound sonography, computed tomography, magnetic resonance imaging, or venography. The median follow-up period was 103 ± 156 [interquartile range (IQR)] mo. RESULTS: The median age of the patients was 47 ± 16 (IQR) years. At diagnosis, 54 patients had cirrhosis, 25 (37.3%) Child-Pugh class A, 23 (34.3%) Child-Pugh class B, and six (9.0%) patients Child-Pugh class C. During the follow-up period, HCC was developed in 17 patients, and the annual incidence of HCC in patients with BCS was 2.8%. Patients in HCC group (n = 17) had higher hepatic venous pressure gradient (HVPG) than those in non-HCC group (n = 50) (21 ± 12 mmHg vs 14 ± 7 mmHg, P = 0.019). The survival rate of BCS patients was 86.2% for 5 years, 73.8% for 10 years, and 61.2% for 15 years. In patients with BCS and HCC, survival was 79% for 5 years, 43.1% for 10 years, and 21.5% for 15 years. CONCLUSION: The incidence of HCC in patients with BCS was similar to that in patients with other etiologic cirrhosis in South Korea. The HVPG is expected to provide additional information for predicting HCC development in BCS patients.ope

A case of isolated metastatic hepatocellular carcinoma arising from the pelvic bone

Reports of metastatic hepatocellular carcinoma (HCC) without a primary liver tumor are rare. Here we present a case of isolated HCC that had metastasized to the pelvic bone without a primary focus. A 73-year-old man presented with severe back and right-leg pain. Radiological examinations, including computed tomography (CT) and magnetic resonance imaging (MRI), revealed a huge mass on the pelvic bone (13×10 cm). He underwent an incisional biopsy, and the results of the subsequent histological examination were consistent with metastatic hepatocellular carcinoma. The tumor cells were positive for cytokeratin (AE1/AE3), hepatocyte paraffin 1, and glypican-3, and negative for CD56, chromogranin A, and synaptophysin on immunohistochemical staining. Examination of the liver by CT, MRI, positron-emission tomography scan, and angiography produced no evidence of a primary tumor. Radiotherapy and transarterial chemoembolization were performed on the pelvic bone, followed by systemic chemotherapy. These combination treatments resulted in tumor regression with necrotic changes. However, multiple lung metastases developed 1 year after the treatment, and the patient was treated with additional systemic chemotherapyope

출혈성 쇼크가 동반된 외상 환자에서 Ulinastatin의 항염증 효과

Dept. of Medicine/석사[한글]배경 및 목적: Ulinastatin은 사람의 소변에서 분리 정제된 당단백질로서 단백 분해 효소를 저해하고 항염증 작용이 있다. 또한 신장의 허혈 손상을 줄이고 수혈 후 주요 장기에 손상을 유발하는 Polymorphonuclear Leukocyte Elastase (PMNE)를 억제하는 효과가 있다고 알려져 있다. 그러나 외상에 의한 출혈성 쇼크에서는 ulinastatin의 효과는 잘 알려져 있지 않다. 본 연구에서는 출혈성 쇼크를 동반한 외상 환자에서 ulinastatin 투여가 환자의 혈청 전염증 시토카인 (cytokine)과 PMNE의 발현을 억제하여 환자의 예후에 좋은 영향을 주는지 알아보고자 한다.대상 및 방법: 2006월 6월부터 10월까지 응급실에 내원한 출혈성 쇼크를 동반한 외상 환자를 대상으로 하였다. 응급실 내원 당시에 출혈성 쇼크가 진단된 환자 중 무작위로 정하여 실험군에 해당하는 환자에게 ulinastatin을 1회에 10만 단위 씩8시간 간격으로 총 3회 투여하였다. 투여 전, 투여 후 24시간, 2일 째, 3일 째, 7일 째에 PMNE, Tumor Necrosis Factor Alpha (TNF-α), Interleukin 6 (IL-6)를 측정하였고, 내원 당시와 내원 후의 Systemic Inflammatory Response Syndrome (SIRS) score, Multiple Organ Dysfunction Syndrome (MODS) score와 Acute Physiology, Age, Chronic Health Evaluation (APACHE) III를 비교하였다.결과: 대상 환자는 모두 19명으로 대조군은 8명, 실험군은 11명이었으며, 두 군의 Injury Severity Scale은 차이가 없었다 (p=0.091). 그리고 두 군의 혈액학적 검사 소견, 치료, 사망률 등에도 유의한 차이가 없었다. PMNE의 농도는 내원 2일 째 대조군이 11.58±5.57 ng/ml, 실험군이 4.33±1.21 ng/ml로 두 군 간의 유의한 차이를 보였다 (p=0.19). TNF-α, IL-6는 내원 1일 째부터 감소하면서 대조군보다 낮은 값을 보였으나 의미 있는 차이는 없었다.결론: Ulinastatin 30만 단위는 출혈성 쇼크를 동반한 외상 환자의 혈청 PMNE 농도를 내원 2일 째에 ulinastatin을 투여하지 않은 환자보다 의미 있게 낮춘다 [영문]Background: Ulinastatin, a glycoprotein from human urine, inhibits the proteolytic action and has an anti-inflammatory effect on tissues. Ulinastatin reduces the renal dysfunction associated with the ischemia-reperfusion of the kidney as well as the blood transfusion-induced Polymorphonuclear Leukocyte Elastase (PMNE) which may injure a variety of tissues and organs. However, the effect of ulinastatin on traumatic hemorrhagic shock has rarely been reported.Purpose: The aim of this study was to investigate the use of ulinastatin in association with the suppression of plasma proinflammatory cytokine and PMNE and the good prognosis in the patients with traumatic hemorrhagic shock.Subjects and Methods: Nineteen patients who were admitted to the emergency department for trauma with hemorrhagic shock from June 2006 to October 2006 were enrolled. Eleven patients received ulinastatin at random. Ulinastatin 100,000 IU was intravenously administered every 8 hours for a total of 300,000 IU. Measurements of serum PMNE, Tumor Necrosis Factor Alpha (TNF-α) and Interleukin 6 (IL-6) were taken before ulinastatin treatment, at 24 hours, 2 days, 3 days and 7 days after admission. We compared the Systemic Inflammatory Response Syndrome (SIRS) score, the Multiple Organ Dysfunction Syndrome (MODS) score and the Acute Physiology, Age, Chronic Health Evaluation (APACHE) III between the control group and the ulinastatin group..Results: There were no significant baseline differences between the control group and the ulinastatin group. Furthermore, there were no significant differences in laboratory data, treatment and mortality between the control group and the ulinastatin group. The serum PMNE levels of the ulinastatin group were lower than the control at the second hospitalized day (11.58±5.57 vs 4.33±1.21, p=0.19). Serum TNF-α and IL-6 levels of the ulinastatin group decreased 24 hours after admission and were lower than the control, however, there were no significant differences.Conclusion: Ulinastatin 300,000 IU leads to decrease the serum PMNE in traumatic patients with a hemorrhagic shock on the second day of hospitalizationope

출혈성 쇼크를 유발한 쥐에서 저체온이 혈액 응고 기능과 생존에 미치는 영향

Dept. of Medical Science/박사Background: Acute coagulopathy, hypothermia, and acidosis are the lethal triad ofconditions manifested by major trauma patients. Recent animal studies have reported thathypothermia improves survival in animals subjected to controlled hemorrhagic shock.Purpose: The objective of this study was to investigate the effect of hypothermia oncoagulation in rats subjected to uncontrolled hemorrhagic shock.Subjects and Methods: Thirty-two male Sprague–Dawley rats were randomly dividedinto four groups: normothermia (control, group N), hypothermia (group H), hypothermichemorrhagic shock (group HS), and normothermic hemorrhagic shock (group NS).Hemorrhagic shock was induced by splenic laceration. Capacity for coagulation was measured by rotation thromboelastometry (ROTEM®), and was measured at baseline aswell as the end of the shock and resuscitation periods. Survival was observed for 48 hourspost-trauma.Results: Baseline parameters were not different among the groups. Rats exposed tohypothermia alone did not differ in coagulation capacity compared to the control group.Clot formation time (CFT) and maximal clot firmness (MCF) in group HS decreased asthe experiment progressed. Maximal clot firmness time (MCFt) in groups H and HS wassignificantly prolonged during shock and resuscitation compared with that in group NS.In group NS, MCF did not change significantly, but MCFt was reduced compared withbaseline. Group HS had poor survival when compared with normovolemic groups.Conclusion: Blood clotted less firmly in traumatic hemorrhagic shock, and hypothermiaprolonged clotting. However, clot firmness maximized rapidly under normothermichemorrhagic shock. Hemorrhage would continue for a longer time in hypothermichemorrhagic shock. Survival of hypothermic shock was not significantly differentcompared to that of normothermic hemorrhagic shock.ope

Effect of teriparatide on pregnancy and lactation-associated osteoporosis with multiple vertebral fractures

Pregnancy and lactation-associated osteoporosis (PLO) is very rare, but it can cause severe vertebral compression fractures with disabling back pain. PLO patients have commonly been treated with antiresorptive agents against high bone turnover. There are, however, some concerns regarding the use of bisphosphonates: (1) PLO occurs during the first pregnancy with a high possibility of recurrence during the second pregnancy, (2) long-term outcomes of bisphosphonates in PLO are lacking, and (3) there is a possibility of bisphosphonates accumulated in the bones crossing the placenta. Therefore, alternative therapies must be considered. We analyzed the effect of teriparatide (TPTD), the human recombinant parathyroid hormone (1-34), for 18 months in three women with PLO. Multiple vertebral fractures with severe back pain appeared within 6 months after their first childbirth. Two of them had a family history of osteoporosis. Lactation was discontinued immediately after diagnosis of PLO. Calcium carbonate, cholecalciferol, and TPTD were prescribed. The back pain immediately resolved. Bone mineral density (BMD) increased by 14.5-25.0% (mean 19.5%) at the lumbar spine and by 9.5-16.7% (mean 13.1%) at the femoral neck, after 18 months of treatment. The final Z scores in these PLO patients were nearly normalized. Two women had a second baby without any complication. BMD significantly improved after 18 months of treatment with TPTD without further fractures. In conclusion, TPTD should be considered to avoid long-term morbidity in young patients with PLO and is highly encouraged for use in PLO patients with multiple vertebral fractures.ope

소포 림프종에 동반된 강직성 척추염 1예

Several autoimmune and chronic inflammatory conditions have been consistently linked with an increased risk of hematologic malignancies. Although ankylosing spondylitis (AS) is a chronic inflammatory disease, previous studies have demonstrated that it is not associated with an increase in risk of malignant lymphomas. Cases of AS accompanied by hematologic malignancies such as multiple myeloma, chronic myelogenous leukemia, and Hodgkin's disease have been reported. In Korea, AS with non-Hodgkin's lymphoma or follicular lymphoma has not been reported. We experienced a 38-year-old male who had been diagnosed with follicular lymphoma with bone metastasis, who achieved complete remission after having been treated with chemotherapy, developed new inflammatory back pain. An MRI of his hip showed an active inflammation of the left sacroiliac joint and a positive HLA-B27. The patient was diagnosed with AS and was treated with naproxen, which improved the pain in his back and buttock.ope

Aortic calcification and bone metabolism: the relationship between aortic calcification, BMD, vertebral fracture, 25-hydroxyvitamin D, and osteocalcin.

The aim of this study was to investigate the relationship between aortic calcification (AC) and low bone mineral density (BMD), 25(OH)D, C-terminal telopeptide (CTx), and osteocalcin levels in Asian women. We also tried to find the association between AC and the risk of vertebral fracture. We included 769 patients in this study. All patients underwent QCT. Aortic calcium score (ACS) was quantified by the Agatston scoring method. Spinal fracture was defined by lumbar spine radiography. Among 769 subjects, 96 had at least one vertebral fracture and 345 had AC. ACS positively correlated with age. Osteocalcin, CTx, 25(OH)D, total-hip trabecular BMD (tBMD), femoral neck tBMD, and vertebral tBMD were inversely related with ACS. However, cortical BMD (cBMD) did not correlate with ACS. Among these parameters, only osteocalcin significantly correlated with ACS, even after adjusting for age. We divided the subjects into two groups based on the presence of AC to determine the association between AC and vertebral fracture. Multivariate logistic regression analysis showed that age, tBMD of each site, and AC were associated with vertebral fractures. After adjusting for confounding factors, patients with AC had more than a threefold increased risk of vertebral fracture (OR = 3.29-3.57, P < 0.05 according to site). This study suggests that high ACS is related to low tBMD but not cBMD. Furthermore, our findings indicate that this relationship is definitely age-dependent. Finally, we found that AC is significantly associated with the prevalence of vertebral fracture in Asian women.ope