Prospective Cohort Study with a 2-Year Follow-up of Clinical Results, Fusion Rate, and Muscle Bulk for Uniportal Full Endoscopic Posterolateral Transforaminal Lumbar Interbody Fusion.

STUDY DESIGN: Retrospective cohort study. PURPOSE: Postoperative evaluation of the cross-sectional area of paraspinal muscle and clinical findings in patients who had interlaminar route uniportal full endoscopic posterolateral transforaminal lumbar interbody fusion (EPTLIF) after 2 years. OVERVIEW OF LITERATURE: There are limited short-term follow-up studies on efficacy, safety, and physiological changes with a 2-year follow-up. There is no study on paraspinal muscle cross-sectional area change in patients who had undergone uniportal EPTLIF. METHODS: We evaluated patients who underwent EPTLIF with a minimum 24-month follow-up. Clinical parameters of the Visual Analog Scale (VAS) and Oswestry Disability Index (ODI) were measured at the preoperative, 1-week postoperative mark, postoperative 3-month mark, and final follow-up. Preoperative and 1-year postoperative magnetic resonance imaging measurement of preoperative and postoperative Kjaer grade, right and left psoas muscle mass area, and right and left paraspinal muscle mass area was performed. RESULTS: EPTLIF with a minimum 24-month follow-up of 35 levels was included. The complication rate was 6%, and the mean Bridwell's fusion grade was 1.37 (1-2). There was statistically significant improvement at 1 week, 3 months, and 2 years in VAS (4.11±1.23, 4.94±1.30, and 5.46±1.29) and in ODI (40.34±10.06, 46.69±9.14, and 49.63±8.68), respectively (p <0.05). Successful operation rate with excellent and good MacNab's criteria at 2 years was 97%. There was an increment of statistically significant bilateral psoas muscle cross-sectional area, right side (70.03±149.1 mm²) and left side (67.59±113.2 mm²) (p <0.05). CONCLUSIONS: Uniportal EPTLIF achieved good fusion and improved clinical outcomes with favorable paraspinal musculature bulk at the 2-year follow-up.

Remodeling of Epidural Fluid Hematoma after Uniportal Lumbar Endoscopic Unilateral Laminotomy with Bilateral Decompression: Comparative Clinical and Radiological Outcomes with a Minimum Follow-up of 2 Years.

STUDY DESIGN: Retrospective cohort study. PURPOSE: To evaluate the clinical and radiological effects of epidural fluid hematoma in the medium term after lumbar endoscopic decompression. OVERVIEW OF LITERATURE: There is limited literature comparing the effect of postoperative epidural fluid hematoma after uniportal endoscopic decompression. METHODS: Magnetic resonance imaging (MRI) and clinical evaluation were performed for patients with single-level uniportal endoscopic lumbar decompression with a minimum follow-up of 2 years. RESULTS: A total of 126 patients were recruited with a minimum follow-up of 26 months. The incidence of epidural fluid hematoma was 27%. Postoperative MRI revealed a significant improvement in the postoperative dura sac area at postoperative day 1 and at the upper endplate at 6 months in the hematoma cohort (39.69±15.72 and 26.89±16.58 mm2) as compared with the nonhematoma cohort (48.92±21.36 and 35.1±20.44 mm2), respectively (p <0.05); and at the lower endplate on postoperative 1 day in the hematoma cohort (51.18±24.69 mm2) compared to the nonhematoma cohort (63.91±27.92 mm2) (p <0.05). No significant difference was observed in the dura sac area at postoperative 1 year in both cohorts. The hematoma cohort had statistically significant higher postoperative 1-week Visual Analog Scale (VAS; 3.32±0.68) pain and Oswestry Disability Index (ODI; 32.65±5.56) scores than the nonhematoma cohort (2.99±0.50 and 30.02±4.84, respectively; p <0.05). No significant difference was found at the final follow-up VAS, ODI, and MRI dura sac area. CONCLUSIONS: Epidural fluid hematoma is a common early postoperative MRI finding in lumbar endoscopic unilateral laminotomy with bilateral decompression. Conservative management is the preferred treatment option for patients who do not have a neurological deficit. Symptoms last only a few days and are self-limiting. A common endpoint is a remodeled fluid hematoma and the subsequent expansion of the dura sac area.

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside.

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

Remodeling Pattern of Spinal Canal after Full Endoscopic Uniportal Lumbar Endoscopic Unilateral Laminotomy for Bilateral Decompression: One Year Repetitive MRI and Clinical Follow-Up Evaluation.

Objective: There is limited literature on repetitive postoperative MRI and clinical evaluation after Uniportal Lumbar Endoscopic Unilateral Laminotomy for Bilateral Decompression. Methods: Clinical visual analog scale, Oswestry Disability Index, McNab's criteria evaluation and MRI evaluation of the axial cut spinal canal area of the upper end plate, mid disc and lower end plate were performed for patients who underwent single-level Uniportal Lumbar Endoscopic Unilateral Laminotomy for Bilateral Decompression. From the evaluation of the axial cut MRI, four types of patterns of remodeling were identified: type A: continuous expanded spinal canal, type B: restenosis with delayed expansion, type C: progressive expansion and type D: restenosis. Result: A total of 126 patients with single-level Uniportal Lumbar Endoscopic Unilateral Laminotomy for Bilateral Decompression were recruited with a minimum follow-up of 26 months. Thirty-six type A, fifty type B, thirty type C and ten type D patterns of spinal canal remodeling were observed. All four types of patterns of remodeling had statistically significant improvement in VAS at final follow-up compared to the preoperative state with type A (5.59 ± 1.58), B (5.58 ± 1.71), C (5.58 ± 1.71) and D (5.27 ± 1.68), p < 0.05. ODI was significantly improved at final follow-up with type A (49.19 ± 10.51), B (50.00 ± 11.29), C (45.60 ± 10.58) and D (45.60 ± 10.58), p < 0.05. A significant MRI axial cut increment of the spinal canal area was found at the upper endplate at postoperative day one and one year with type A (39.16 ± 22.73; 28.00 ± 42.57) mm2, B (47.42 ± 18.77; 42.38 ± 19.29) mm2, C (51.45 ± 18.16; 49.49 ± 18.41) mm2 and D (49.10 ± 23.05; 38.18 ± 18.94) mm2, respectively, p < 0.05. Similar significant increment was found at the mid-disc at postoperative day one, 6 months and one year with type A (55.16 ± 27.51; 37.23 ± 25.88; 44.86 ± 25.73) mm2, B (72.83 ± 23.87; 49.79 ± 21.93; 62.94 ± 24.43) mm2, C (66.85 ± 34.48; 54.92 ± 30.70; 64.33 ± 31.82) mm2 and D (71.65 ± 16.87; 41.55 ± 12.92; 49.83 ± 13.31) mm2 and the lower endplate at postoperative day one and one year with type A (49.89 ± 34.50; 41.04 ± 28.56) mm2, B (63.63 ± 23.70; 54.72 ± 24.29) mm2, C (58.50 ± 24.27; 55.32 ± 22.49) mm2 and D (81.43 ± 16.81; 58.40 ± 18.05) mm2 at postoperative day one and one year, respectively, p < 0.05. Conclusions: After full endoscopic lumbar decompression, despite achieving sufficient decompression immediately postoperatively, varying severity of asymptomatic restenosis was found in postoperative six months MRI without clinical significance. Further remodeling with a varying degree of increment of the spinal canal area occurs at postoperative one year with overall good clinical outcomes.

CRISPRi-Guided Metabolic Flux Engineering for Enhanced Protopanaxadiol Production in Saccharomyces cerevisiae.

Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in Panax ginseng and a long cultivation time (4-6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in Saccharomyces cerevisiae and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in S. cerevisiae. Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in S. cerevisiae. With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in S. cerevisiae, we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.

Ginsenoside Rh2 Ameliorates Atopic Dermatitis in NC/Nga Mice by Suppressing NF-kappaB-Mediated Thymic Stromal Lymphopoietin Expression and T Helper Type 2 Differentiation.

Ginsenosides are known to have various highly pharmacological activities, such as anti-cancer and anti-inflammatory effects. However, the search for the most effective ginsenosides against the pathogenesis of atopic dermatitis (AD) and the study of the effects of ginsenosides on specific cytokines involved in AD remain unclear. In this study, ginsenoside Rh2 was shown to exert the most effective anti-inflammatory action on thymic stromal lymphopoietin (TSLP) and interleukin 8 in tumor necrosis factor-alpha and polyinosinic: polycytidylic acid induced normal human keratinocytes by inhibiting proinflammatory cytokines at both protein and transcriptional levels. Concomitantly, Rh2 also efficiently alleviated 2,4-dinitrochlorobenzene-induced AD-like skin symptoms when applied topically, including suppression of immune cell infiltration, cytokine expression, and serum immunoglobulin E levels in NC/Nga mice. In line with the in vitro results, Rh2 inhibited TSLP levels in AD mice via regulation of an underlying mechanism involving the nuclear factor κB pathways. In addition, in regard to immune cells, we showed that Rh2 suppressed not only the expression of TSLP but the differentiation of naïve CD4+ T-cells into T helper type 2 cells and their effector function in vitro. Collectively, our results indicated that Rh2 might be considered as a good therapeutic candidate for the alternative treatment of AD.

mRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in Escherichia coli.

The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3' untranslated region of a target mRNA by fusing with an RNA-binding domain in the chaperone-recruiting mRNA scaffold (CRAS) system, or coupled the expression of DnaJ and a target recombinant protein using the overlapping stop-start codons 5'-TAATG-3' between the two genes in a chaperone-substrate co-localized expression (CLEX) system. By engineering the untranslated and intergenic sequences of the mRNA transcript, bacterial molecular chaperones are spatially constrained to the location of protein translation, expressing selected aggregation-prone proteins in their functionally active, soluble form. Our mRNA engineering methods surpassed the in-vivo solubilization efficiency of the simple DnaJ chaperone co-overexpression method, thus providing more effective tools for producing soluble therapeutic proteins and enzymes.

Gypenoside LXXV Promotes Cutaneous Wound Healing In Vivo by Enhancing Connective Tissue Growth Factor Levels Via the Glucocorticoid Receptor Pathway.

Cutaneous wound healing is a well-orchestrated event in which many types of cells and growth factors are involved in restoring the barrier function of skin. In order to identify whether ginsenosides, the main active components of Panax ginseng, promote wound healing, the proliferation and migration activities of 15 different ginsenosides were tested by MTT assay and scratched wound closure assay. Among ginsenosides, gypenoside LXXV (G75) showed the most potent wound healing effects. Thus, this study aimed to investigate the effects of G75 on wound healing in vivo and characterize associated molecular changes. G75 significantly increased proliferation and migration of keratinocytes and fibroblasts, and promoted wound closure in an excision wound mouse model compared with madecassoside (MA), which has been used to treat wounds. Additionally, RNA sequencing data revealed G75-mediated significant upregulation of connective tissue growth factor (CTGF), which is known to be produced via the glucocorticoid receptor (GR) pathway. Consistently, the increase in production of CTGF was confirmed by western blot and ELISA. In addition, GR-competitive binding assay and GR translocation assay results demonstrated that G75 can be bound to GR and translocated into the nucleus. These results demonstrated that G75 is a newly identified effective component in wound healing.

Adaptive laboratory evolution of a genome-reduced Escherichia coli.

Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain's systems biology.

Improved n-butanol tolerance in Escherichia coli by controlling membrane related functions.

As the increasing demand from both chemical and fuel markets, the interest in producing n-butanol using biological route has been rejuvenated to engineer an economical fermentation process, competing with the currently-dominant chemical synthesis. n-Butanol has been traditionally produced from the ABE fermentation of Clostridium acetobutylicum. This system, however, is not economically feasible due to its limited efficiency and the lack of genetic modification tools for further improvements. Alternatively, n-butanol synthesis pathway was successfully transferred into Escherichia coli and rapidly improved to reach a level of production comparable to the native producer. Nevertheless, the toxicity of n-butanol has become a common issue that either approach has to deal with. Previously, we reported our success in improving n-butanol tolerance in E. coli by engineering an Artificial Transcription Factor (ATF) that can modify the expression level of multiple targets simultaneously and improved the n-butanol tolerance of MG1655 strain to 1.5% (vol/vol) n-butanol. However, it was observed that some possible n-butanol tolerance mechanisms did not occurred upon the ATF expression, especially the membrane-related functions such as the homeoviscous adaptation, iron uptaking system, and efflux pump system. In this work, we attempted to enhance the n-butanol tolerance associated with the ATF by combining it with the membrane-related functions in E. coli, including the overexpression of fatty acid synthesis genes, iron-uptaking protein FeoA, and introducing a SrpABC efflux pump from Pseudomonas putida into E. coli. The synergistic effect of this combinatorial approach led to 4, 5, and 9-fold improved growths in the cultures containing 1, 1.5, and 2% (vol/vol) n-butanol, respectively, of an MG1655 knockout strain engineered for n-butanol production, and expanded the tolerance limit to 2% (vol/vol) n-butanol.

Enhanced production of n-alkanes in Escherichia coli by spatial organization of biosynthetic pathway enzymes.

Alkanes chemically mimic hydrocarbons found in petroleum, and their demand as biofuels is steadily increasing. Biologically, n-alkanes are produced from fatty acyl-ACPs by acyl-ACP reductases (AARs) and aldehyde deformylating oxygenases (ADOs). One of the major impediments in n-alkane biosynthesis is the low catalytic turnover rates of ADOs. Here, we studied n-alkane biosynthesis in Escherichia coli using a chimeric ADO-AAR fusion protein or zinc finger protein-guided ADO/AAR assembly on DNA scaffolds to control their stoichiometric ratios and spatial arrangements. Bacterial production of n-alkanes with the ADO-AAR fusion protein was increased 4.8-fold (24 mg/L) over a control strain expressing ADO and AAR separately. Optimal n-alkane biosynthesis was achieved when the ADO:AAR binding site ratio on a DNA scaffold was 3:1, yielding an 8.8-fold increase (44 mg/L) over the control strain. Our findings indicate that the spatial organization of alkane-producing enzymes is critical for efficient n-alkane biosynthesis in E. coli.

Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd.

Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 4-6 years, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Dammarene-type ginsenosides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hydroxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, dammarenediol-II synthase (DS) and two P450 enzymes, protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), have been reported, but glycosyltransferases that are necessary to synthesize specific ginsenosides have not yet been fully identified. To discover glycosyltransferases responsible for ginsenoside biosynthesis, we sequenced and assembled the ginseng transcriptome de novo and characterized two UDP-glycosyltransferases (PgUGTs): PgUGT74AE2 and PgUGT94Q2. PgUGT74AE2 transfers a glucose moiety from UDP-glucose (UDP-Glc) to the C3 hydroxyl groups of PPD and compound K to form Rh2 and F2, respectively, whereas PgUGT94Q2 transfers a glucose moiety from UDP-Glc to Rh2 and F2 to form Rg3 and Rd, respectively. Introduction of the two UGT genes into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3. Our results indicate that these two UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation.

Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences.

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10(-8)/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10(-8)/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.

Improved production of L-threonine in Escherichia coli by use of a DNA scaffold system.

Despite numerous approaches for the development of l-threonine-producing strains, strain development is still hampered by the intrinsic inefficiency of metabolic reactions caused by simple diffusion and random collisions of enzymes and metabolites. A scaffold system, which can promote the proximity of metabolic enzymes and increase the local concentration of intermediates, was reported to be one of the most promising solutions. Here, we report an improvement in l-threonine production in Escherichia coli using a DNA scaffold system, in which a zinc finger protein serves as an adapter for the site-specific binding of each enzyme involved in l-threonine production to a precisely ordered location on a DNA double helix to increase the proximity of enzymes and the local concentration of metabolites to maximize production. The optimized DNA scaffold system for l-threonine production significantly increased the efficiency of the threonine biosynthetic pathway in E. coli, substantially reducing the production time for l-threonine (by over 50%). In addition, this DNA scaffold system enhanced the growth rate of the host strain by reducing the intracellular concentration of toxic intermediates, such as homoserine. Our DNA scaffold system can be used as a platform technology for the construction and optimization of artificial metabolic pathways as well as for the production of many useful biomaterials.

Clinical and angiographic outcomes of wingspan stent placement for treatment of symptomatic intracranial stenosis: single center experience with 19 cases.

OBJECTIVE: The limitations of medical management of symptomatic intracranial arterial stenosis (ICS) have prompted development of new strategies, including endovascular treatment. However, stenting of symptomatic ICS remains investigational. Here, we have reported and analyzed a series of 19 endovascular procedures involving placement of a Wingspan stent. METHODS: We conducted a retrospective review of a series of ICS in which patients were treated with percutaneous transarterial balloon angioplasty and stent placement (PTAS). Patients included in the study were diagnosed as symptomatic ICS between May 2010 and September 2011. RESULTS: Nineteen patients (median age, 65 years; 12 males, seven women) were treated with the Wingspan stent system for symptomatic ICS ranging from 50% to 99%. The technical success rate was 100%. The location of ICS included the internal carotid (n = 5; 1 petrous, 3 cavernous, and 1 clinoid segments), vertebral (n = 1; V4 segment), basilar (n = 1), and middle cerebral (n = 12; 9 M1, 3 M2) arteries. There was no occurrence of procedure-related mortality. Periprocedural morbidity occurred in two cases (10.5%), including carotid-cavernous fistula (n = 1) and subarachnoid hemorrhage (n = 1). No ipsilateral stroke was recorded beyond 30 days during a mean follow-up period of 13.2 months (range 9-19 months). Restenosis (> 50%) was observed in one patient (6.3%), who was asymptomatic, on follow-up imaging. CONCLUSION: Wingspan stent for symptomatic ICS can be performed with a high rate of technical success and acceptable periprocedural morbidity rates. Our initial experience indicates that this procedure represents a viable treatment option for this patient population.

Stereotactic burr hole aspiration surgery for spontaneous hypertensive cerebellar hemorrhage.

OBJECTIVE: Patients with severe spontaneous cerebellar hemorrhage typically undergo treatment with suboccipital craniectomy and hematoma evacuation. However, this is a stressful procedure for patients due to the long operating time and operation-induced tissue damage. In addition, the durotomy can result in pseudomeningocele. We investigated the efficacy of stereotactic or navigation-guided burr hole aspiration surgery as a treatment for spontaneous hypertensive cerebellar hemorrhage (SHCH). METHODS: Between January 2002 and December 2011, 26 patients with SHCH underwent surgery using the stereotactic or navigation-guided burr hole aspiration and catheter insertion technique in our institution. RESULTS: Mean hematoma volume was 21.8 ± 5.8 cc at admission and 13.1 ± 5.4 cc immediately following surgery. Preoperative Glasgow Coma Scale (GCS) score was 12.5 ± 1.3 and postoperative GCS score was 13.1 ± 1.2. Seven days after surgery, the mean hematoma volume was 4.3 ± 5.6 cc, and there was no occurrence of surgery-related complications during the six-month follow-up period. The mean operation time for catheter insertion was 43.1 ± 8.9 min, and a mean 31.3 ± 6.0 min was also added for extra-ventricular drainage. The mean Glasgow Outcome Scale (GOS) score after six months was 4.6 ± 1.0. CONCLUSION: Stereotactic burr hole aspiration surgery for treatment of SHCH is less time-consuming and invasive than other interventions, and resulted in no surgery-related complications. Therefore, we suggest that this surgical method could be a safe and effective treatment option for selected patients with SHCH.

Scarless chromosomal gene knockout methods.

An improved and rapid genomic engineering method has been developed for the construction of -custom-designed microorganisms by scarless chromosomal gene knockouts. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via scarless deletion of selected genomic regions. The deletion process is mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (1) an arabinose-inducible promoter that drives expression of λ-RED recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (2) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which accomplishes deletion of the introduced marker by double-strand breakage - mediated intramolecular recombination. This genomic deletion is performed simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette are nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.

Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production.

BACKGROUND: Deletion of large blocks of nonessential genes that are not needed for metabolic pathways of interest can reduce the production of unwanted by-products, increase genome stability, and streamline metabolism without physiological compromise. Researchers have recently constructed a reduced-genome Escherichia coli strain MDS42 that lacks 14.3% of its chromosome. RESULTS: Here we describe the reengineering of the MDS42 genome to increase the production of the essential amino acid L-threonine. To this end, we over-expressed a feedback-resistant threonine operon (thrA*BC), deleted the genes that encode threonine dehydrogenase (tdh) and threonine transporters (tdcC and sstT), and introduced a mutant threonine exporter (rhtA23) in MDS42. The resulting strain, MDS-205, shows an ~83% increase in L-threonine production when cells are grown by flask fermentation, compared to a wild-type E. coli strain MG1655 engineered with the same threonine-specific modifications described above. And transcriptional analysis revealed the effect of the deletion of non-essential genes on the central metabolism and threonine pathways in MDS-205. CONCLUSION: This result demonstrates that the elimination of genes unnecessary for cell growth can increase the productivity of an industrial strain, most likely by reducing the metabolic burden and improving the metabolic efficiency of cells.

Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli.

Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 x 10(4) ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure and cold shock. Genes associated with the heat-shock resistance phenotype were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the phenotypic engineering of microorganisms and can facilitate microbial functional genomic studies.