CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids.

Antibiotics have been widely used for plasmid-mediated cell engineering. However, continued use of antibiotics increases the metabolic burden, horizontal gene transfer risks, and biomanufacturing costs. There are limited approaches to maintaining multiple plasmids without antibiotics. Herein, we developed an inverter cascade using CRISPRi by building a plasmid containing a single guide RNA (sgRNA) landing pad (pSLiP); this inhibited host cell growth by repressing an essential cellular gene. Anti-sgRNAs on separate plasmids restored cell growth by blocking the expression of growth-inhibitory sgRNAs in pSLiP. We maintained three plasmids in Escherichia coli with a single antibiotic selective marker. To completely avoid antibiotic use and maintain the CRISPRi-based logic inverter cascade, we created a novel d-glutamate auxotrophic E. coli. This enabled the stable maintenance of the plasmid without antibiotics, enhanced the production of the terpenoid, (-)-α-bisabolol, and generation of an antibiotic-resistance gene-free plasmid. CRISPRi is therefore widely applicable in genetic circuits and may allow for antibiotic-free biomanufacturing.

Proximal row carpectomy with interposition arthroplasty using both capsular flap and acellular human dermal matrix.

BACKGROUND: In cases of wrist arthritis, proximal row carpectomy (PRC) has been widely utilized and shown favorable long-term outcomes. However, its applicability is limited in cases where arthritis extends to the lunate fossa or capitate. Recently, surgical approaches combining various methods of interposition arthroplasty have been introduced to overcome these drawbacks. The purpose of this study was to perform PRC and interposition arthroplasty with dorsal capsule and acellular dermal matrix(ADM),and analyze the clinical outcomes of these procedures. METHODS: Fourteen cases who underwent PRC and interposition arthroplasty using both dorsal capsular flap and ADM were retrospectively recruited. The researchers assessed the patients' Visual Analog Scale (VAS) pain score, Disabilities of the Arm, Shoulder and Hand (DASH) scores, range of motion (ROM), retear, and radiocarpal distance (RCD). RESULTS: One year post-surgery, both the VAS pain scores, DASH scores, and ROM showed statistically significant improvement compared to before the surgery. Upon reviewing the radiological results, the postoperative mean RCD was 4.8 ± 0.8 mm and one year follow up mean RCD was 3.6 ± 0.5 mm at one year post-surgery. Moreover, in the one year follow-up, there was no observed failure of the allodermis graft in any of the cases. CONCLUSION: The PRC and interposition arthroplasty with ADM demonstrated significantly improved clinical outcomes after surgery, showing a maintain of RCD without graft failure effectively.

Hericerin derivatives activates a pan-neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory.

The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA-12 only partly prevented the NDPIH-induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK-293T cells, an effect that was not sensitive to ANA-12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan-neurotrophic signaling pathway, leading to improved cognitive performance.

Scientific evidence of foods that improve the lifespan and healthspan of different organisms.

Age is a risk factor for numerous diseases. Although the development of modern medicine has greatly extended the human lifespan, the duration of relatively healthy old age, or 'healthspan', has not increased. Targeting the detrimental processes that can occur before the onset of age-related diseases can greatly improve health and lifespan. Healthspan is significantly affected by what, when and how much one eats. Dietary restriction, including calorie restriction, fasting or fasting-mimicking diets, to extend both lifespan and healthspan has recently attracted much attention. However, direct scientific evidence that consuming specific foods extends the lifespan and healthspan seems lacking. Here, we synthesized the results of recent studies on the lifespan and healthspan extension properties of foods and their phytochemicals in various organisms to confirm how far the scientific research on the effect of food on the lifespan has reached.

Does the anteromedial plate position affect proximal screw length and worsen the clinical outcomes in medial opening wedge high tibial osteotomy?

BACKGROUND: During medial opening wedge high tibial osteotomy (MOWHTO), sometimes the plate tends to be positioned anteromedially. The plate position can affect the length of the proximal screw, which significantly affects stability after osteotomy. Therefore, research on the correlation among plate position, screw length, and clinical outcomes is needed. METHODS: This retrospective review examines 196 knees in 175 patients who underwent MOWHTO from May 2012 to December 2018, for symptomatic medial compartment osteoarthritis with a varus alignment of > 5°. We evaluated the anteroposterior plate position, length of proximal screw, and postoperative computed tomography (CT). We reviewed patients' clinical outcome scores, presence of lateral hinge fracture, neurovascular complications, and infection. The correlation among proximal plate position, proximal screw length, and clinical outcomes was evaluated using Pearson's correlation analysis. A subgroup analysis by screw angle (> 48 ° or < 48 °) was also performed using chi-square test and Student t-test. RESULTS: The mean proximal plate position was 16.28% (range, 5.17-44.74) of the proximal tibia's anterior-to-posterior distance ratio, and the proximal screw length averaged 63.8 mm (range, 44-80 mm). Proximal posteromedial plate position and proximal screw length were significantly correlated (r2 = 0.667, P < .001), as were screw angle and length (r2 = 0.746, P < .001). Medial plating (< 48°) can use a longer proximal screw; nevertheless, no significant difference occurred in clinical outcomes between the two groups. Also, no differences occurred in complication rate, including hinge fracture. CONCLUSION: With more medially positioned plating during MOWHTO, we can use longer proximal screws. However, there was no significant difference in clinical outcomes and the incidence of lateral hinge fractures regardless of plate position and screw length.

Sectioning of the Anterolateral Ligaments in Anterior Cruciate Ligament Sectioned Knees Increases Internal Rotation of the Knee Joint: A Systematic Review and Meta-analysis of Cadaveric Studies.

PURPOSE: To investigate whether anterolateral ligament (ALL) sectioning (sALL) in the anterior cruciate ligament (ACL)-sectioned (sACL) knee increases the anterior tibial translation (ATT) or internal rotation (IR) of the knee from previous cadaveric biomechanical studies. METHODS: Multiple comprehensive literature databases, including PubMed (MEDLINE), EMBASE, and Cochrane Library, were searched for studies evaluating the in vitro biomechanical function of ALL. This meta-analysis compared the increased ATT and IR between the sACL and sACL + sALL knees at 30°, 60°, and 90° of knee flexion. Thresholds of 2 mm for the difference in ATT and 2° for the difference in IR were considered to be clinically significant. RESULTS: Thirteen cadaveric biomechanical studies were included. All 13 studies satisfied the threshold for a satisfactory methodological quality (Quality Appraisal for Cadaveric Studies score >75%). At 30° of knee flexion, the meta-analysis showed a greater increase in ATT in the sACL + sALL knees than in the sACL knees by 1.23 mm (95% confidence interval [CI], 0.62-1.84; P < .0001). However, the mean difference was less than the minimal clinically significant difference (<2 mm). The meta-analysis also showed a greater increase in IR in the sACL + sALL knees than in the sACL knees at 30° (mean difference [MD]: 2.24°; 95% CI: 1.39-3.09; P < .00001), 60° (MD: 2.77°; 95% CI: 1.88-3.67; P < .00001), and 90° (MD: 2.29°; 95% CI: 1.42-3.15; P < .00001) of knee flexion. The differences in IR at 30°, 60°, and 90° of knee flexion were clinically relevant (>2°). CONCLUSIONS: Despite the different experimental setups and protocols between studies, the meta-analysis of biomechanical cadaveric studies showed that sectioning of the ALL in sACL knees increased IR at 30°, 60°, and 90° of knee flexion. CLINICAL RELEVANCE: The results of this systematic review and meta-analysis suggest that ALL contributes to IR in ACL-deficient knees at 30°, 60°, and 90° of flexion.

Serial change of femoral and tibial tunnel width after anterior cruciate ligament reconstruction with allograft.

PURPOSE: To investigate progressive tunnel widening and its correlation with postoperative outcomes after anterior cruciate ligament (ACL) reconstruction using allografts. METHODS: Sixty-five patients who underwent ACL reconstruction using a tibialis anterior allograft between 2015 and 2017 were enrolled. Femoral and tibial tunnel widths were measured on anteroposterior (AP) and lateral radiographs immediately and at 3, 6, 12, and 24 months postoperatively. Average femoral and tibial tunnel widths in AP and lateral views were calculated at three different measurement points. Tunnel widening was calculated as the difference in tunnel width immediately and 2 years postoperatively. The correlation between tunnel widening and the postoperative results was analysed. RESULTS: Tunnel width changes between immediate and 2 years postoperatively were as follows, in AP and lateral views, respectively: femur, 3.0 mm ± 1.5 mm and 2.4 mm ± 1.4 mm; and tibia, 2.8 mm ± 1.4 mm and 2.9 mm ± 1.5 mm. Femoral tunnel widths significantly increased until 1 year, but not from 1 to 2 years postoperatively. Tibial tunnel width significantly increased until 2 years postoperatively. In all tunnels, the increments in tunnel widening decreased over time. Increased knee laxity significantly correlated with greater femoral tunnel widening in AP (r = 0.346, P = 0.006) and lateral views (r = 0.261, P = 0.049). CONCLUSION: Femoral tunnel widths gradually increased until 1 year postoperatively, and tibial tunnel widths increased until 2 years after ACL reconstruction with allografts. The tunnel widening rate gradually decreased over time. Femoral tunnel widening of 3.7 mm and 3.2 mm on AP and lateral views, respectively, were the cut-off values for postoperative knee laxity. LEVEL OF EVIDENCE: Level III.

Changes in ankle and hip joints following medial opening-wedge high tibial osteotomy affect knee joint line obliquity.

PURPOSE: This study aimed to measure the change in knee joint line obliquity (KJLO) and the changes in radiologic parameters of the ankle and hip joints after medial opening-wedge high tibial osteotomy (MOWHTO), and to evaluate the correlation and causal relationship between these parameters. METHODS: This study evaluated 109 patients who underwent MOWHTO between April 2015 and December 2021. Radiologic parameters, including KJLO, medial proximal tibial angle (MPTA), ankle joint line obliquity (AJLO), and hip abduction angle (HAA), were analysed before and 1 year after MOWHTO. Multiple linear regression analysis was used to identify independent variables that significantly affected the change in KJLO after MOWHTO. Receiver operating characteristic (ROC) analysis was used to evaluate the cutoff value for a change in KJLO that exceeded 5° postoperatively, and the predicting values of radiologic parameters. RESULTS: Multiple linear regression analysis showed that changes in MPTA, AJLO, and HAA (ß = 0.440, P < 0.001; ß = - 0.310, P < 0.001; ß = 0.164, P = 0.035, respectively) were predictors of the change in KJLO after MOWHTO. ROC analysis showed that the threshold value for a change in KJLO which exceeded 5° postoperatively was 4.6° (66.7% sensitivity, 63.8% specificity, P = 0.025). Moreover, ROC curves for predicting a change in KJLO of > 4.6° showed that the AUC was significantly higher for the change in MPTA than that of the other two parameters (P = 0.011 for AJLO and P < 0.001 for HAA). CONCLUSION: MOWHTO increases the KJLO by valgization of the proximal tibia and causes hip adduction and ankle valgization. The postoperative ankle valgization after MOWHTO could reduce the increase in KJLO, counteracting the effects of proximal tibial valgization and hip adduction. Therefore, the effects of the hip and ankle joints should be considered to achieve an optimal KJLO and satisfactory clinical outcomes after MOWHTO. LEVEL OF STUDY: Cohort study, IV.

Adaptive laboratory evolution of Escherichia coli W enhances gamma-aminobutyric acid production using glycerol as the carbon source.

The microbial conversion of glycerol into value-added commodity products has emerged as an attractive means to meet the demands of biosustainability. However, glycerol is a non-preferential carbon source for productive fermentation because of its low energy density. We employed evolutionary and metabolic engineering in tandem to construct an Escherichia coli strain with improved GABA production using glycerol as the feedstock carbon. Adaptive evolution of E. coli W under glycerol-limited conditions for 1300 generations harnessed an adapted strain with a metabolic system optimized for glycerol utilization. Mutation profiling, enzyme kinetic assays, and transcriptome analysis of the adapted strain allowed us to decipher the basis of glycerol adaptation at the molecular level. Importantly, increased substrate influx mediated by the mutant glpK and modulation of intracellular cAMP levels were the key drivers of improved fitness in the glycerol-limited condition. Leveraging the enhanced capability of glycerol utilization in the strain, we constructed a GABA-producing E. coli W-derivative with superior GABA production compared to the wild-type. Furthermore, rationally designed inactivation of the non-essential metabolic genes, including ackA, mgsA, and gabT, in the glycerol-adapted strain improved the final GABA titer and specific productivity by 3.9- and 4.3-fold, respectively, compared with the wild-type.

Total Synthesis of Isohericerinol A and Its Analogues to Access Their Potential Neurotrophic Effects.

The secondary metabolites from Hericium erinaceus are well-known to have neurotrophic and neuroprotective effects. Isohericerinol A (1), isolated by our colleagues from its fruiting parts has a strong ability to increase the nerve growth factor secretion in C6 glioma cells. The current work describes the total synthesis of 1 and its regioisomer 5 in a few steps. We present two different approaches to 1 and a regiodivergent approach for both 1 and 5 by utilizing easily accessible feedstocks. Interestingly, the natural product 1, regioisomer 5, and their intermediates exhibited potent neurotrophic activity in in vitro experimental systems. Thus, these synthetic strategies provide access to a systematic structure-activity relationship study of natural product 1.

Distal femoral phenotypes in Asian varus osteoarthritic knees.

PURPOSE: There has been a general consensus regarding the varus phenotype of the proximal tibia in osteoarthritic patients with varus knee alignment of the whole limb. However, a valgus phenotype of the distal femur may occur in osteoarthritic patients with varus knee alignment. This study evaluated the distal femur phenotype in varus osteoarthritic knees. METHODS: This study included 128 patients who underwent primary total knee arthroplasty (TKA) by computer-assisted navigation for primary medial osteoarthrosis with varus knee alignment. The hip-knee-ankle (HKA) angle, medial proximal tibial angle (MPTA), lateral distal femoral angle (LDFA), and joint line convergence angle (JLCA) were measured on which radiographs preoperatively. The radiographic parameters were compared between groups with HKA angle varus ≥ 10° and < 10°. RESULTS: The MPTA was significantly lower (4°) in the HKA angle varus ≥ 10° group than in the < 10° group (82.13° vs. 86.13° P = 0.001), but the LDFA did not differ significantly between the groups (89.81° vs. 89.19° P = 0.181). Regarding the JLCA, the varus ≥ 10° group showed a 1.3° greater lateral widening than the varus < 10° group (4.87 vs. 3.56, P = 0.002). The MPTA was the only independent predictor of the MA of the lower limb (ß = -  0.353, P < 0.001). CONCLUSION: One-third of varus osteoarthritic knees had a distal femur valgus phenotype. Varus knee alignment was mainly affected by proximal tibia varus rather than by distal femur varus. LEVEL OF EVIDENCE: Level III, consecutive case series.

Comparison of Anatomical Conformity between TomoFix Anatomical Plate and TomoFix Conventional Plate in Open-Wedge High Tibial Osteotomy.

Background and Objectives: The TomoFix anatomical plate was developed to improve plate position, proximal screw direction, and post-correction tibial contouring. The purpose of this study was to compare postoperative configurations between the TomoFix anatomical plate and the TomoFix conventional plate. It was hypothesized that the new modified plate provides a better fixative coaptation than the conventional plate. Materials and Methods: A total of 116 cases (112 patients) were enrolled in this study from March 2015 to February 2021. Among them, 63 patients underwent surgery using the TomoFix conventional plate, and 53 underwent surgery using the TomoFix anatomical plate. The radiographic outcomes, including the hip−knee−ankle (HKA) angle, medial proximal tibial angle (MPTA), tibial slope, plate angle, proximal screw angles, and plate-to-cortex distance at #1 hole (just below the osteotomy site) were compared between the two groups. Results: Patients with the TomoFix anatomical plate showed similar results in terms of the pre- and postoperative HKA angle, MPTA, and tibial slope. The TomoFix anatomical group showed a significantly greater plate angle (39.2° ± 8.1° vs. 31.7° ± 7.0°, p < 0.001) and less screw angles, indicating that the TomoFix anatomical plates allowed a more posterior plate position than the conventional plate. The plate-to-cortex distance was significantly less in the TomoFix anatomical group than in the TomoFix conventional group (p < 0.001). Conclusion: The TomoFix anatomical plate showed a more posteromedial plating position, better proximal screw direction to the lateral hinge, and improved post-correction tibial contour compared to the TomoFix conventional plate.

Engineering Bacteroides thetaiotaomicron to produce non-native butyrate based on a genome-scale metabolic model-guided design.

Bacteroides thetaiotaomicron represents a major symbiont of the human gut microbiome that is increasingly viewed as a promising candidate strain for microbial therapeutics. Here, we engineer B. thetaiotaomicron for heterologous production of non-native butyrate as a proof-of-concept biochemical at therapeutically relevant concentrations. Since B. thetaiotaomicron is not a natural producer of butyrate, we heterologously expressed a butyrate biosynthetic pathway in the strain, which led to the production of butyrate at the final concentration of 12 mg/L in a rich medium. Further optimization of butyrate production was achieved by a round of metabolic engineering guided by an expanded genome-scale metabolic model (GEM) of B. thetaiotaomicron. The in silico knock-out simulation of the expanded model showed that pta and ldhD were the potent knock-out targets to enhance butyrate production. The maximum titer and specific productivity of butyrate in the pta-ldhD double knockout mutant increased by nearly 3.4 and 4.8 folds, respectively. To our knowledge, this is the first engineering attempt that enabled butyrate production from a non-butyrate producing commensal B. thetaiotaomicron. The study also highlights that B. thetaiotaomicron can serve as an effective strain for live microbial therapeutics in human.

Syntrophic co-culture of a methanotroph and heterotroph for the efficient conversion of methane to mevalonate.

As the bioconversion of methane becomes increasingly important for bio-industrial and environmental applications, methanotrophs have received much attention for their ability to convert methane under ambient conditions. This includes the extensive reporting of methanotroph engineering for the conversion of methane to biochemicals. To further increase methane usability, we demonstrated a highly flexible and efficient modular approach based on a synthetic consortium of methanotrophs and heterotrophs mimicking the natural methane ecosystem to produce mevalonate (MVA) from methane. In the methane-conversion module, we used Methylococcus capsulatus Bath as a highly efficient methane biocatalyst and optimized the culture conditions for the production of high amounts of organic acids. In the MVA-synthesis module, we used Escherichia coli SBA01, an evolved strain with high organic acid tolerance and utilization ability, to convert organic acids to MVA. Using recombinant E. coli SBA01 possessing genes for the MVA pathway, 61 mg/L (0.4 mM) of MVA was successfully produced in 48 h without any addition of nutrients except methane. Our platform exhibited high stability and reproducibility with regard to cell growth and MVA production. We believe that this versatile system can be easily extended to many other value-added processes and has a variety of potential applications.

Neurotrophic isoindolinones from the fruiting bodies of Hericium erinaceus.

Four compounds, hericerin (1), isohericerinol A (2), N-de-phenylethyl isohericerin (3) and corallocin A (4) were isolated from the fruiting bodies of Hericium erinaceus, a lion's mane mushroom (Hericiaceae). Among them, isohericerinol A (2) was newly reported in nature. Further investigation of the neurotrophic effect of isolated compounds demonstrated that isohericerinol A (2) strongly increased the nerve growth factor (NGF) production in C6 glioma cells followed by corallocin A (4) and hericerin (1). Increased NGF production by these compounds promoted the neurite outgrowth in N2a neuronal cells. Western blot analysis also showed the increased protein expression of NGF, brain-derived neurotrophic factor (BDNF) and synaptophysin (SYP) in C6-N2a cells. Taken together, our present study characterized the neurotrophic constituents of H. erinaceus, which may support the potential use of memory improvement.

Proteomic Analysis of the Meniscus Cartilage in Osteoarthritis.

The distribution of differential extracellular matrix (ECM) in the lateral and medial menisci can contribute to knee instability, and changes in the meniscus tissue can lead to joint disease. Thus, deep proteomic identification of the lateral and medial meniscus cartilage is expected to provide important information for treatment and diagnosis of various knee joint diseases. We investigated the proteomic profiles of 12 lateral/medial meniscus pairs obtained from excess tissue of osteoarthritis patients who underwent knee arthroscopy surgery using mass spectrometry-based techniques and measured 75 ECM protein levels in the lesions using a multiple reaction monitoring (MRM) assay we developed. A total of 906 meniscus proteins with a 1% false discovery rate (FDR) was identified through a tandem mass tag (TMT) analysis showing that the lateral and medial menisci had similar protein expression profiles. A total of 131 ECM-related proteins was included in meniscus tissues such as collagen, fibronectin, and laminin. Our data showed that 14 ECM protein levels were differentially expressed in lateral and medial lesions (p < 0.05). We present the proteomic characterization of meniscal tissue with mass spectrometry-based comparative proteomic analysis and developed an MRM-based assay of ECM proteins correlated with tissue regeneration. The mass spectrometry dataset has been deposited to the MassIVE repository with the dataset identifier MSV000087753.

Comparison of proprioception between osteoarthritic and age-matched unaffected knees: a systematic review and meta-analysis.

PURPOSE: Theoretically, proprioceptive acuity could decrease in patients with knee osteoarthritis. However, there have been conflicting results in terms of proprioceptive deficit in osteoarthritic knees. The purpose of this systematic review and meta-analysis was to compare knee proprioception between osteoarthritic and healthy control knees. METHODS: Studies comparing proprioception in osteoarthritic and healthy knees of age-matched control group using thresholds to detect passive motion (TTDPM) or joint position sense (JPS) tests were identified. JPS was assessed by measuring the reproduction of passive positioning (RPP) or active positioning (RAP) of the knees. RESULTS: Seventeen studies were finally included in this meta-analysis. The pooled results of the analyses of the TTDPM for both 30° and 45° knee flexion showed that the mean angle of error was 0.83° greater (95% confidence interval: 0.44 to 1.23°; p < 0.001) in the osteoarthritic knees than in control knees. The pooled data of the RAP and RPP also showed that the mean angle of error was 1.89° greater in the osteoarthritic knees than in the control group. The mean difference in the angle of error between the osteoarthritic knees and control group was 1.06° greater in the JPS test than in the TTDPM (p < 0.001). CONCLUSION: The knee proprioceptive acuity of the patients with knee osteoarthritis was poorer than that of the patients with unaffected knees in the age-matched control group both in terms of the TTDPM and JPS; clinical relevance of these deficits needs to be clarified in further studies. LEVEL OF EVIDENCE: Meta-analysis, Level II.

The unc-51 like autophagy activating kinase 1-autophagy related 13 complex has distinct functions in tunicamycin-treated cells.

Endoplasmic reticulum (ER) stress and autophagy are regulated by shared signaling pathways, and their dysfunction is directly related to pathological conditions. This study investigated the function of the unc-51 like autophagy activating kinase 1 (ULK1)-autophagy related 13 (ATG13) complex in ER stress conditions through a knockout (KO) approach. Unlike other autophagy genes, KO of ULK1 or ATG13 attenuated ER stress and promoted mammalian target of rapamycin complex 1 (mTORC1) activation. Compared with wild type (WT) cells, ULK1 and ATG13 KO cells displayed increased viability, while beclin 1, ATG14, and ULK1/2 KO cells did not. Tunicamycin treatment upregulated the expression of ER stress markers (DNA damage inducible transcript 3, heat shock protein family A (Hsp70) member 5, and phosphorylated eukaryotic translation initiation factor 2 alpha kinase 3, eukaryotic translation initiation factor 2 subunit alpha, and endoplasmic reticulum to nucleus signaling 1); however, these were decreased in ULK1 and ATG13 KO cells. Insulin treatment upregulates the phosphorylation of ribosomal protein S6 kinase B1 (RPS6KB1) and AKT serine/threonine kinase 1 (AKT1), which was suppressed by tunicamycin. Notably, ATG13 and ULK1 deficiency ameliorated tunicamycin-induced insulin resistance, with enhanced RPS6KB1 and AKT1 phosphorylation in KO cells compared to WT cells. Although ULK1 and ATG13 are necessary for autophagy induction after tunicamycin-induced ER stress, autophagy does not seem to directly affect tunicamycin-induced cell death, ER stress, or insulin resistance. Our results indicate that loss of the ULK1-ATG13 complex attenuates ER stress and cell death and increases mTORC1 signaling.

Adaptive laboratory evolution of Escherichia coli lacking cellular byproduct formation for enhanced acetate utilization through compensatory ATP consumption.

Acetate has attracted great attention as a carbon source to develop economically feasible bioprocesses for sustainable bioproducts. Acetate is a less-preferred carbon source and a well-known growth inhibitor of Escherichia coli. In this study, we carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, and frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. Four genomic mutations were found in the evolved strain through whole-genome sequencing, and two major mutations (in cspC and patZ) mainly contributed to efficient utilization of acetate and tolerance to acetate. Transcriptomic reprogramming was examined by analyzing the genome-wide transcriptome with different carbon sources. The evolved strain showed high levels of intracellular ATP by upregulation of genes involved in NADH and ATP biosynthesis, which facilitated the production of enhanced green fluorescent protein, mevalonate, and n-butanol using acetate alone. This new strain, given its high acetate tolerance and high ATP levels, has potential as a starting host for cell factories targeting the production of acetyl-CoA-derived products from acetate or of products requiring high ATP levels.

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins.

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.