Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone.

The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP-citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

Understanding Consequences and Tradeoffs of Melt Processing as a Pretreatment for Enzymatic Depolymerization of Poly(ethylene terephthalate).

Melt extrusion pretreatment of poly(ethylene terephthalate) (PET) prior to enzymatic depolymerization with an unpurified leaf branch compost cutinase enzyme cocktail is explored to ascertain the efficiency gained by different processing methods on the enzymatic depolymerization of PET. Specific surface area (SSA) is investigated as a key factor in reducing depolymerization time. Higher SSA substrates (>5.6 mm2  mg-1 ) show higher depolymerization rates (≈0.88 g L-1 terephthalic acid [TPA] per day) and no induction phase, while lower SSA substrates (≈4.3, 4.4, and 5.6 mm2  mg-1 ) show, after an initial induction phase, similar depolymerization rates (≈0.46, 0.45, and 0.44 g L-1 TPA per day) despite increases in SSA of up to 30%. The mechanism of enzymatic depolymerization manifests in the appearance of anisotropic pitting. Longer incubation time used to overcome the induction phase in low SSA substrates allows for nearly full recovery of monomeric products, but manual pregrinding of extruded PET sharply increases SSA, depolymerization rate, and substrate crystallinity which may decrease the maximum recycled yield of the product materials. An estimate of the energy cost of increasing SSA is made and its effects on material properties are discussed. This work highlights key material structure and pretreatment aspects influencing the enzymatic recycling of PET.

Microbial synthesis of wax esters.

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7-4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C36, C34 and C32, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.

Biomanufacturing of value-added products from oils or fats: A case study on cellular and fermentation engineering of Yarrowia lipolytica.

The United States produces more than 10 million tons of waste oils and fats each year. This paper aims to establish a new biomanufacturing platform that converts waste oils or fats into a series of value-added products. Our research employs the oleaginous yeast Yarrowia lipolytica as a case study for citric acid (CA) production from waste oils. First, we conducted the computational fluid dynamics (CFD) simulation of the bioreactor system and identified that the extracellular mixing and mass transfer is the first limiting factor of an oil fermentation process due to the insolubility of oil in water. Based on the CFD simulation results, the bioreactor design and operating conditions were optimized and successfully enhanced oil uptake and bioconversion in fed-batch fermentation experiments. After that, we investigated the impacts of cell morphology on oil uptake, intracellular lipid accumulation, and CA formation by overexpressing and deleting the MHY1 gene in the wild type Y. lipolytica ATCC20362. Fairly good linear correlations (R2 > 0.82) were achieved between cell morphology and productivities of biomass, lipid, and CA. Finally, fermentation kinetics with both glucose and oil substrates were compared and the oil fermentation process was carefully evaluated. Our study suggests that waste oils or fats can be economical feedstocks for biomanufacturing of many high-value products.

Differences in the clinical presentation, management, and in-hospital outcomes of acute aortic dissection in patients with and without end-stage renal disease.

BACKGROUND: Few studies have evaluated the clinical presentation, management, and outcomes of patients with end-stage renal disease (ESRD) presenting with acute aortic dissection (AAD) in real-world clinical practice. Thus, this study investigated the clinical characteristics, management, and outcomes of AAD patients with ESRD. METHODS: A total of 217 patients were included. We evaluated the differences in the clinical features, management, and in-hospital outcomes of patients with and without a history of ESRD presenting with AAD. RESULTS: A history of ESRD was present in 71 of 217 patients. Patients with ESRD had atypical clinical manifestations (p < 0.001) and were more likely to be managed medically compared with patients without ESRD (p = 0.002). Hypertension and type B aortic dissection were significantly more common among patients with ESRD. Moreover, patients with ESRD had lower leucocyte and platelet counts than patients without ESRD in laboratory findings (p < 0.001). However, hospitalization days and in-hospital mortality were similar between the two groups (p > 0.05). Multivariate analysis identified Type A aortic dissection as an independent predictor of in-hospital mortality among patients without ESRD (OR, 13.68; 95% CI, 1.92 to 98.90; P = 0.006). CONCLUSIONS: This study highlights differences in the clinical characteristics, management, and outcomes of AAD patients with ESRD. These patients usually have atypical symptoms and more comorbid conditions and are managed more conservatively. However, these patients have no in-hospital survival disadvantage over those without ESRD. Further studies are needed to better understand and optimize care for patients with ESRD presenting with AAD.

Alterations in gut microbiota of abdominal aortic aneurysm mice.

BACKGROUND: The gut microbiome plays an important role in various cardiovascular diseases, such as atherosclerosis and hypertension, which are associated with abdominal aortic aneurysms (AAAs). METHODS: Here, we used 16S rRNA sequencing to explore gut microbiota in C57BL ApoE-/- mice with AAAs. A mouse model of abdominal aortic aneurysms was induced with angiotensin II (Ang II) (1000 ng/min per kg). On day 28 after the operation, fecal samples were collected and stored at - 80 °C until DNA extraction. We determined the relative abundances of bacterial taxonomic groups using 16S rRNA amplicon metabarcoding, and sequences were analyzed using a combination of mother software and UPARSE. RESULTS: We found that the gut microbiome was different between control and AAA mice. The results of correlation analysis between AAA diameter and the gut microbiome as well as LEfSe of the genera Akkermansia, Odoribacter, Helicobacter and Ruminococcus might be important in the progression of AAAs. CONCLUSIONS: AAA mice is subjected to gut microbial dysbiosis, and gut microbiota might be a potential target for further investigation.

Omega-3 production by fermentation of Yarrowia lipolytica: From fed-batch to continuous.

The omega-3 fatty acid, cis-5,8,11,14,17-eicosapentaenoic acid (C20:5; EPA) has wide-ranging benefits in improving heart health, immune function, and mental health. A sustainable source of EPA production through fermentation of metabolically engineered Yarrowia lipolytica has been developed. In this paper, key fed-batch fermentation conditions were identified to achieve 25% EPA in the yeast biomass, which is so far the highest EPA titer reported in the literature. Dynamic models of the EPA fermentation process were established for analyzing, optimizing, and scaling up the fermentation process. In addition, model simulations were used to develop a two-stage continuous process and compare to single-stage continuous and fed- batch processes. The two stage continuous process, which is equipped with a smaller growth fermentor (Stage 1) and a larger production fermentor (Stage 2), was found to be a superior process to achieve high titer, rate, and yield of EPA. A two-stage continuous fermentation experiment with Y. lipolytica strain Z7334 was designed using the model simulation and then tested in a 2 L and 5 L fermentation system for 1,008 h. Compared with the standard 2 L fed-batch process, the two-stage continuous fermentation process improved the overall EPA productivity by 80% and EPA concentration in the fermenter by 40% while achieving comparable EPA titer in biomass and similar conversion yield from glucose. During the long-term experiment it was also found that the Y. lipolytica strain evolved to reduce byproduct and increase lipid production. This is one of the few continuous fermentation examples that demonstrated improved productivity and concentration of a final product with similar conversion yield compared with a fed-batch process. This paper suggests the two-stage continuous fermentation could be an effective process to achieve improved production of omega-3 and other fermentation products where non-growth or partially growth associated kinetics characterize the process. Biotechnol. Bioeng. 2017;114: 798-812. © 2016 Wiley Periodicals, Inc.

Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.

The omega-3 fatty acids, cis-5, 8, 11, 14, and 17-eicosapentaenoic acid (C20:5; EPA) and cis-4, 7, 10, 13, 16, and 19-docosahexaenoic acid (C22:6; DHA), have wide-ranging benefits in improving heart health, immune function, mental health, and infant cognitive development. Currently, the major source for EPA and DHA is from fish oil, and a minor source of DHA is from microalgae. With the increased demand for EPA and DHA, DuPont has developed a clean and sustainable source of the omega-3 fatty acid EPA through fermentation using metabolically engineered strains of Yarrowia lipolytica. In this mini-review, we will focus on DuPont's technology for EPA production. Specifically, EPA biosynthetic and supporting pathways have been introduced into the oleaginous yeast to synthesize and accumulate EPA under fermentation conditions. This Yarrowia platform can also produce tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acid mixtures in the cellular lipid profiles. Fundamental research such as metabolic engineering for strain construction, high-throughput screening for strain selection, fermentation process development, and process scale-up were all needed to achieve the high levels of EPA titer, rate, and yield required for commercial application. Here, we summarize how we have combined the fundamental bioscience and the industrial engineering skills to achieve large-scale production of Yarrowia biomass containing high amounts of EPA, which led to two commercial products, New Harvest™ EPA oil and Verlasso® salmon.

Design of a two-degree-of-freedom magnetic levitation vibration energy harvester for bridge vibration response analysis.

For bridges with high automobile traffic, a large amount of vibration is generated daily due to cars driving over imperfectly level roads, and a vibration energy harvester can convert this energy into electrical energy, thus providing energy for devices such as bridge health sensors. However, the traditional single degree of freedom magnetic levitation vibration energy harvester (SMEH) has the disadvantage of low output power, so this research designs an improved dual degree of freedom magnetic levitation vibration energy harvester (DMEH), and a mathematical model of the energy harvester is built for simulation tests and an optimization model based on NSGA-II algorithm is developed for optimizing the structural parameters of the energy harvester. The experimental results show that the maximum total output power of DMEH and SMEH on CSSBB1, CSSBB2 and CSSBB3 are 48.7 mW, 36.8 mW, 25.4 mW and 27.2 mW, 21.5 mW, 14.9 mW, respectively, and the minimum total magnet volumes of both on CSSBB1, CSSBB2 and CSSBB3 are 268 cm3, 132 cm3, 219 cm3, 214 cm3, 86.2 cm3, 156 cm3. Based on the experimental data, it is found that the maximum output power of the optimal solution of DMEH is larger than that of SMEH for the selected simply supported girder bridge project, and the volume of the former is also larger than that of the latter, but the degree of increase can still be adapted to the application environment. The research results have some reference significance for improving the energy harvesting efficiency of bridge vibration energy harvesters.

CFD evaluation of hydrophobic feedstock bench-scale fermenters for efficient high agitation volumetric mass transfer.

A new biomanufacturing platform combining intracellular metabolic engineering of the oleaginous yeast Yarrowia lipolytica and extracellular bioreaction engineering provides efficient bioconversion of plant oils/animal fats into high-value products. However, predicting the hydrodynamics and mass transfer parameters is difficult due to the high agitation and sparging required to create dispersed oil droplets in an aqueous medium for efficient yeast fermentation. In the current study, commercial computational fluid dynamic (CFD) solver Ansys CFX coupled with the MUSIG model first predicts two-phase system (oil/water and air/water) mixing dynamics and their particle size distributions. Then, a three-phase model (oil, air, and water) utilizing dispersed air bubbles and a polydispersed oil phase was implemented to explore fermenter mixing, gas dispersion efficiency, and volumetric mass transfer coefficient estimations (kL a). The study analyzed the effect of the impeller type, agitation speed, and power input on the tank's flow field and revealed that upward-pumping pitched blade impellers (PBI) in the top two positions (compared to Rushton-type) provided advantageous oil phase homogeneity and similar estimated kL a values with reduced power. These results show good agreement with the experimental mixing and kL a data.

Association of genetic variants in soy isoflavones metabolism-related genes with decreased lung cancer risk.

BACKGROUND: Soy isoflavones have been reported to exhibit anti-tumor effects. We hypothesize that genetic variants in soy isoflavone metabolism-related genes are associated with the risk of lung cancer. METHODS: A two-stage case-control study design was conducted in this study. The discovery stage included 300 lung cancer cases and 600 healthy controls to evaluate the association of candidate genetic variants with lung cancer risk. The validation stage involved 1200 cases and 1200 controls to validate the associations found. Furthermore, qPCR was performed to assess the mRNA expression levels of different genotypes of the SNP. ELISA was used to explore the association between genotype and soy isoflavone levels, as well as the association between soy isoflavone levels and lung cancer risk. RESULTS: A nonlinear association was observed between plasma soy isoflavone levels and lung cancer risk, with higher soy isoflavone levels associated with lower lung cancer risk (P < 0.001). The two-stage case-control study identified that UGT1A1 rs3755319 A > C was associated with decreased lung cancer risk (Recessive model: adjusted OR = 0.69, 95 %CI = 0.57-0.84, P < 0.001). Moreover, eQTL analysis showed that the expression level of UGT1A1 in the rs3755319 CC genotype was lower than in the AA + AC genotype (P < 0.05). The plasma concentration of soy isoflavones in the rs3755319 CC genotype was higher than in the AA + AC genotype (P = 0.008). CONCLUSIONS: We identified a potentially functional SNP, UGT1A1 rs3755319 A > C, as being associated with decreased lung cancer risk. Further experiments will be needed to explore the mechanisms underlying the observed associations.

Robust positive association between serum urate and the risk of chronic obstructive pulmonary disease: hospital-based cohort and Mendelian randomisation study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) and hyperuricaemia are both characterised by systemic inflammation. Preventing chronic diseases among the population with common metabolic abnormality is an effective strategy. However, the association of hyperuricaemia with the higher incidence and risk of COPD remains controversial. Therefore, replicated researches in populations with distinct characteristics or demographics are compellingly warranted. METHODS: This cohort study adopted a design of ambispective hospital-based cohort. We used propensity score matching (PSM) and inverse probability of treatment weighting (IPTW) to minimise the effects of potential confounding factors. A Cox regression model and restricted cubic spline (RCS) model were applied further to assess the effect of serum urate on the risk of developing COPD. Finally, we conducted a two-sample Mendelian randomisation (MR) analysis to explore evidence of causal association. RESULTS: There is a higher incidence in the population with hyperuricaemia compared with the population with normal serum urate (22.29/1000 person-years vs 8.89/1000 person-years, p=0.009). This result is robust after performing PSM (p=0.013) and IPTW (p<0.001). The Cox model confirms that hyperuricaemia is associated with higher risk of developing COPD (adjusted HR=3.35 and 95% CI=1.61 to 6.96). Moreover, RCS shows that the risk of developing COPD rapidly increases with the concentration of serum urate when it is higher than the reference (420 µmol/L). Finally, in MR analysis, the inverse variance weighted method evidences that a significant causal effect of serum urate on COPD (OR=1.153, 95% CI=1.034 to 1.289) is likely to be true. The finding of MR is robust in the repeated analysis using different methods and sensitivity analysis. CONCLUSIONS: Our study provides convincing evidence suggesting a robust positive association between serum urate and the risk of developing COPD, and indicates that the population with hyperuricaemia is at high risk of COPD in the Chinese population who seek medical advice or treatment in the hospital.

[A meta-analysis on the efficacy of stem cells transplantation therapy on the prevention of cardiac remodeling and main adverse cardiovascular event in patients with chronic heart failure].

OBJECTIVE: Stem cells transplantation is a promising strategy in cardiology. This meta-analysis summarizes the efficacy and safety of stem cells transplantation on top of standard medication on chronic heart failure patients. METHODS: The following databases were searched, including Cochrane Library (Issue 4, 2011), PubMed (1980 to 2011), Embase (1990 to 2011), CBM (1978 to 2011), CNKI (1994 to 2011), VIP (1989 to 2011), and WanFang Data (1998 to 2011). Search criteria:studies were screened and the quality was evaluated according to predefined inclusion and exclusion criteria. Intervention measures: the treatment group using stem cell transplantation therapy on top of standard drug treatment, while the control group using standard drug treatments. RESULTS: A total of 31 studies involving 2375 patients were included. The results show that the improvement of LVEDV in the stem cell treatment group is greater than in the control group [SMD = -11.8% (95%CI: -0.223 - 0.013), P = 0.027] and the relative-risk of cardiac events is lower in stem cell treatment group [RR = 0.77 (95%CI: 0.66 - 0.90), P < 0.01] than in control group. CONCLUSION: Stem cells therapy is effective in improving cardiac remodeling and reducing the relative-risk of cardiac events in patients with chronic heart failure.

Biotechnological production of omega-3 fatty acids: current status and future perspectives.

Omega-3 fatty acids, including alpha-linolenic acids (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have shown major health benefits, but the human body's inability to synthesize them has led to the necessity of dietary intake of the products. The omega-3 fatty acid market has grown significantly, with a global market from an estimated USD 2.10 billion in 2020 to a predicted nearly USD 3.61 billion in 2028. However, obtaining a sufficient supply of high-quality and stable omega-3 fatty acids can be challenging. Currently, fish oil serves as the primary source of omega-3 fatty acids in the market, but it has several drawbacks, including high cost, inconsistent product quality, and major uncertainties in its sustainability and ecological impact. Other significant sources of omega-3 fatty acids include plants and microalgae fermentation, but they face similar challenges in reducing manufacturing costs and improving product quality and sustainability. With the advances in synthetic biology, biotechnological production of omega-3 fatty acids via engineered microbial cell factories still offers the best solution to provide a more stable, sustainable, and affordable source of omega-3 fatty acids by overcoming the major issues associated with conventional sources. This review summarizes the current status, key challenges, and future perspectives for the biotechnological production of major omega-3 fatty acids.

Perspectives for Using CO2 as a Feedstock for Biomanufacturing of Fuels and Chemicals.

Microbial cell factories offer an eco-friendly alternative for transforming raw materials into commercially valuable products because of their reduced carbon impact compared to conventional industrial procedures. These systems often depend on lignocellulosic feedstocks, mainly pentose and hexose sugars. One major hurdle when utilizing these sugars, especially glucose, is balancing carbon allocation to satisfy energy, cofactor, and other essential component needs for cellular proliferation while maintaining a robust yield. Nearly half or more of this carbon is inevitably lost as CO2 during the biosynthesis of regular metabolic necessities. This loss lowers the production yield and compromises the benefit of reducing greenhouse gas emissions-a fundamental advantage of biomanufacturing. This review paper posits the perspectives of using CO2 from the atmosphere, industrial wastes, or the exhausted gases generated in microbial fermentation as a feedstock for biomanufacturing. Achieving the carbon-neutral or -negative goals is addressed under two main strategies. The one-step strategy uses novel metabolic pathway design and engineering approaches to directly fix the CO2 toward the synthesis of the desired products. Due to the limitation of the yield and efficiency in one-step fixation, the two-step strategy aims to integrate firstly the electrochemical conversion of the exhausted CO2 into C1/C2 products such as formate, methanol, acetate, and ethanol, and a second fermentation process to utilize the CO2-derived C1/C2 chemicals or co-utilize C5/C6 sugars and C1/C2 chemicals for product formation. The potential and challenges of using CO2 as a feedstock for future biomanufacturing of fuels and chemicals are also discussed.

A strontium ferrite modified separator for adsorption and catalytic conversion of polysulfides for excellent lithium-sulfur batteries.

Lithium-sulfur batteries (LSBs) have emerged as one of the ideal contenders for the upcoming generation of high energy storage devices due to their superb energy density. Nonetheless, the shuttle effect generated by intermediate lithium polysulfides (LiPSs) during cell cycling brings about capacity degradation and poor cycling stability of LSBs. Here, a versatile SrFe12O19 (FSO) and acetylene black (AB) modified PP separator is first presented to inhibit the shuttle effect. Thanks to the strong chemical interaction of Fe and Sr with polysulphides in FSO, it can trap LiPSs and provide catalytic sites for their conversion. Therefore, the cell using the FSO/AB@PP separator has a high initial discharge specific capacity (930 mA h g-1) at 2 C and lasts for 1000 cycles with a remarkably low fading rate (0.036% per cycle), while those using PE and AB@PP separators have inferior initial specific capacities (255 mA h g-1 and 652 mA h g-1, respectively) and fail within 600 cycles. This work proposes a novel approach for addressing the shuttle of LiPSs from a bimetallic oxide modified separator.

Using oils and fats to replace sugars as feedstocks for biomanufacturing: Challenges and opportunities for the yeast Yarrowia lipolytica.

More than 200 million tons of plant oils and animal fats are produced annually worldwide from oil, crops, and the rendered animal fat industry. Triacylglycerol, an abundant energy-dense compound, is the major form of lipid in oils and fats. While oils or fats are very important raw materials and functional ingredients for food or related products, a significant portion is currently diverted to or recovered as waste. To significantly increase the value of waste oils or fats and expand their applications with a minimal environmental footprint, microbial biomanufacturing is presented as an effective strategy for adding value. Though both bacteria and yeast can be engineered to use oils or fats as the biomanufacturing feedstocks, the yeast Yarrowia lipolytica is presented as one of the most attractive platforms. Y. lipolytica is oleaginous, generally regarded as safe, demonstrated as a promising industrial producer, and has unique capabilities for efficient catabolism and bioconversion of lipid substrates. This review summarizes the major challenges and opportunities for Y. lipolytica as a new biomanufacturing platform for the production of value-added products from oils and fats. This review also discusses relevant cellular and metabolic engineering strategies such as fatty acid transport, fatty acid catabolism and bioconversion, redox balances and energy yield, cell morphology and stress response, and bioreaction engineering. Finally, this review highlights specific product classes including long-chain diacids, wax esters, terpenes, and carotenoids with unique synthesis opportunities from oils and fats in Y. lipolytica.

Bidirectional effects of oral anticoagulants on gut microbiota in patients with atrial fibrillation.

Background: The imbalance of gut microbiota (GM) is associated with a higher risk of thrombosis in patients with atrial fibrillation (AF). Oral anticoagulants (OACs) have been found to significantly reduce the risk of thromboembolism and increase the risk of bleeding. However, the OAC-induced alterations in gut microbiota in patients with AF remain elusive. Methods: In this study, the microbial composition in 42 AF patients who received long-term OAC treatment (AF-OAC group), 47 AF patients who did not (AF group), and 40 volunteers with the risk of AF (control group) were analyzed by 16S rRNA gene sequencing of fecal bacterial DNA. The metagenomic functional prediction of major bacterial taxa was performed using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) software package. Results: The gut microbiota differed between the AF-OAC and AF groups. The abundance of Bifidobacterium and Lactobacillus decreased in the two disease groups at the genus level, but OACs treatment mitigated the decreasing tendency and increased beneficial bacterial genera, such as Megamonas. In addition, OACs reduced the abundance of pro-inflammatory taxa on the genus Ruminococcus but increased certain potential pathogenic taxa, such as genera Streptococcus, Escherichia-Shigella, and Klebsiella. The Subgroup Linear discriminant analysis effect size (LEfSe) analyses revealed that Bacteroidetes, Brucella, and Ochrobactrum were more abundant in the anticoagulated bleeding AF patients, Akkermansia and Faecalibacterium were more abundant in the non-anticoagulated-bleeding-AF patients. The neutrophil-to-lymphocyte ratio (NLR) was lower in the AF-OAC group compared with the AF group (P < 0.05). Ruminococcus was positively correlated with the NLR and negatively correlated with the CHA2DS2-VASc score (P < 0.05), and the OACs-enriched species (Megamonas and Actinobacteria) was positively correlated with the prothrombin time (PT) (P < 0.05). Ruminococcus and Roseburia were negatively associated with bleeding events (P < 0.05). Conclusions: Our study suggested that OACs might benefit AF patients by reducing the inflammatory response and modulating the composition and abundance of gut microbiota. In particular, OACs increased the abundance of some gut microbiota involved in bleeding and gastrointestinal dysfunction indicating that the exogenous supplementation with Faecalibacterium and Akkermansia might be a prophylactic strategy for AF-OAC patients to lower the risk of bleeding after anticoagulation.

Enzyme selection, optimization, and production toward biodegradation of post-consumer poly(ethylene terephthalate) at scale.

Poly(ethylene terephthalate) (PET) is one of the world's most widely used polyester plastics. Due to its chemical stability, PET is extremely difficult to hydrolyze in a natural environment. Recent discoveries in new polyester hydrolases and breakthroughs in enzyme engineering strategies have inspired enormous research on biorecycling of PET. This study summarizes our research efforts toward large-scale, efficient, and economical biodegradation of post-consumer waste PET, including PET hydrolase selection and optimization, high-yield enzyme production, and high-capacity enzymatic degradation of post-consumer waste PET. First, genes encoding PETase and MHETase from Ideonella sakaiensis and the ICCG variant of leaf-branch compost cutinase (LCCICCG ) were codon-optimized and expressed in Escherichia coli BL21(DE3) for high-yield production. To further lower the enzyme production cost, a pelB leader sequence was fused to LCCICCG so that the enzyme can be secreted into the medium to facilitate recovery. To help bind the enzyme on the hydrophobic surface of PET, a substrate-binding module in a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) was fused to the C-terminus of LCCICCG . The resulting four different LCCICCG variants (LCC, PelB-LCC, LCC-PBM, and PelB-LCC-PBM), together with PETase and MHETase, were compared for PET degradation efficiency. A fed-batch fermentation process was developed to produce the target enzymes up to 1.2 g L-1 . Finally, the best enzyme, PelB-LCC, was selected and used for the efficient degradation of 200 g L-1 recycled PET in a well-controlled, stirred-tank reactor. The results will help develop an economical and scalable biorecycling process toward a circular PET economy.

Continuous biomanufacturing with microbes - upstream progresses and challenges.

Current biomanufacturing facilities are mainly built for batch or fed-batch operations, which are subject to low productivities and do not achieve the great bioconversion potential of the rewired cells generated via modern biotechnology. Continuous biomanufacturing should be the future directions for high-yield and low-cost manufacturing of various fermentation products. This review discusses the major challenges and the strategies for continuous biomanufacturing with microbes, which include minimizing contamination risk, enhancing genetic stability over a long-term continuous operation, achieving high product titer, rate, and yield simultaneously by decoupling cell growth from product formation, and using modeling approach to accelerate research and development of continuous biomanufacturing. New strain designs and process engineering strategies, including integration with artificial intelligence, are also discussed for intelligent and the next generation of continuous biomanufacturing.