Strain Improvement Research Articles

Evolutionary engineering and chemical mutagenesis of Propionibacterium acidipropionici for improved propionic acid production from sugarcane-derived saccharides

Propionic acid (PA) is a 3-carbon carboxylic acid widely used in the chemical, food and pharmaceutical industries. Current production of PA is primarily via petrochemical routes and alternative sources using renewable feedstocks are desirable. The goal of the present study was to develop bacterial strains for efficient PA production by fermentation of sugarcane-derived saccharides in inverted sugarcane syrup. Adaptive evolution and random chemical mutagenesis of Propionibacterium acidipropionici were individually evaluated. Although evolutionary engineering proved to be a suitable strategy for producing strains tolerant to acid stress, chemical mutagenesis coupled with a novel screening methodology proved to be a superior strategy for strain improvement. The selected strain presented high PA productivity (0.55 g L−1 h−1) and titer (40 g L−1) in relation to the parental strain (0.38 g L−1 h−1 and 27 g L−1, respectively), in fermentations using inverted sugarcane syrup.

Efficient Production of Glucaric Acid by Engineered Saccharomyces cerevisiae.

Glucaric acid is a valuable chemical with applications in the detergent, polymer, pharmaceutical and food industries. In this study, two key enzymes for glucaric acid biosynthesis, MIOX4 (myo-inositol oxygenase) and Udh (uronate dehydrogenase), were fused and expressed with different peptide linkers. It was found that a strain harboring the fusion protein MIOX4-Udh linked by the peptide (EA3K)3 produced the highest glucaric acid titer and thereby resulted in glucaric acid production that was 5.7-fold higher than that of the free enzymes. Next, the fusion protein MIOX4-Udh linked by (EA3K)3 was integrated into delta sequence sites of the Saccharomyces cerevisiae opi1 mutant, and a strain, GA16, that produced a glucaric acid titer of 4.9 g/L in a shake flask fermentation was identified by a high-throughput screening method using an Escherichia coli glucaric acid biosensor. Strain improvement by further engineering was performed to regulate the metabolic flux of myo-inositol to increase the supply of glucaric acid precursors. The downregulation of ZWF1 and the overexpression of INM1 and ITR1 increased glucaric acid production significantly, and glucaric acid production was increased to 8.49 g/L in the final strain GA-ZII in a shake flask fermentation. Finally, in a 5-L bioreactor, GA-ZII produced a glucaric acid titer of 15.6 g/L through fed-batch fermentation. IMPORTANCE Glucaric acid is a value-added dicarboxylic acid that was synthesized mainly through the oxidation of glucose chemically. Due to the problems of the low selectivity, by-products, and highly polluting waste of this process, producing glucaric acid biologically has attracted great attention. The activity of key enzymes and the intracellular myo-inositol level were both rate-limiting factors for glucaric acid biosynthesis. To increase glucaric acid production, this work improved the activity of the key enzymes in the glucaric acid biosynthetic pathway through the expression of a fusion of Arabidopsis thaliana MIOX4 and Pseudomonas syringae Udh as well as a delta sequence-based integration. Furthermore, intracellular myo-inositol flux was optimized by a series of metabolic strategies to increase the myo-inositol supply, which improved glucaric acid production to a higher level. This study provided a way for constructing a glucaric acid-producing strain with good synthetic performance, making glucaric acid production biologically in yeast cells much more competitive.

Regulation of bimodal structure and mechanical properties of powder-thixoformed GO/ZK60 magnesium matrix composite through adjusting GO content

Although remarkable strength enhancements can be achieved in graphene oxide (GO)/ graphene nanoplatelets (GNPs) reinforced Mg matrix composites by using the available techniques, their ductility is always quite poor due to the difficultly avoided strength-ductility trade-off. To conquer this dilemma, GO/ZK60 composites with bimodal-grain structure were fabricated using powder thixoforming in this work. The results indicate that the grain size and volume fraction of coarse grains (CGs) first decrease as the GO content increases to 0.2 wt.% and then increase again as the content increases to 0.3 wt.%, while the grain size in the fine grains (FGs) almost does not change. Consequently, the strength of the composites is improved with increasing GO content and reaches the peak values at the content of 0.2 wt.%. The composite with 0.1 wt.% GO content exhibits significantly increased tensile yield strength up to 177 ± 2 MPa while maintaining a high elongation of 23.1% ± 2.5%, being equivalent to that of the ZK60 matrix alloy. The increased FGs volume fraction, together with the promoted dislocation accumulation and storage via GO and grain refinement of large-sized CGs lead to the improvement of strain hardening ability, thus rendering the composite an excellent ductility. Furthermore, the deformation of the GO/ZK60 composites occurs progressively from the FGs to the CGs, which is opposite to the status of the milled ZK60 matrix alloy. In view of the microstructure characteristics of the composites, a new complex calculation model was proposed and it could well predict the strength of the bimodal GO/ZK60 composites. This study provides a new insight into the microstructure design and fabrication technology of GO/GNPs reinforced metal-based composites with high strength and ductility.

Lignocellulose-Derived Arabinose for Energy and Chemicals Synthesis through Microbial Cell Factories: A Review

The exploration of natural substrates for microbial conversion to synthesize industrial platform and fuel chemicals seems to be inevitable within a circular bioeconomy context. Hemicellulose is a natural carbohydrate polymer consisting of a variety of pentose (C5) sugar monomers such as arabinose, mannose, erythrose, and xylose. Among the C5 sugars, L-arabinose (L-Ara) is the second-most-abundant pentose sugar in the lignocellulosic biomass after xylose. L-Ara has been used as an industrial carbon source to produce several value-added chemicals such as putrescine, which is used to synthesize polymers in the textile industry; sugar alcohols that are used as sweeteners in diet foods; and amino acids such as L-lysine, L-glutamate, L-arginine, and L-ornithine, which are used in nutritional supplements, fertilizers, and other products in the food and beverage industries. L-Ara, a natural non-caloric sweetener, is used as a substitute in the food and beverage industry, when the risk of blood sugar and lipid levels could be reduced. Major use of L-Ara is also found in the medical and pharmaceutical sectors to treat several conditions, including mineral absorption disorder, constipation, and diabetes, among others. In recent years, there has been a rising interest in synthesizing various sugar alcohols and derivatives, including arabitol, xylitol, and 2,3-butanediol, through the modification of producer organisms either genetically or metabolically to produce value-added products. Understanding the current demand and the need to increase the diversified production of industrial green chemicals with the reduced waste of useful lignocellulosic resources, this review focuses on the background of L-Ara and its various sources, microbes that utilize L-Ara to produce high-value-added products, and the future prospects for strain improvements to increase the yield of high-value-added products.

Mutagenesis and Selection Strategies of SCO and Carotenoid Producing Microorganisms

Biotechnologies of microorganisms for producing various industrially important products, such as single-cell oil (SCO), single-cell protein (SCP), carotenoid, enzymes, etc., have been studied since the last century and are of current actuality to the present day. The SCO and carotenoids are an alternative to oil of plant and animal origin and to pigment of plant and synthetic origin, respectively. These cellular components of microorganisms can be used in the food, aquaculture and livestock feed, pharmaceutical, and cosmetic industries. However, microorganisms-based technologies have not found ubiquitous practice due to a number of limitations. One of them is the threshold of the oil and carotenoids content in the biomass of microorganisms due to their nature. Therefore, the development of the fast-growing strains with a high level of these product accumulation is required. Random mutagenesis, adaptive laboratory evolution (ALE), and genetic engineering are used for strain improvement. This paper reviews random mutagenesis as a simple, cost-effective tool for improving single-cell oil and carotenoid synthesis in microorganisms, followed by the selecting of mutants with preferable characteristics. Nevertheless, it should be considered that random mutagenesis accelerates naturally occurring nonspecific mutations by exposure to a physical or chemical mutagenic agent. Despite the result of a large variety of created mutants, the characteristics of the mutants can be unstable and reversible. Therefore, different cultivation strategies for developing traits of interest and testing their persistence are reviewed in this study. Choosing effective mutagenesis techniques, screening, and selection methods is essential for creating suitable mutant strains. Various strengths and drawbacks of such tools are discussed in this review, and the main directions for further development are highlighted.

Heterologous Expression of Inulinase Gene in Bacillus licheniformis 24 for 2,3-Butanediol Production from Inulin

Inulin is a renewable and cheap carbon source used in microbial fermentations. Bacillus licheniformis 24 is known as an excellent 2,3-butanediol (2,3-BD) producer from fructose; therefore, the cloning and expression of a robust heterologous inulinase could enhance its 2,3-BD production from inulin. The inu gene of Lacticaseibacillus paracasei DSM 23505 encoding fructan-β-fructosidase (EC 3.2.1.80) was chosen for the purpose. PCR fragments containing the complete inu (3.6 kb) and its truncated variant inu-tr (2.2 kb, lacking Big3 cell wall attachment domains) were cloned into Escherichia coli StellarTM and B. licheniformis 24. The high quality of the recombinant constructs was confirmed by restriction analysis, PCR, sequencing, and phenotypic tests. The results showed that the inulinase activity of B. licheniformis cells harboring the full-length inu variant (T26) was eightfold higher compared to the wild type, retaining cell wall attachment in the B. licheniformis host. In contrast, the truncated variant inu-tr (T14) showed mostly extracellular but weak activity, thus suggesting that the Big3 domains are also important for the enzyme’s function. During flask-batch fermentation of 100 g/L raw chicory flour (containing 90% inulin), T26 produced acetoin and 2,3-BD from inulin. Contrariwise, T14 and the wild type formed products only from the mono- and disaccharides naturally found in the chicory flour. In the fermenter, from 200 g/L of raw chicory flour, the recombinant T26 degraded approximately 140 g/L of the inulin. However, the final concentrations of the produced 2,3-BD and acetoin were 18.5 g/L and 8.2 g/L, respectively, because of the accumulation of unconverted sucrose. To conclude, further strain improvement is necessary to make the process efficient for obtaining 2,3-BD from inulin by simultaneous saccharification and fermentation (SSF).

Strain improvement, artificial intelligence optimization, and sensitivity analysis of asparaginase-mediated acrylamide reduction in sweet potato chips.

The online version contains supplementary material available at 10.1007/s13197-023-05757-5.

PO-03-008 CORRECTION OF DYSSYNCHRONY BY CONDUCTION SYSTEM PACING: RESULTS OF LEVEL-AT TRIAL

Correction of dyssynchrony measured with echo strain in conduction system pacing (CSP) vs biventricular pacing (BIVP) has not been reported. Our purpose was to compare echocardiographic dyssynchrony correction and strain improvement during follow-up between CSP and BIVP. A treatment received analysis was performed in patients included in the LEVEL-AT trial (NCT04054895) (n=29 CSP; BIVP 40). Patients were evaluated at baseline (ON and OFF programming) and at 6 and 12 months. All echocardiograms were completed by operators blinded to the type of device implanted. Intraventricular (septal flash), interventricular (difference between left and right ventricular outflow times), and auriculoventricular (diastolic filling time) dyssynchrony, as well as strain parameters (septal rebound, global longitudinal strain (GLS), and Risum pattern (typical left bundle branch contraction pattern)) were analyzed. 32% (22/69) were women; baseline LVEF was 27.5 ± 7% and left ventricular end-systolic volume (LVESV) 138 ± 77ml without differences between groups. At the 12-month follow-up, there were no differences in CSP vs. BIVP in LVEF (45.8 ± 10% vs. 42.7 ± 12%, p=0.25) and LVESV (81.5 ± 41 ml vs. 101.7 ± 56 ml, p=0.08). Longitudinal analyses showed that the improvement of all echocardiographic dyssynchrony parameters over time (p<0.001) was similar between groups (Table 1 and Fig 1A). A representative example of GLS at baseline and follow-up is shown in Figure 1B. GLS acute correction significantly correlated with LVEF (Fig 1C) and LVESV at the 12-month follow-up. CSP and BIVP provided similar dyssynchrony and strain correction over time. Acute strain correction predicted ventricular remodeling at 12 months follow-up.Tabled 1Table 1. (PO-03-008)Conduction system pacingBiventricular pacingEffects15 days OFF12 Months15 days OFF12 MonthsGroupTimeInteraction(Group*Time)Categorical variablesAdjusted prevalence +/- SEAdjusted prevalence +/- SEp-valueSeptal rebound0.769 +/- 0.0830.150 +/- 0.080.763 +/- 0.070.312 +/- 0.0820.13<0.0010.77Septal flash0.759 +/- 0.0790.310 +/- 0.0860.750 +/- 0.0680.108 +/- 0.0510.10<0.0010.49Risum pattern0.919 +/- 0.0730.0150 +/- 0.0210.84 +/- 0.1000.070 +/- 0.0590.81<0.0010.48Quantitative variablesAdjusted mean +/- SEAdjusted mean +/- SEp-valueGlobal longitudinal strain, %-9.62 +/- 0.68-11.28 +/- 0.73-8.69 +/- 0.57-11.28 +/- 0.590.32<0.0010.55Diastolic filling time, %47.6 +/- 1.5753.2 +/- 1.5746.0 +/- 1.3448.8 +/- 1.370.10<0.0010.57Difference in left and right ventricular outflow times, ms45.7 +/- 3.4816.6 +/- 3.4835.5 +/- 2.9621.4 +/- 3.110.76<0.0010.06Treatment received longitudinal analysis comparing CSP and BiVP in LEVEL-AT trial. Treatment effect over time but not different between groups. SE: standard error. Open table in a new tab

Effects of Edge Heating and Strain Gradient on Stretch Flange Deformation Limit of Steel Sheet

To improve the stretch flange deformation limit of ultrahigh-strength steels, a partial edge heating method was investigated. The effect of the strain gradient on the stretch flange deformation limit when a partial edge heating method was applied was investigated by the hole expansion test and FEM analysis. The stretch flange formability was improved by application of a partial edge heating method because of the recovery of the shearing microstructure and the reduction of the hardness difference of microstructure. It was found that the improvement of the deformation limit strain for the strain gradient with edge heating depends on the strain gradient region. In the low strain gradient region, the improvement margin of the deformation limit strain with edge heating was small. On the other hand, in the high strain gradient region, the deformation limit strain was markedly improved compared with that without heating. It was confirmed that the strain gradient in the direction perpendicular to the maximum principal strain could be used for the prediction of the deformation limit and also the determination of the stretch flange fracture by the FEM analysis of actual parts even under edge heating conditions.

Development of high cell density Limosilactobacillus reuteri KUB-AC5 for cell factory using oxidative stress reduction approach

BackgroundExpression systems for lactic acid bacteria have been developed for metabolic engineering applications as well as for food-grade recombinant protein production. But the industrial applications of lactic acid bacteria as cell factories have been limited due to low biomass formation resulted in low efficiency of biomanufacturing process. Limosilactobacillus reuteri KUB-AC5 is a safe probiotic lactic acid bacterium that has been proven as a gut health enhancer, which could be developed as a mucosal delivery vehicle for vaccines or therapeutic proteins, or as expression host for cell factory applications. Similar to many lactic acid bacteria, its oxygen sensitivity is a key factor that limits cell growth and causes low biomass production. The aim of this study is to overcome the oxidative stress in L. reuteri KUB-AC5. Several genes involved in oxidative and anti-oxidative stress were investigated, and strain improvement for higher cell densities despite oxidative stress was performed using genetic engineering.ResultsAn in-silico study showed that L. reuteri KUB-AC5 genome possesses an incomplete respiratory chain lacking four menaquinone biosynthesis genes as well as a complete biosynthesis pathway for the production of the precursor. The presence of an oxygen consuming enzyme, NADH oxidase (Nox), leads to high ROS formation in aerobic cultivation, resulting in strong growth reduction to approximately 25% compared to anaerobic cultivation. Recombinant strains expressing the ROS scavenging enzymes Mn-catalase and Mn-superoxide dismutase were successfully constructed using the pSIP expression system. The Mn-catalase and Mn-SOD-expressing strains produced activities of 873 U/ml and 1213 U/ml and could minimize the ROS formation in the cell, resulting in fourfold and sevenfold higher biomass formation, respectively.ConclusionsExpression of Mn-catalase and Mn-SOD in L. reuteri KUB-AC5 successfully reduced oxidative stress and enhanced growth. This finding could be applied for other lactic acid bacteria that are subject to oxidative stress and will be beneficial for applications of lactic acid bacteria for cell factory applications.

Processing and characterizing repulping reject of liquid package board

AbstractLiquid package board (LPB) packages typically have the smallest global warming potential compared to other package solutions, but recycling of the integrated plastic and aluminum fractions of the packages is still limited. In the fiber recycling process the plastic and aluminum fractions with some residual fibers are concentrated to the repulping reject (Polyal). This study investigates composition and properties of repulping rejects from recycling of separately collected LPB packages. The rejects were modified in different ways, hot pressed, grinded and finally compounded with a twin screw extruder. Compositions of the compounds were characterized with Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) and their properties were studied with tensile testing and melt flow rate (MFR) measurements. All studied compounds had relatively good processability and mechanical properties. Properties of compounds were improved by extraction of residual fibers and melt filtering of polyethylene terephthalate (PET) flakes. Especially melt filtering offered a significant improvement in strain at break from 11% to 33%. It was also found that high density polyethylene (HDPE) should be extracted from the reject and recycled separately since the extraction is relatively easy, does not cause drastic changes in the compound properties and recycled HDPE has more potential applications than the Polyal compounds.

Multiple strategies for the development of multienzyme complex for one-pot reactions

The main intention in the modern era is to makelife and activities on earth more comfortable by adding necessary products through biological machinery. Millions of tons of biological raw materials and lignocellulosic biomass are wasted by burning each year without providing benefits to living organisms. Instead of being the cause of disturbing the natural environment by increasing global warming and pollutants worldwide, now, it is the need of the hour to develop an advanced strategy to utilize these biological raw materials to produce renewable energy resources to meet the energy crisis. The review presents the idea of multiple enzymes in one step to hydrolyze complex biomaterials into useful products. The paper discusses how multiple enzymes are arranged in a cascade for complete hydrolysis of raw material in one-pot to prevent multistep, time consuming, and expensive methods. Furthermore, there was the immobilization of multiple enzymes in a cascade system with in vitro and in vivo conditions for reusability of enzymes. The role of genetic engineering, metabolic engineering, and random mutation techniques is described for the development of multiple enzyme cascades. Techniques that are involved in the improvement of native strain to recombinant strain for the enhancement of hydrolytic capacity were used. The preparative steps, before enzymatic hydrolysis like acid, and base treatment methods are more effective for improving the hydrolysis of biomass by multiple enzymes in a one-pot system. Finally, the applications of one-pot multienzyme complexes in biofuel production from lignocellulosic biomass, biosensor production, medicine, food industry, and the conversion of biopolymers into useful products are described.

Transcriptome Analysis Reveals an Eicosapentaenoic Acid Accumulation Mechanism in a Schizochytrium sp. Mutant.

Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. Schizochytrium is a marine eukaryote that has been widely utilized for the synthesis of PUFAs. The current low potency and performance of EPA production by fermentation of Schizochytrium spp. limits its prospect in commercial production of EPA. Since the synthesis pathway of EPA in Schizochytrium spp. is still unclear, mutagenesis combined with efficient screening methods are still desirable. In this study, a novel screening strategy was developed based on a two-step progressive mutagenesis method based on atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) after multiple stresses (sethoxydim, triclosan and 2,2'-bipyridine) compound screening. Finally, the mutant strain DBT-64 with increased lipid (1.57-fold, 31.71 g/L) and EPA (5.64-fold, 1.86 g/L) production was screened from wild-type (W) strains; the docosahexaenoic acid (DHA) content of mutant DBT-64 (M) was 11.41% lower than that of wild-type strains. Comparative transcriptomic analysis showed that the expression of genes related to the polyketide synthase, fatty acid prolongation, and triglyceride synthesis pathways was significantly upregulated in the mutant strain, while the expression of genes involved in the β-oxidation pathway and fatty acid degradation pathway was downregulated in favor of EPA biosynthesis in Schizochytrium. This study provides an effective strain improvement method to enhance EPA accumulation in Schizochytrium spp. IMPORTANCE Schizochytrium, a marine eukaryotic microorganism, has emerged as a candidate for the commercial production of PUFAs. EPA is an omega-3 PUFA with preventive and therapeutic effects against cardiovascular diseases, schizophrenia, and other disorders. Currently, the low potency and performance of EPA production by Schizochytrium spp. limits its commercialization. In this study, we performed two-step progressive mutagenesis based on ARTP and DES and screened multiple stresses (sethoxydim, triclosan, and 2,2'-bipyridine) to obtain the EPA-high-yielding Schizochytrium mutant. In addition, high expression of the polyketide synthase pathway, fatty acid elongation pathway, and triglyceride synthesis pathway in the mutants was confirmed by transcriptomic analysis. Therefore, the multistress screening platform established in this study is important for breeding EPA-producing Schizochytrium spp. and provides valuable information for regulating the proportion of EPA in microalgal lipids by means of genetic engineering.

Strain Analysis in Patients with Frequent Premature Ventricular Complexes and Preserved Left Ventricular Function Undergoing Ablation.

Frequent premature ventricular complexes (PVCs) can cause PVC-induced cardiomyopathy. The value of PVC ablation in patients with preserved left ventricular function in the low-normal range (ejection fraction: 50-55%) is not established. Strain analysis has been used to estimate changes in left ventricular function beyond assessment of the ejection fraction (EF). Longitudinal strain has been proposed as a method to detect changes over time in the setting of frequent asymptomatic premature ventricular complexes and preserved left ventricular (LV) function. A decrease in strain may be evidence of PVC-induced cardiomyopathy. In this study, we assessed the role of PVC ablation in patients with low-normal EF and the effect on EF and myocardial strain before and after PVC ablation. A total of 70 consecutive patients with either low-normal EF (0.5-<0.55, n = 35) or high-normal EF (≥0.55; n = 35), using available imaging and Holter data, were referred for ablation due to frequent PVCs. EF and longitudinal strain were assessed pre- and post-ablation. There was a significant increase in EF (53.2 ± 0.4% to 58.3 ± 0.5%, p < 0.001) and improvement in longitudinal strain (-15.2 ± 3.3 to -16.6 ± 3, p = 0.007) post-ablation in patients with low-normal EF and successful ablation. There was no change in EF or longitudinal strain in patients with high-normal EF and a successful ablation pre- vs. post-ablation. Patients with frequent PVCs and low-normal LV EF compared to patients with frequent PVCs and high-normal LV EF have evidence of PVC-induced cardiomyopathy and may benefit from ablation despite a preserved left ventricular EF.

Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening.

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based microfluidics technology, single cells can be encapsulated in droplets. The screening can be carried out by detecting the fluorescence intensity of secreted riboflavin. Thus, an efficient and high-throughput screening method suitable for riboflavin production strain improvement could be established. In this study, droplet-based microfluidics screening was applied, and a more competitive riboflavin producer U3 was selected from the random mutation library of strain S1. The riboflavin production and biomass of U3 were higher than that of S1 in flask fermentation. In addition, the results of fed-batch fermentation showed that the riboflavin production of U3 was 24.3 g/L, an 18% increase compared with the parent strain S1 (20.6 g/L), and the yield (g riboflavin/100 g glucose) increased by 19%, from 7.3 (S1) to 8.7 (U3). Two mutations of U3 (sinRG89R and icdD28E) were identified through whole genome sequencing and comparison. Then they were introduced into BS168DR (parent of S1) for further analysis, which also caused riboflavin production to increase. This paper provides protocols for screening riboflavin-producing B. subtilis with droplet-based microfluidics technology and reveals mutations in riboflavin overproduction strains.

Enhanced Pentostatin Production in Actinomadura sp. by Combining ARTP Mutagenesis, Ribosome Engineering and Subsequent Fermentation Optimization

The special structure of pentostatin causes it to possess a wide spectrum of biological and pharmacological properties, and it has been extensively employed to treat malignant tumors and is the first-line treatment for hairy cell leukemia. Pentostatin is mainly distributed in several actinomycetes and fungi species. However, its low titer in microbes is not able to meet medical needs. Here, we report a strain improvement strategy based on combined atmospheric and room-temperature plasma (ARTP) mutagenesis and ribosome engineering screening, as well as fermentation optimization, for enhanced pentostatin production. The original strain, Actinomadura sp. ATCC 39365, was treated with ARTP and screened by ribosome engineering to obtain one stable pentostatin high-yield mutant Actinomadura sp. S-15, which produced 86.35 mg/L pentostatin, representing a 33.79% increase compared to Actinomadura sp. ATCC 39365. qRT-PCR analysis revealed that pentostatin biosynthesis-related gene expression was significantly upregulated in Actinomadura sp. S-15. Then, to further enhance pentostatin production, the fermentation medium was optimized in flask culture and the pentostatin production of Actinomadura sp. S-15 reached 152.06 mg/L, which is the highest pentostatin production reported so far. These results demonstrate the effectiveness of combined ARTP mutation, ribosome engineering screening, and medium optimization for the enhancement of pentostatin production, and provide a methodology enabling the sustainable production of pentostatin on an industrial scale.

Strain improvement of Trichoderma harzianum for enhanced biocontrol capacity: Strategies and prospects.

In the control of plant diseases, biocontrol has the advantages of being efficient and safe for human health and the environment. The filamentous fungus Trichoderma harzianum and its closely related species can inhibit the growth of many phytopathogenic fungi, and have been developed as commercial biocontrol agents for decades. In this review, we summarize studies on T. harzianum species complex from the perspective of strain improvement. To elevate the biocontrol ability, the production of extracellular proteins and compounds with antimicrobial or plant immunity-eliciting activities need to be enhanced. In addition, resistance to various environmental stressors should be strengthened. Engineering the gene regulatory system has the potential to modulate a variety of biological processes related to biocontrol. With the rapidly developing technologies for fungal genetic engineering, T. harzianum strains with increased biocontrol activities are expected to be constructed to promote the sustainable development of agriculture.

Sacubitril/Valsartan Improves Left Atrial and Ventricular Strain and Strain Rate in Patients with Heart Failure with Reduced Ejection Fraction.

Data on the impact of sacubitril/valsartan (SV) therapy on phasic left atrial (LA) and left ventricular (LV) strain in heart failure with reduced ejection fraction (HFrEF) are limited. The aim of this study was to evaluate changes in two-dimensional speckle tracking (2D-STE) parameters with SV therapy in HFrEF patients. Prospective evaluation of HFrEF patients receiving optimized medical therapy. Two-dimensional speckle tracking (2D-STE) parameters were assessed at baseline and after 6 months of SV therapy. LA strain and strain rate (SR) in reservoir, conduit, and contraction phases were compared with LV longitudinal, radial, and circumferential strain and SR and stratified according to heart rhythm and HFrEF etiology. A total of 35 patients completed the 6-month follow-up, with a mean age of 59 ± 11 years, 40% in atrial fibrillation, 43% with ischemic etiology, and LVEF of 29 ± 6%. There were significant improvements in LA reservoir, conduit, and contractile strain and SR following SV therapy, particularly among patients in sinus rhythm. There were significant improvements in longitudinal, radial, and circumferential LV function indices. SV therapy in HFrEF was associated with improved longitudinal, radial, and circumferential function, particularly among patients in sinus rhythm. These findings can provide insights into the mechanisms underlying the improvement of cardiac function and help assess subclinical responses to the treatment.

Radiofrequency catheter ablation for improving myocardial work in patients with ventricular pre-excitation.

Previous studies have not consistently found significant improvements in left ventricular ejection fraction or global longitudinal strain (GLS) after radiofrequency catheter ablation (RFCA) in patients with ventricular pre-excitation. The aim of this study was thus to explore the effects of RFCA on left ventricular function in patients with ventricular pre-excitation using a new noninvasive echocardiographic method of myocardial work. A total of 34 patients with ventricular pre-excitation who underwent RFCA and 18 healthy controls were prospectively included in this study. Before and after participants underwent RFCA, electrocardiographic and echocardiographic data of the patients were collected at resting and pacing heart rates (HRs) of 100 beats per minute (bpm) and 120 bpm (controlled by high right atrial pacing during the procedure). Clinical data of the healthy controls at resting HR were also collected. A self-controlled paired sample t test was used to compare the differences before and after participants underwent RFCA. After participants underwent RFCA, the global wasted work (GWW) of the included patients decreased (resting HR: 165.3±68.8 vs. 92.6±42.5 mmHg%, P<0.001; HR of 100 bpm: 276.3±121.2 vs. 187.9±96.0 mmHg%, P<0.001; HR of 120 bpm: 323.9±126.7 vs. 181.0%±74.3 mmHg%. P<0.001), while the global work efficiency (GWE) increased (resting HR: 91.5%±3.8% vs. 94.9%±1.6%; P<0.001; HR of 100 bpm: 87.0%±5.2% vs. 91.0%±3.3%, P<0.001; HR of 120 bpm: 85.0%±5.1% vs. 90.3%±3.7%, P<0.001). In patients with ventricular pre-excitation, impaired GWW and GWE can be improved with RFCA. In clinical practice, noninvasive myocardial work assessment can be used in patients with ventricular pre-excitation.

Challenges and advancements in bioprocess intensification of fungal secondary metabolite: kojic acid

Kojic acid is a fungal secondary metabolite commonly known as a tyrosinase inhibitor, that acts as a skin-whitening agent. Its applications are widely distributed in the area of cosmetics, medicine, food, and chemical synthesis. Renewable resources are the alternative feedstocks that can fulfill the demand for free sugars which are fermented for the production of kojic acid. This review highlights the current progress and importance of bioprocessing of kojic acid from various types of competitive and non-competitive renewable feedstocks. The bioprocessing advancements, secondary metabolic pathway networks, gene clusters and regulations, strain improvement, and process design have also been discussed. The importance of nitrogen sources, amino acids, ions, agitation, and pH has been summarized. Two fungal species Aspergillus flavus and Aspergillus oryzae are found to be extensively studied for kojic acid production due to their versatile substrate utilization and high titer ability. The potential of A. flavus to be a competitive industrial strain for large-scale production of kojic acid has been studied.