Molecular weight control of poly-γ-glutamic acid reveals novel insights into extracellular polymeric substance synthesis in Bacillus licheniformis.

BACKGROUND: The structural diversity of extracellular polymeric substances produced by microorganisms is attracting particular attention. Poly-gamma-glutamic acid (γ-PGA) is a widely studied extracellular polymeric substance from Bacillus species. The function of γ-PGA varies with its molecular weight (Mw). RESULTS: Herein, different endogenous promoters in Bacillus licheniformis were selected to regulate the expression levels of pgdS, resulting in the formation of γ-PGA with Mw values ranging from 1.61 × 103 to 2.03 × 104 kDa. The yields of γ-PGA and exopolysaccharides (EPS) both increased in the pgdS engineered strain with the lowest Mw and viscosity, in which the EPS content was almost tenfold higher than that of the wild-type strain. Subsequently, the compositions of EPS from the pgdS engineered strain also changed. Metabolomics and RT-qPCR further revealed that improving the transportation efficiency of EPS and the regulation of carbon flow of monosaccharide synthesis could affect the EPS yield. CONCLUSIONS: Here, we present a novel insight that increased pgdS expression led to the degradation of γ-PGA Mw and changes in EPS composition, thereby stimulating EPS and γ-PGA production. The results indicated a close relationship between γ-PGA and EPS in B. licheniformis and provided an effective strategy for the controlled synthesis of extracellular polymeric substances.

Disease-induced changes in bacterial and fungal communities from plant below- and aboveground compartments.

The plant microbes are an integral part of the host and play fundamental roles in plant growth and health. There is evidence indicating that plants have the ability to attract beneficial microorganisms through their roots in order to defend against pathogens. However, the mechanisms of plant microbial community assembly from below- to aboveground compartments under pathogen infection remain unclear. In this study, we investigated the bacterial and fungal communities in bulk soil, rhizosphere soil, root, stem, and leaf of both healthy and infected (Potato virus Y disease, PVY) plants. The results indicated that bacterial and fungal communities showed different recruitment strategies in plant organs. The number and abundance of shared bacterial ASVs between bulk and rhizosphere soils decreased with ascending migration from below- to aboveground compartments, while the number and abundance of fungal ASVs showed no obvious changes. Field type, plant compartments, and PVY infection all affected the diversity and structures of microbial community, with stronger effects observed in the bacterial community than the fungal community. Furthermore, PVY infection, rhizosphere soil pH, and water content (WC) contributed more to the assembly of the bacterial community than the fungal community. The analysis of microbial networks revealed that the bacterial communities were more sensitive to PVY infection than the fungal communities, as evidenced by the lower network stability of the bacterial community, which was characterized by a higher proportion of positive edges. PVY infection further increased the bacterial network stability and decreased the fungal network stability. These findings advance our understanding of how microbes respond to pathogen infections and provide a rationale and theoretical basis for biocontrol technology in promoting sustainable agriculture. KEY POINTS: • Different recruitment strategies between plant bacterial and fungal communities. • Bacterial community was more sensitive to PVY infection than fungal community. • pH and WC drove the microbial community assembly under PVY infection.

Self-Buffering system for Cost-Effective production of lactic acid from glucose and xylose using Acid-Tolerant Issatchenkia orientalis.

This study presents a cost-effective strategy for producing organic acids from glucose and xylose using the acid-tolerant yeast, Issatchenkia orientalis. I. orientalis was engineered to produce lactic acid from xylose, and the resulting strain, SD108XL, successfully converted sorghum hydrolysates into lactic acid. In order to enable low-pH fermentation, a self-buffering strategy, where the lactic acid generated by the SD108XL strain during fermentation served as a buffer, was developed. As a result, the SD108 strain produced 67 g/L of lactic acid from 73 g/L of glucose and 40 g/L of xylose, simulating a sugar composition of sorghum biomass hydrolysates. Moreover, techno-economic analysis underscored the efficiency of the self-buffering strategy in streamlining the downstream process, thereby reducing production costs. These results demonstrate the potential of I. orientalis as a platform strain for the cost-effective production of organic acids from cellulosic hydrolysates.

Multidimensional Optimization of Saccharomyces cerevisiae for Carotenoid Overproduction.

Microbial synthesis of carotenoids is a highly desirable alternative to plant extraction and chemical synthesis. In this study, we investigated multidimensional strategies to improve the carotenoid synthesis in the industrial workhorse of Saccharomyces cerevisiae. First, we rewired the yeast central metabolism by optimizing non-oxidative glycolysis pathway for an improved acetyl-CoA supply. Second, we restricted the consumption of farnesyl pyrophosphate (FPP) by the down-regulation of squalene synthase using the PEST degron. Third, we further explored the human lipid binding/transfer protein saposin B (hSapB)-mediated metabolic sink for an enhanced storage of lipophilic carotenoids. Last, the copper-induced GAL expression system was engineered to function in the yeast-peptone-dextrose medium for an increased biomass accumulation. By combining the abovementioned strategies, the final engineered yeast produced 166.79 ± 10.43 mg/l ß-carotene in shake flasks, which was nearly 5-fold improvement of the parental carotenoid-producing strain. Together, we envision that multidimensional strategies reported here might be applicable to other hosts for the future industrial development of carotenoid synthesis from renewable feedstocks.

Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile.

BACKGROUND: The hydrolysis and transphosphatidylation of phospholipase D (PLD) play important roles in the interconversion of phospholipids (PLs), which has been shown to profoundly impact lipid metabolism in plants. In this study, the effect of the PLD1 gene of Schizochytrium limacinum SR21 (S. limacinum SR21) on lipid metabolism was investigated. RESULTS: PLD1 knockout had little impact on cell growth and lipid production, but it significantly improved the percentage of polyunsaturated fatty acids in lipids, of which docosahexaenoic acid (DHA) content increased by 13.3% compared to the wild-type strain. Phospholipomics and real-time quantitative PCR analysis revealed the knockout of PLD1 reduced the interexchange and increased de novo synthesis of PLs, which altered the composition of PLs, accompanied by a final decrease in phosphatidylcholine (PC) and an increase in phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid levels. PLD1 knockout also increased DHA content in triglycerides (TAGs) and decreased it in PLs. CONCLUSIONS: These results indicate that PLD1 mainly performs the transphosphatidylation activity in S. limacinum SR21, and its knockout promotes the migration of DHA from PLs to TAGs, which is conducive to DHA accumulation and storage in TAGs via an acyl CoA-independent pathway. This study provides a novel approach for identifying the mechanism of DHA accumulation and metabolic regulation strategies for DHA production in S. limacinum SR21.

Metabolic engineering strategies to enable microbial electrosynthesis utilization of CO2: recent progress and challenges.

Microbial electrosynthesis (MES) is a promising technology that mainly utilizes microbial cells to convert CO2 into value-added chemicals using electrons provided by the cathode. However, the low electron transfer rate is a solid bottleneck hindering the further application of MES. Thus, as an effective strategy, genetic tools play a key role in MES for enhancing the electron transfer rate and diversity of production. We describe a set of genetic strategies based on fundamental characteristics and current successes and discuss their functional mechanisms in driving microbial electrocatalytic reactions to fully comprehend the roles and uses of genetic tools in MES. This paper also analyzes the process of nanomaterial application in extracellular electron transfer (EET). It provides a technique that combines nanomaterials and genetic tools to increase MES efficiency, because nanoparticles have a role in the production of functional genes in EET although genetic tools can subvert MES, it still has issues with difficult transformation and low expression levels. Genetic tools remain one of the most promising future strategies for advancing the MES process despite these challenges.

Mixed Selectivity Coding of Content-Temporal Detail by Dorsomedial Posterior Parietal Neurons.

The dorsomedial posterior parietal cortex (dmPPC) is part of a higher-cognition network implicated in elaborate processes underpinning memory formation, recollection, episode reconstruction, and temporal information processing. Neural coding for complex episodic processing is however under-documented. Here, we recorded extracellular neural activities from three male rhesus macaques (Macaca mulatta) and revealed a set of neural codes of "neuroethogram" in the primate parietal cortex. Analyzing neural responses in macaque dmPPC to naturalistic videos, we discovered several groups of neurons that are sensitive to different categories of ethogram items, low-level sensory features, and saccadic eye movement. We also discovered that the processing of category and feature information by these neurons is sustained by the accumulation of temporal information over a long timescale of up to 30 s, corroborating its reported long temporal receptive windows. We performed an additional behavioral experiment with additional two male rhesus macaques and found that saccade-related activities could not account for the mixed neuronal responses elicited by the video stimuli. We further observed monkeys' scan paths and gaze consistency are modulated by video content. Taken altogether, these neural findings explain how dmPPC weaves fabrics of ongoing experiences together in real time. The high dimensionality of neural representations should motivate us to shift the focus of attention from pure selectivity neurons to mixed selectivity neurons, especially in increasingly complex naturalistic task designs.

Emerging nonmodel eukaryotes for biofuel production.

Microbial synthesis of biofuels offers a promising solution to the global environmental and energy concerns. However, the main challenge of microbial cell factories is their high fermentation costs. Model hosts, such as Escherichia coli and Saccharomyces cerevisiae, are typically used for proof-of-concept studies of producing different types of biofuels, however, they have a limited potential for biofuel production at an industrially relevant scale due to the weak stability/robustness and narrow substrate scope. With the advancements of synthetic biology and metabolic engineering, nonmodel eukaryotes, with naturally favorable phenotypic and metabolic features, have been emerging as promising biofuel producers. Here, we introduce the emerging nonmodel eukaryotes for the biofuel production and discuss their specific advantages, especially those with the capacity of producing cellulosic ethanol, higher alcohols, and fatty acid-/terpene-derived biofuel molecules. We also propose the challenges and prospects for developing nonmodel eukaryotic as the ideal hosts for future biofuel production.

Metabolic regulation of Shewanella oneidensis for microbial electrosynthesis: From extracellular to intracellular.

Shewanella oneidensis MR-1 (S. oneidensis MR-1) has been shown to benefit from microbial electrosynthesis (MES) due to its exceptional electron transfer efficiency. In this study, genes involved in both extracellular electron uptake (EEU) and intracellular CO2 conversion processes were examined and regulated to enhance MES performance. The key genes identified for MES in the EEU process were mtrB, mtrC, mtrD, mtrE, omcA and cctA. Overexpression of these genes resulted in 1.5-2.1 times higher formate productivity than that of the wild-type strains (0.63 mmol/(L·µg protein)), as 0.94-1.61 mmol/(L·µg protein). In the intracellular CO2 conversion process, overexpression of the nadE, nadD, nadR, nadV, pncC and petC genes increased formate productivity 1.3-fold-3.4-fold. Moreover, overexpression of the formate dehydrogenase genes fdhA1, fdhB1 and fdhX1 in modified strains led to a 2.3-fold-3.1-fold increase in formate productivity compared to wild-type strains. The co-overexpression of cctA, fdhA1 and nadV in the mutant strain resulted in 5.59 times (3.50 mmol/(L·µg protein)) higher formate productivity than that of the wild-type strains. These findings revealed that electrons of MES derived from the electrode were utilized in the energy module for synthesizing ATP and NADH, followed by the synthesis of formate in formate dehydrogenase by the combinatorial effects of ATP, NADH, electrons and CO2. The results provide new insights into the mechanism of MES in S. oneidensis MR-1 and pave the way for genetic improvements that could facilitate the further application of MES.

A Layered Genetic Design Enables the Yeast Galactose Regulon to Respond to Cyanamide.

The commonly used expression systems in Saccharomyces cerevisiae typically rely on either constitutive or galactose-regulated promoters. The lack of inducible systems in S. cerevisiae limits the precise temporal regulation of protein function and yeast metabolism. We herein repurposed the galactose-regulated system to make it respond to cyanamide. By using a cyanamide-inducible DDI2 promoter to control Gal4 expression in CEN.PK2-1C with Δgal80, a tight and graded cyanamide-inducible GAL system with an enhanced signal output was constructed. Subsequently, we demonstrated that the cyanamide-inducible GAL system was capable of tightly regulating the pentafunctional Aro1 protein to achieve conditional shikimate pathway activity. Taken together, the cyanamide-inducible GAL system could be implemented for both fundamental research and applied biotechnology.

Emodin attenuates diabetic kidney disease by inhibiting ferroptosis via upregulating Nrf2 expression.

Diabetic kidney disease (DKD) poses a threat to people's health. The current treatments only provide partial relief of symptoms. Therefore, seeking a promising therapeutic medication for the prevention and control on DKD will benefit patients. Recently, a novel iron-dependent and non-apoptotic regulated mode of cell death, termed as ferroptosis, is expected to offer us a novel insight into the mechanism of DKD. We conducted experiments to investigate the role of ferroptosis in the development of DKD. Iron accumulation, weakened antioxidant capacity and ROS overproduction were observed in the renal tissues of STZ-induced diabetic rats. A persistent high glucose condition contributed to down regulated levels of Glutathione Peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11 (SLC7A11) which marked the occurrence of ferroptosis. Treatment of Emodin in DKD models could significantly attenuated these changes and reduced renal injury. Besides, NFE2-related factor 2 (Nrf2), an important antioxidant regulator, was inhibited in both in vivo and in vitro assay, which contributes to Reactive Oxygen Species (ROS) generation that further promoted the expression of ferroptosis related protein. These unwanted effects were offset by the intervention of Emodin. The specific Nrf2 knock out enhanced cell's sensitivity to ferroptosis by being exposed to high glucose culture, which was improved by treatment of Emodin via restoring activity of Nrf2. In conclusion, our research demonstrated that Emodin exerted renal protection against DKD via inhibiting ferroptosis and restoring Nrf2 mediated antioxidant capacity, which could be employed as a novel therapeutic medication against DKD.

Advanced strategies for metabolic engineering of Bacillus to produce extracellular polymeric substances.

Extracellular polymeric substances are mainly synthesized via a variety of biosynthetic pathways in bacteria. Bacilli-sourced extracellular polymeric substances, such as exopolysaccharides (EPS) and poly-γ-glutamic acid (γ-PGA), can serve as active ingredients and hydrogels, and have other important industrial applications. However, the functional diversity and widespread applications of these extracellular polymeric substances, are hampered by their low yields and high costs. Biosynthesis of extracellular polymeric substances is very complex in Bacillus, and there is no detailed elucidation of the reactions and regulations among various metabolic pathways. Therefore, a better understanding of the metabolic mechanisms is required to broaden the functions and increase the yield of extracellular polymeric substances. This review systematically summarizes the biosynthesis and metabolic mechanisms of extracellular polymeric substances in Bacillus, providing an in-depth understanding of the relationships between EPS and γ-PGA synthesis. This review provides a better clarification of Bacillus metabolic mechanisms during extracellular polymeric substance secretion and thus benefits their application and commercialization.

Copper-inducible expression system for metabolic engineering of Escherichia coli.

AIMS: The inducible expression system plays an important role in engineering Escherichia coli for chemical production. However, it still heavily relies on expensive chemical inducers, like IPTG. There is a pressing need to develop alternative expression systems with more affordable inducers. MATERIALS AND RESULTS: We herein report a copper-inducible expression system in E. coli based on the two-component Cus system and T7 RNA polymerase (RNAP). By integrating the gene encoding T7 RNAP at the CusC locus, we managed to program eGFP expression under the T7 promoter in response to different concentrations of Cu2+ (0-20 µM). Subsequently, we demonstrated that the copper-inducible expression system was suitable for the metabolic engineering of E. coli toward protocatechuic acid overproduction, and the resulting strain with combined manipulation of the central metabolism via CRISPRi produced 4.12 g L-1 PCA under the optimal copper concentration and induction time. CONCLUSIONS: We have established a copper-inducible T7 RNAP expression system in E. coli. The copper-inducible expression system could rationally control metabolic pathways in a temporal and dose-dependent manner. The gradient expression system based on copper inducer could be widely used in E. coli cell factories, and the design principle reported here would also be applicable in other prokaryotes.

Meta-analysis of the characteristic expression of circulating microRNA in type 2 diabetes mellitus with acute ischemic cerebrovascular disease.

Objective: To comprehensively evaluate the characteristics of the circulating microRNA expression profile in type 2 diabetic patients with acute ischemic cerebrovascular disease by systematic evaluation and meta-analysis. Methods: The literatures up to March 2022 related to circulating microRNA and acute ischemic cerebrovascular disease in type 2 diabetes mellitus were searched and screened from multiple databases. The NOS quality assessment scale was used to evaluate methodological quality. Heterogeneity tests and statistical analyses of all data were performed by Stata 16.0. The differences in microRNA levels between groups were illustrated by the standardized mean difference (SMD) and 95% confidence interval (95% CI). Results: A total of 49 studies on 12 circulating miRNAs were included in this study, including 486 cases of type 2 diabetes complicated with acute ischemic cerebrovascular disease and 855 controls. Compared with the control group (T2DM group), miR-200a, miR-144, and miR-503 were upregulated and positively correlated with acute ischemic cerebrovascular disease in type 2 diabetes mellitus patients. Their comprehensive SMD and 95% CI were 2.71 (1.64~3.77), 5.77 (4.28~7.26) and 0.73 (0.27~1.19), respectively. MiR-126 was downregulated and negatively correlated with acute ischemic cerebrovascular disease in type 2 diabetes mellitus patients, its comprehensive SMD and 95% CI were -3.64 (-5.56~-1.72). Conclusion: In type 2 diabetes mellitus patients with acute ischemic cerebrovascular disease, the expression of serum miR-200a, miR-503, plasma and platelet miR-144 was upregulated and the expression of serum miR-126 was downregulated. It may have diagnostic value in the early identification of type 2 diabetes mellitus with acute ischemic cerebrovascular disease.

Expanding sugar alcohol industry: Microbial production of sugar alcohols and associated chemocatalytic derivatives.

Sugar alcohols are polyols that are widely employed in the production of chemicals, pharmaceuticals, and food products. Chemical synthesis of polyols, however, is complex and necessitates the use of hazardous compounds. Therefore, the use of microbes to produce polyols has been proposed as an alternative to traditional synthesis strategies. Many biotechnological approaches have been described to enhancing sugar alcohols production and microbe-mediated sugar alcohol production has the potential to benefit from the availability of inexpensive substrate inputs. Among of them, microbe-mediated erythritol production has been implemented in an industrial scale, but microbial growth and substrate conversion rates are often limited by harsh environmental conditions. In this review, we focused on xylitol, mannitol, sorbitol, and erythritol, the four representative sugar alcohols. The main metabolic engineering strategies, such as regulation of key genes and cofactor balancing, for improving the production of these sugar alcohols were reviewed. The feasible strategies to enhance the stress tolerance of chassis cells, especially thermotolerance, were also summarized. Different low-cost substrates like glycerol, molasses, cellulose hydrolysate, and CO2 employed for producing these sugar alcohols were presented. Given the value of polyols as precursor platform chemicals that can be leveraged to produce a diverse array of chemical products, we not only discuss the challenges encountered in the above parts, but also envisioned the development of their derivatives for broadening the application of sugar alcohols.

Mass Spectrometry-Based High-Throughput Quantification of Bioproducts in Liquid Culture.

To meet the ever-increasing need for high-throughput screening in metabolic engineering, information-rich, fast screening methods are needed. Mass spectrometry (MS) provides an efficient and general approach for metabolite screening and offers the capability of characterizing a broad range of analytes in a label-free manner, but often requires a range of sample clean-up and extraction steps. Liquid extraction surface analysis (LESA) coupled MS is an image-guided MS surface analysis approach that directly samples and introduces metabolites from a surface to MS. Here, we combined the advantages of LESA-MS and an acoustic liquid handler with stable isotope-labeled internal standards. This approach provides absolute quantitation of target chemicals from liquid culture-dried droplets and enables high-throughput quantitative screening for microbial metabolites. In this study, LESA-MS was successfully applied to quantify several different metabolites (itaconic acid, triacetic acid lactone, and palmitic acid) from different yeast strains in different mediums, demonstrating its versatility, accuracy, and efficiency across a range of microbial engineering applications.

Hyperproduction of extracellular polymeric substance in Pseudomonas fluorescens for efficient chromium (VI) absorption.

A novel extracellular polymeric substance (EPS) with flocculating activity produced by Pseudomonas fluorescein isolated from soil was studied in this paper. Firstly, atmospheric and room temperature plasma (ARTP) was applied to get a mutant of P. fluorescein with higher EPS production. A mutant T4-2 exhibited a 106.48% increase in flocculating activity compared to the original strain. The maximum EPS yield from T4-2 was enhanced up to 6.42 g/L, nearly 10 times higher than the original strain on a 3.6-L bioreactor with optimized fermentation conditions. Moreover, the flocculating activity of the mutant reached 3023.4 U/mL, 10.96-fold higher than that of T4. Further identification showed that EPS from mutant T4-2 was mainly composed of polysaccharide (76.67%) and protein (15.8%) with a molecular weight of 1.17 × 105 Da. The EPS showed excellent adsorption capacities of 80.13 mg/g for chromium (VI), which was much higher than many reported adsorbents such as chitosan and cellulose. The adsorption results were described by Langmuir isotherm and pseudo-second-order kinetic model. The thermodynamic parameters (ΔG0, ΔH0 and ΔS0) revealed that the adsorption process was spontaneous and exothermic. Adsorption mechanisms were speculated to be electrostatic interaction, reduction, and chelation.