Exploration of the Native Sucrose Operon Enables the Development of an Inducible T7 Expression System in Paenibacillus polymyxa.

The use of Paenibacillus polymyxa as an industrial producer is limited by the lack of suitable synthetic biology tools. In this study, we identified a native sucrose operon in P. polymyxa. Its structural and functional relationship analysis revealed the presence of multiple regulatory elements, including four ScrR-binding sites and a catabolite-responsive element (CRE). In P. polymyxa, we established a cascade T7 expression system involving an integrated T7 RNA polymerase (T7P) regulated by the sucrose operon and a T7 promoter. It enables controllable gene expression by sucrose and regulatory elements, and a 5-fold increase in expression efficiency compared with the original sucrose operon was achieved. Further deletion of SacB in P. polymyxa resulted in a 38.95% increase in the level of thermophilic lipase (TrLip) production using the cascade T7 induction system. The results highlight the effectiveness of sucrose regulation as a novel synthetic biology tool, which facilitates exploring gene circuits and enables their dynamic regulation.

Emulsion for stabilizing ß-carotene and curcumin prepared directly using a continuous phase of polysaccharide-rich Schizophyllum commune fermentation broth.

In this study, we examined the effect of Schizophyllum commune fermentation broth (SCFB) rich in polysaccharides (SCFP) on the stability and bioaccessibility of ß-carotene and curcumin. An SCFB-stabilized oil-in-water (o/w) emulsion (SCFBe) was prepared using SCFB as the continuous phase, and then evaluated for storage stability using an SCFP-based emulsion (SCFPe) as the control. The findings revealed that SCFBe is more stable at 60 °C than SCFPe, and stratification or droplet size varied at differing pH levels (3-9) and concentrations of Na+ (0.1-0.5 M) and Ca2+ (0.01-0.05 M). Since the absolute value of the zeta potential of SCFBe is much lower at 60 °C than that at 4 °C and 25 °C, a higher temperature (60 °C) may enhance the reactivity of polysaccharides and proteins in SCFB to improve the stability of SCFBe. Both the protective impact of SCFB on functional food molecules and their capacity to block lipid oxidation increased as polysaccharide content improved. The bioaccessibility of ß-carotene after in vitro simulated gastrointestinal digestion is 11.18 %-12.28 %, whereas that of curcumin is 31.64 %-33.00 %. By fermenting edible and medicinal fungi in liquid, we created a unique and environmentally friendly approach for getting food-grade emulsifiers without extraction.

Lignin-degrading enzyme production was enhanced by the novel transcription factor Ptf6 in synergistic microbial co-culture.

Synergistic microbial co-culture has been an efficient and energy-saving strategy to produce lignin-degrading enzymes (LDEs), including laccase, manganese peroxidase, and versatile peroxidase. However, the regulatory mechanism of microbial co-culture is still unclear. Herein, the extracellular LDE activities of four white-rot fungi were significantly increased by 88-544% over monoculture levels when co-cultured with Rhodotorula mucilaginosa. Ptf6 was demonstrated from the 9 million Y1H clone library to be a shared GATA transcription factor in the four fungi, and could directly bind to the laccase gene promoter. Ptf6 exists in two alternatively spliced isoforms under monoculture, namely Ptf6-α (1078 amino acids) containing Cys2/Cys2-type zinc finger and Ptf6-ß (963 amino acids) lacking the complete domain. Ptf6 responded to co-culture by up-regulation of both its own transcripts and the proportion of Ptf6-α. Ptf6-α positively activated the production of most LDE isoenzymes and bound to four GATA motifs on the LDEs' promoter with different affinities. Moreover, Ptf6-regulation mechanism can be applicable to a variety of microbial co-culture systems. This study lays a theoretical foundation for further improving LDEs production and providing an efficient way to enhance the effects of biological and enzymatic pretreatment for lignocellulosic biomass conversion.

Identification of a Novel Lactose-Specific PTS Operon in Bacillus licheniformis and Development of Derivative Artificial Operon Modules.

Bacillus licheniformis plays a crucial role as a microbial host in the food industry and shows promising potential as a probiotic for human intestinal regulation. It exhibits a remarkable ability to utilize lactose as its sole carbon source. Despite its significance, the lactose-related metabolic pathway in this strain remains unclear. In this study, we identified a novel lactose-specific operon (lacDCAB) in B. licheniformis, consisting of the lacD gene that encodes a unique 6-phospho-ß-galactosidase belonging to the GH4 family, and the lacCAB genes encoding a lactose-specific PTS1 system. Notably, we constructed and assessed an array library of transport and catabolic modules specifically for lactose utilization. Among these modules, PDS-lacD-P2-pts1 demonstrated the highest specific lactose consumption rate of 0.64 g/(L·h·OD), which was 8 times higher than that of the control strain. Furthermore, we developed a dual carbon source transport model based on the PDS-lacD-P2-pts1 assembly module, which highlighted efficient coutilization of glucose/sucrose, lactose/sucrose, lactose/galactose, and lactose/2,3-butanediol. This study provides insight into the lactose-specific metabolic pathway of B. licheniformis and presents a promising strategy for enhancing lactose utilization efficiency and mixed carbon source coutilization.

Design of a sorbitol-activated nitrogen metabolism-dependent regulatory system for redirection of carbon metabolism flow in Bacillus licheniformis.

Synthetic regulation of metabolic fluxes has emerged as a common strategy to improve the performance of microbial cell factories. The present regulatory toolboxes predominantly rely on the control and manipulation of carbon pathways. Nitrogen is an essential nutrient that plays a vital role in growth and metabolism. However, the availability of broadly applicable tools based on nitrogen pathways for metabolic regulation remains limited. In this work, we present a novel regulatory system that harnesses signals associated with nitrogen metabolism to redirect excess carbon flux in Bacillus licheniformis. By engineering the native transcription factor GlnR and incorporating a sorbitol-responsive element, we achieved a remarkable 99% inhibition of the expression of the green fluorescent protein reporter gene. Leveraging this system, we identified the optimal redirection point for the overflow carbon flux, resulting in a substantial 79.5% reduction in acetoin accumulation and a 2.6-fold increase in acetate production. This work highlight the significance of nitrogen metabolism in synthetic biology and its valuable contribution to metabolic engineering. Furthermore, our work paves the way for multidimensional metabolic regulation in future synthetic biology endeavors.

Remarkable response to PD-1 inhibitor in a patient with extensive-stage small cell lung cancer: a case report and literature review.

We report a case of a 59-year-old male diagnosed with extensive-stage small cell lung cancer (SCLC). He received first-line platinum doublet chemotherapy and second-line topotecan-based regimen, but experienced disease progression after each line of therapy. He was then treated with Sintilimab, a PD-1 inhibitor, in combination with nab-paclitaxel in the third-line setting, which resulted in significant tumor shrinkage. Restaging scans showed a partial response per RECIST criteria with 62% reduction in tumor burden. This case highlights the application and efficacy of immune checkpoint inhibitors in extensive-stage SCLC.

Mechanisms and biotechnological applications of transcription factors.

Transcription factors play an indispensable role in maintaining cellular viability and finely regulating complex internal metabolic networks. These crucial bioactive functions rely on their ability to respond to effectors and concurrently interact with binding sites. Recent advancements have brought innovative insights into the understanding of transcription factors. In this review, we comprehensively summarize the mechanisms by which transcription factors carry out their functions, along with calculation and experimental-based methods employed in their identification. Additionally, we highlight recent achievements in the application of transcription factors in various biotechnological fields, including cell engineering, human health, and biomanufacturing. Finally, the current limitations of research and provide prospects for future investigations are discussed. This review will provide enlightening theoretical guidance for transcription factors engineering.

A New Mechanism of Carbon Metabolism and Acetic Acid Balance Regulated by CcpA.

Catabolite control protein A (CcpA) is a critical regulator in Gram-positive bacteria that orchestrates carbon metabolism by coordinating the utilization of different carbon sources. Although it has been widely proved that CcpA helps prioritize the utilization of glucose over other carbon sources, this global regulator's precise mechanism of action remains unclear. In this study, a mutant Bacillus licheniformis deleted for CcpA was constructed. Cell growth, carbon utilization, metabolites and the transcription of key enzymes of the mutant strain were compared with that of the wild-type one. It was found that CcpA is involved in the regulation of glucose concentration metabolism in Bacillus. At the same time, CcpA regulates glucose metabolism by inhibiting acetic acid synthesis and pentose phosphate pathway key gene zwF. The conversion rate of acetic acid is increased by about 3.5 times after ccpA is deleted. The present study provides a new mechanism of carbon metabolism and acetic acid balance regulated by CcpA. On the one hand, this work deepens the understanding of the regulatory function of CcpA and provides a new view on the regulation of glucose metabolism. On the other hand, it is helpful to the transformation of B. licheniformis chassis microorganisms.

Highly Efficient Production of UDP-Glucose from Sucrose via the Semirational Engineering of Sucrose Synthase and a Cascade Route Design.

Nucleotide sugars are essential precursors for carbohydrate synthesis but are in scarce supply. Uridine diphosphate (UDP)-glucose is a core building block in nucleotide sugar preparation, making its efficient synthesis critical. Here, a process for producing valuable UDP-glucose and functional mannose from sucrose was established and improved via a semirational sucrose synthase (SuSy) design and the accurate D-mannose isomerase (MIase) cascade. Engineered SuSy exhibited enzyme activity 2.2-fold greater than that of the WT. The structural analysis identified a latch-hinge combination as the hotspot for enhancing enzyme activity. Coupling MIase, process optimization, and reaction kinetic analysis revealed that MIase addition during the high-speed UDP-glucose synthesis phase distinctly accelerated the entire process. The simultaneous triggering of enzyme modules halved the reaction time and significantly increased the UDP-glucose yield. A maximum UDP-glucose yield of 83%, space-time yield of 70 g/L/h, and mannose yield of 32% were achieved. This novel and efficient strategy for sucrose value-added exploitation has industrial promise.

Engineering Saccharomyces cerevisiae YPH499 for Overproduction of Geranylgeraniol.

Optimization of supply and conversion efficiency of geranylgeranyl diphosphate (GGPP) is important for enhancing geranylgeraniol (GGOH) production in Saccharomyces cerevisiae. In this study, first, a strain producing 26.92 ± 1.59 mg/g of dry cell weight squalene was constructed with overexpression of all genes of the mevalonate (MVA) pathway, and an engineered strain producing 597.12 mg/L GGOH at the shake flask level was obtained. Second, through additional expression of PaGGPPs-ERG20 and PaGGPPs-DPP1, and downregulating expression of ERG9, the GGOH titer was increased to 1221.96 mg/L. Then, a NADH HMG-CoA reductase from Silicibacter pomeroyi (SpHMGR) was introduced to alleviate the high dependence of the strain upon NADPH, and the GGOH production was further increased to 1271.14 mg/L. Finally, the GGOH titer reached 6.33 g/L after optimizing the fed-batch fermentation method in a 5 L bioreactor, with a 24.9% improvement from the previous report. This study might accelerate the process of developing S. cerevisiae cell factories for diterpenoid and tetraterpenoid production.

Improved laccase production by Trametes versicolor using Copper-Glycyl-L-Histidyl-L-Lysine as a novel and high-efficient inducer.

A highly efficient strategy using Copper-Glycyl-L-Histidyl-L-Lysine (GHK-Cu) as a novel inducer was developed to enhance laccase production by Trametes versicolor. After medium optimization, laccase activity increased by 12.77-fold compared to that without GHK-Cu. The laccase production of 1113.8 U L-1 was obtained by scaling-up culture in 5-L stirring tank. The laccase production induced by CuSO4 was poorer than that of GHK-Cu at the same mole concentration. GHK-Cu could increase the permeability of cell membrane with less damage, and it facilitated the adsorption, accumulation, and utilization of copper by fungal cells, which was beneficial for laccase synthesis. GHK-Cu induced better expression of laccase related genes than that of CuSO4, resulting in higher laccase production. This study provided a useful method for induced production of laccase by applying GHK chelated metal ion as a non-toxic inducer, which reduced the safety risk of laccase broth and provided the potential application of crude laccase in food industry. In addition, GHK can be used as the carrier of different metal ions to enhance the production of other metalloenzymes.

Biotechnological and food synthetic biology potential of platform strain: Bacillus licheniformis.

Bacillus licheniformis is one of the most characteristic Gram-positive bacteria. Its unique genetic background and safety characteristics make it have important biologic applications in the food industry, including, the biosynthesis of high value-added bioproducts, probiotic functions, biological treatment of wastes derived from food production, etc. In this review, these recent advances are summarized and presented systematically for the first time. In addition, we highlight synthetic biology strategies as a potential driver of developing this strain for wider and more efficient application in the food industry. Finally, we present the current challenges faced and provide our unique perspective on relevant future research directions. In summary, this review will provide an illuminating and comprehensive perspective that will allow an in-depth understanding of B. licheniformis and promote its more effective development in the food industry.

Novel Insights into the Mechanism Underlying High Polysaccharide Yield in Submerged Culture of Ganoderma lucidum Revealed by Transcriptome and Proteome Analyses.

Polysaccharides are crucial dietary supplements and traditional pharmacological components of Ganoderma lucidum; however, the mechanisms responsible for high polysaccharide yields in G. lucidum remain unclear. Therefore, we investigated the mechanisms underlying the high yield of polysaccharides in submerged cultures of G. lucidum using transcriptomic and proteomic analyses. Several glycoside hydrolase (GH) genes and proteins, which are associated with the degradation of fungal cell walls, were significantly upregulated under high polysaccharide yield conditions. They mainly belonged to the GH3, GH5, GH16, GH17, GH18, GH55, GH79, GH128, GH152, and GH154 families. Additionally, the results suggested that the cell wall polysaccharide could be degraded by GHs, which is beneficial for extracting more intracellular polysaccharides from cultured mycelia. Furthermore, some of the degraded polysaccharides were released into the culture broth, which is beneficial for obtaining more extracellular polysaccharides. Our findings provide new insights into the mechanisms underlying the roles that GH family genes play to regulate high polysaccharide yields in G. lucidum.

Engineering the Thermostability of Sucrose Synthase by Reshaping the Subunit Interaction Contributes to Efficient UDP-Glucose Production.

The restricted availability of UDP-glucose, an essential precursor that targets oligo/polysaccharide and glycoside synthesis, makes its practical application difficult. Sucrose synthase (Susy), which catalyzes one-step UDP-glucose synthesis, is a promising candidate. However, due to poor thermostability of Susy, mesophilic conditions are required for synthesis, which slow down the process, limit productivity, and prevent scaled and efficient UDP-glucose preparation. Here, we obtained an engineered thermostable Susy (mutant M4) from Nitrosospira multiformis through automated prediction and greedy accumulation of beneficial mutations. The mutant improved the T1/2 value at 55 °C by 27-fold, resulting in UDP-glucose synthesis at 37 g/L/h of space-time yield that met industrial biotransformation standards. Furthermore, global interaction between mutant M4 subunits was reconstructed by newly formed interfaces according to molecular dynamics simulations, with residue Trp162 playing an important role in strengthening the interface interaction. This work enabled effective, time-saving UDP-glucose production and paved the way for rational thermostability engineering of oligomeric enzymes.

Selective Immobilization of His-Tagged Enzyme on Ni-Chelated Ion Exchange Resin and Its Application in Protein Purification.

Ion exchange resins are suitable as carriers for immobilized enzymes because of their stable physicochemical properties, appropriate particle size and pore structure, and lower loss in continuous operation. In this paper, we report the application of the Ni-chelated ion exchange resin in the immobilization of His-tagged enzyme and protein purification. Acrylic weak acid cation exchange resin (D113H) was selected from four cationic macroporous resins that could chelate the transition metal ion Ni. The maximum adsorption capacity of Ni was ~198 mg/g. Phosphomannose isomerase (PMI) can be successfully immobilized on Ni-chelated D113H from crude enzyme solution through chelation of transition metal ions with the His-tag on the enzyme. The maximum amount of immobilized PMI on the resin was ~143 mg/g. Notably, the immobilized enzyme showed excellent reusability and maintained 92% of its initial activity with 10 cycles of catalytic reaction. In addition, PMI was successfully purified using an affinity chromatography column prepared by Ni-chelated D113H, which showed the potential for the immobilization and purification process to be realized in one step.

Insight into the Cold Adaptation Mechanism of an Aerobic Denitrifying Bacterium: Bacillus simplex H-b.

Psychrophilic bacteria with aerobic denitrification ability have promising potential for application in nitrogen-contaminated wastewater treatment, especially under cold conditions. A better understanding of the cold adaptation mechanism during aerobic denitrification would be beneficial for the practical application of this type of functional bacterium. In this study, Bacillus simplex H-b with good denitrification performance at 5°C was used to investigate the corresponding cold tolerance mechanism. Transcriptomics and nitrogen removal characterization experiments were conducted at different temperatures (5°C, 20°C, and 30°C). At low temperatures, more nitrogen was utilized for assimilation, accompanied by the accumulation of ATP and extracellular polymeric substances (EPS), rather than transforming inorganic nitrogen in the dissimilation pathway. In addition, the proportion of unsaturated fatty acids was higher in strains cultured at low temperatures. At the molecular level, the adjustment of membrane transport, synthesis of cofactors and vitamins, and transcriptional regulators might contribute to the survival of the strain under cold conditions. Moreover, nucleotide precursor synthesis, translation, and oxidative and temperature stress response mechanisms also enhanced the resistance of strain H-b to low temperatures. The results suggest that combining multiple regulatory mechanisms and synergistic adaptation to cold stress enabled the growth and relatively high nitrogen removal rate (27.22%) of strain H-b at 5°C. By clarifying the mechanism of regulation and cold resistance of strain H-b, a theoretical foundation for enhancing the application potential of this functional bacterium for nitrogen-contaminated wastewater treatment was provided. IMPORTANCE The newly isolated aerobic denitrifying bacterium Bacillus simplex H-b removed various forms of inorganic nitrogen (nitrate, nitrite, and ammonium) from wastewater, even when the temperature was as low as 5°C. Although this environmentally functional bacterium has been suggested as a promising candidate for nitrogen-contaminated water treatment at low temperatures, understanding its cold adaptation mechanism during aerobic denitrification is limited. In this study, the cold tolerance mechanism of this strain was comprehensively explained. Furthermore, a theoretical basis for the practical application of this type of functional bacterium for nitrogen removal in cold regions is provided. The study expands our understanding of the survival strategy of psychrophilic bacteria and hence supports their further utilization in wastewater treatment applications.

Efficient extraction of phycobiliproteins from dry biomass of Spirulina platensis using sodium chloride as extraction enhancer.

An efficient strategy for phycobiliprotein extraction from Spirulina platensis dry biomass has been developed by using NaCl as an enhancer. Different sodium ion and chloride ion salts were screened, and NaCl was selected as the most appropriate solvent for phycobiliprotein extraction. The extraction parameters with NaCl were optimized using response surface methodology. Under optimal operating conditions, a phycobiliprotein extraction rate of 74.8 % and a phycocyanin extraction yield of 102.4 mg/g with a purity of 74.0 % were achieved. Adding NaCl resulted in smaller fragments and destroyed the cell integrity of S. platensis, facilitating phycobiliprotein exudation. The secondary structure and antioxidant activity of phycobiliproteins were not affected by NaCl extraction. The stability of the phycobiliproteins was improved by adding NaCl. This study provides a potential method for phycobiliprotein extraction with high efficiency and good quality using an inexpensive extraction enhancer.

Pivotal biological processes and proteins for selenite reduction and methylation in Ganoderma lucidum.

Microbial transformations, especially the reduction and methylation of Se oxyanion, have gained significance in recent years as effective detoxification methods. Ganoderma lucidum is a typical Se enrichment resource that can reduce selenite to elemental Se and volatile Se metabolites under high selenite conditions. However, the detailed biological processes and reduction mechanisms are unclear. In this study, G. lucidum reduced selenite to elemental Se and further aggregated it into Se nanoparticles with a diameter of < 200 nm, simultaneously accompanied by the production of pungent, odorous, and volatile methyl-selenium metabolites. Tandem mass tag-based quantitative proteomic analysis revealed thioredoxin 1, thioredoxin reductase (NADPH), glutathione reductase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and cystathionine gamma-lyase as proteins involved in selenite reduction and methylation. Furthermore, the high expression of proteins associated with cell structures that prompted cell lysis may have facilitated Se release. The upregulation of proteins involved in the defense reactions was also detected, reflecting their roles in the self-defense mechanism. This study provides novel insights into the vital role of G. lucidum in mediating Se transformation in the biogeochemical Se cycle and contributes to the application of fungi in Se bioremediation.

Understanding and application of Bacillus nitrogen regulation: A synthetic biology perspective.

BACKGROUND: Nitrogen sources play an essential role in maintaining the physiological and biochemical activity of bacteria. Nitrogen metabolism, which is the core of microorganism metabolism, makes bacteria able to autonomously respond to different external nitrogen environments by exercising complex internal regulatory networks to help them stay in an ideal state. Although various studies have been put forth to better understand this regulation in Bacillus, and many valuable viewpoints have been obtained, these views need to be presented systematically and their possible applications need to be specified. AIM OF REVIEW: The intention is to provide a deep and comprehensive understanding of nitrogen metabolism in Bacillus, an important industrial microorganism, and thereby apply this regulatory logic to synthetic biology to improve biosynthesis competitiveness. In addition, the potential researches in the future are also discussed. KEY SCIENTIFIC CONCEPT OF REVIEW: Understanding the meticulous regulation process of nitrogen metabolism in Bacillus not only could facilitate research on metabolic engineering but also could provide constructive insights and inspiration for studies of other microorganisms.

Construction of the genetic switches in response to mannitol based on artificial MtlR box.

Synthetic biology has rapidly advanced from the setup of native genetic devices to the design of artificial elements able to provide organisms with highly controllable functions. In particular, genetic switches are crucial for deploying new layers of regulation into the engineered organisms. While the assembly and mutagenesis of native elements have been extensively studied, limited progress has been made in rational design of genetic switches due to a lack of understanding of the molecular mechanism by which a specific transcription factor interacts with its target gene. Here, a reliable workflow is presented for designing two categories of genetic elements, one is the switch element-MtlR box and the other is the transcriptional regulatory element- catabolite control protein A (CcpA) box. The MtlR box was designed for ON/OFF-state selection and is controlled by mannitol. The rational design of MtlR box-based molecular structures can flexibly tuned the selection of both ON and OFF states with different output switchability in response to varied kind effectors. Different types of CcpA boxes made the switches with more markedly inducer sensitivities. Ultimately, the OFF-state value was reduced by 90.69%, and the maximum change range in the presence of two boxes was 15.31-fold. This study presents a specific design of the switch, in a plug-and-play manner, which has great potential for controlling the flow of the metabolic pathway in synthetic biology.