Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Temporal drivers of tryptophan-like fluorescent dissolved organic matter along a river continuum.

Tryptophan-like fluorescence (TLF) is used to indicate anthropogenic inputs of dissolved organic matter (DOM), typically from wastewater, in rivers. We hypothesised that other sources of DOM, such as groundwater and planktonic microbial biomass can also be important drivers of riverine TLF dynamics. We sampled 19 contrasting sites of the River Thames, UK, and its tributaries. Multivariate mixed linear models were developed for each site using 15 months of weekly water quality observations and with predictor variables selected according to the statistical significance of their linear relationship with TLF following a stepwise procedure. The variables considered for inclusion in the models were potassium (wastewater indicator), nitrate (groundwater indicator), chlorophyll-a (phytoplankton biomass), and Total bacterial Cells Counts (TCC) by flow cytometry. The wastewater indicator was included in the model of TLF at 89 % of sites. Groundwater was included in 53 % of models, particularly those with higher baseflow indices (0.50-0.86). At these sites, groundwater acted as a negative control on TLF, diluting other potential sources. Additionally, TCC was included positively in the models of six (32 %) sites. The models on the Thames itself using TCC were more rural sites with lower sewage inputs. Phytoplankton biomass (Chlorophyll-a) was only used in two (11 %) site models, despite the seasonal phytoplankton blooms. It is also notable that, the wastewater indicator did not always have the strongest evidence for inclusion in the models. For example, there was stronger evidence for the inclusion of groundwater and TCC than wastewater in 32 % and 5 % of catchments, respectively. Our study underscores the complex interplay of wastewater, groundwater, and planktonic microbes, driving riverine TLF dynamics, with their influence determined by site characteristics.

Biosynthesis of fragrance 2-phenylethanol from sugars by Pseudomonas putida.

BACKGROUND: Petrochemicals contribute to environmental issues, with concerns ranging from energy consumption and carbon emission to pollution. In contrast, microbial biorefineries offer eco-friendly alternatives. The solvent-tolerant Pseudomonas putida DOT-T1E serves as a suitable host for producing aromatic compounds, specifically L-phenylalanine and its derivative, 2-phenylethanol (2-PE), which find widespread applications in various industries. RESULTS: This study focuses on enhancing 2-PE production in two L-phenylalanine overproducing strains of DOT-T1E, namely CM12-5 and CM12-5Δgcd (xylABE), which grow with glucose and glucose-xylose, respectively. To synthesize 2-PE from L-phenylalanine, these strains were transformed with plasmid pPE-1, bearing the Ehrlich pathway genes, and it was found higher 2-PE production with glucose (about 50-60 ppm) than with xylose (< 3 ppm). To understand the limiting factors, we tested the addition of phenylalanine and intermediates from the Ehrlich and shikimate pathways. The results identified intracellular L-phenylalanine as a key limiting factor for 2-PE production. To overcame this limitation, a chorismate mutase/prephenate dehydratase variant-insentive to feedback inhibition by aromatic amino acids-was introduced in the producing strains. This led to increased L-phenylalanine production and subsequently produced more 2-PE (100 ppm). Random mutagenesis of the strains also produced strains with higher L-phenylalanine titers and increased 2-PE production (up to 120 ppm). The improvements resulted from preventing dead-end product accumulation from shikimate and limiting the catabolism of potential pathway intermediates in the Ehrlich pathway. The study explored agricultural waste substrates, such as corn stover, sugarcane straw and corn-syrup as potential C sources. The best results were obtained using 2G substrates at 3% (between 82 and 100 ppm 2-PE), with glucose being the preferred sugar for 2-PE production among the monomeric sugars in these substrates. CONCLUSIONS: The findings of this study offer strategies to enhance phenylalanine production, a key substrate for the synthesis of aromatic compounds. The ability of P. putida DOT-T1E to thrive with various C-sources and its tolerance to substrates, products, and potential toxicants in industrial wastes, are highlighted. The study identified and overcome possible bottlenecks for 2-PE production. Ultimately, the strains have potential to become efficient microbial platforms for synthesizing 2-PE from agro-industrial waste materials.

Microbial production of sulfur-containing amino acids using metabolically engineered Escherichia coli.

L-Cysteine and L-methionine, as the only two sulfur-containing amino acids among the canonical 20 amino acids, possess distinct characteristics and find wide-ranging industrial applications. The use of different organisms for fermentative production of L-cysteine and L-methionine is gaining increasing attention, with Escherichia coli being extensively studied as the preferred strain. This preference is due to its ability to grow rapidly in cost-effective media, its robustness for industrial processes, the well-characterized metabolism, and the availability of molecular tools for genetic engineering. This review focuses on the genetic and molecular mechanisms involved in the production of these sulfur-containing amino acids in E. coli. Additionally, we systematically summarize the metabolic engineering strategies employed to enhance their production, including the identification of new targets, modulation of metabolic fluxes, modification of transport systems, dynamic regulation strategies, and optimization of fermentation conditions. The strategies and design principles discussed in this review hold the potential to facilitate the development of strain and process engineering for direct fermentation of sulfur-containing amino acids.

Recent advances in the biosynthesis and industrial biotechnology of Gamma-amino butyric acid.

GABA (Gamma-aminobutyric acid), a crucial neurotransmitter in the central nervous system, has gained significant attention in recent years due to its extensive benefits for human health. The review focused on recent advances in the biosynthesis and production of GABA. To begin with, the investigation evaluates GABA-producing strains and metabolic pathways, focusing on microbial sources such as Lactic Acid Bacteria, Escherichia coli, and Corynebacterium glutamicum. The metabolic pathways of GABA are elaborated upon, including the GABA shunt and critical enzymes involved in its synthesis. Next, strategies to enhance microbial GABA production are discussed, including optimization of fermentation factors, different fermentation methods such as co-culture strategy and two-step fermentation, and modification of the GABA metabolic pathway. The review also explores methods for determining glutamate (Glu) and GABA levels, emphasizing the importance of accurate quantification. Furthermore, a comprehensive market analysis and prospects are provided, highlighting current trends, potential applications, and challenges in the GABA industry. Overall, this review serves as a valuable resource for researchers and industrialists working on GABA advancements, focusing on its efficient synthesis processes and various applications, and providing novel ideas and approaches to improve GABA yield and quality.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases.

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2'-fucosyllactose (2'-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2'-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2'-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2'-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2'-FL production. Therefore, to decrease the formation of byproduct 2'-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2'-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154-171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154-171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2'-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2'-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2'-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

Production of conjugated linoleic acid by lactic acid bacteria; important factors and optimum conditions.

Conjugated linoleic acid (CLA) has recently attracted significant attention as a health-promoting compound. CLA is a group of positional isomers of linoleic acid (LA) with a conjugated double bond naturally occurring in dairy and ruminant meat products. Microbial biosynthesis of CLA is a practical approach for commercial production due to its high safety and purity. There are some factors for the microbial CLA production such as strain type, microbial growth phase, pH, temperature and incubation time, based on which the amount and type of CLA can be controlled. Understanding the interplay of these factors is essential in optimizing the quantity and composition of microbial CLA, as discussed in the current study. Further exploration of CLA and its influences on human health remains a dynamic and evolving area of study.

p-Nitrobenzoate production from glucose by utilizing p-aminobenzoate N-oxygenase: AurF.

Nitroaromatic compounds are widely used in industry, but their production is associated with issues such as the hazardousness of the process and low regioselectivity. Here, we successfully demonstrated the production of p-nitrobenzoate (PNBA) from glucose by constructing p-aminobenzoate N-oxygenase AurF-expressing E. coli. We generated this strain, which we named PN-1 by disrupting four genes involved in PNBA degradation: nfsA, nfsB, nemA, and azoR. We then expressed AurF from Streptomyces thioluteus in this strain, which resulted in the production of 945 mg/L PNBA in the presence of 1 g/L p-aminobenzoate. Direct production of PNBA from glucose was achieved by co-expressing the pabA, pabB, and pabC, as well as aurF, resulting in the production of 393 mg/L PNBA from 20 g/L glucose. To improve the PNBA titer, we disrupted genes involved in competing pathways: pheA, tyrA, trpE, pykA, and pykF. The resultant strain PN-4Ap produced 975 mg/L PNBA after 72 h of cultivation. These results highlight the potential of using microorganisms to produce other nitroaromatic compounds.

Recent advances in the production of malic acid by native fungi and engineered microbes.

Malic acid is mainly produced by chemical methods which lead to various environmental sustainability concerns associated with CO2 emissions and resulting global warming. Since malic acid is naturally synthesized, microorganisms offer an eco-friendly and cost-effective alternative for its production. An additional advantage of microbial production is the synthesis of pure L-form of malic acid. Due to its numerous applications, biotechnologically- produced L-malic acid is a much sought-after platform chemical. Malic acid can be produced by microbial fermentation via oxidative/reductive TCA and glyoxylate pathways. This article elaborates the potential and limitations of high malic acid producing native fungi belonging to Aspergillus, Penicillium, Ustilago and Aureobasidium spp. The utilization of industrial side streams and low value renewable substrates such as crude glycerol and lignocellulosic biomass is also discussed with a view to develop a competitive bio-based production process. The major impediments present in the form of toxic compounds from lignocellulosic residues or synthesized during fermentation along with their remedial measures are also described. The article also focuses on production of polymalic acid from renewable substrates which opens up a cost-cutting dimension in production of this biodegradable polymer. Finally, the recent strategies being employed for its production in recombinant organisms have also been covered.

Alginate: Microbial production, functionalization, and biomedical applications.

Alginates are natural polysaccharides widely participating in food, pharmaceutical, and environmental applications due to their excellent gelling capacity. Their excellent biocompatibility and biodegradability further extend their application to biomedical fields. The low consistency in molecular weight and composition of algae-based alginates may limit their performance in advanced biomedical applications. It makes microbial alginate production more attractive due to its potential for customizing alginate molecules with stable characteristics. Production costs remain the primary factor limiting the commercialization of microbial alginates. However, carbon-rich wastes from sugar, dairy, and biodiesel industries may serve as potential substitutes for pure sugars for microbial alginate production to reduce substrate costs. Fermentation parameter control and genetic engineering strategies may further improve the production efficiency and customize the molecular composition of microbial alginates. To meet the specific needs of biomedical applications, alginates may need functionalization, such as functional group modifications and crosslinking treatments, to achieve enhanced mechanical properties and biochemical activities. The development of alginate-based composites incorporated with other polysaccharides, gelatin, and bioactive factors can integrate the advantages of each component to meet multiple requirements in wound healing, drug delivery, and tissue engineering applications. This review provided a comprehensive insight into the sustainable production of high-value microbial alginates. It also discussed recent advances in alginate modification strategies and alginate-based composites for representative biomedical applications.

Rational design of GDP­D­mannose mannosyl hydrolase for microbial L­fucose production.

BACKGROUND: L­Fucose is a rare sugar that has beneficial biological activities, and its industrial production is mainly achieved with brown algae through acidic/enzymatic fucoidan hydrolysis and a cumbersome purification process. Fucoidan is synthesized through the condensation of a key substance, guanosine 5'­diphosphate (GDP)­L­fucose. Therefore, a more direct approach for biomanufacturing L­fucose could be the enzymatic degradation of GDP­L­fucose. However, no native enzyme is known to efficiently catalyze this reaction. Therefore, it would be a feasible solution to engineering an enzyme with similar function to hydrolyze GDP­L­fucose. RESULTS: Herein, we constructed a de novo L­fucose synthetic route in Bacillus subtilis by introducing heterologous GDP­L­fucose synthesis pathway and engineering GDP­mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP­L­fucose, therefore, a substrate simulation­based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP­L­fucose­splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield L­fucose. WcaHR36Y/N38R was found to produce 1.6 g/L L­fucose during shake­flask growth, which was 67.3% higher than that achieved by wild­type WcaH. The accumulated L­fucose concentration in a 5 L bioreactor reached 6.4 g/L. CONCLUSIONS: In this study, we established a novel microbial engineering platform for the fermentation production of L­fucose. Additionally, we found an efficient GDP­mannose mannosyl hydrolase mutant for L­fucose biosynthesis that directly hydrolyzes GDP­L­fucose. The engineered strain system established in this study is expected to provide new solutions for L­fucose or its high value­added derivatives production.

A highly efficient transcriptome-based biosynthesis of non-ethanol chemicals in Crabtree negative Saccharomyces cerevisiae.

BACKGROUND: Owing to the Crabtree effect, Saccharomyces cerevisiae produces a large amount of ethanol in the presence of oxygen and excess glucose, leading to a loss of carbon for the biosynthesis of non-ethanol chemicals. In the present study, the potential of a newly constructed Crabtree negative S. cerevisiae, as a chassis cell, was explored for the biosynthesis of various non-ethanol compounds. RESULTS: To understand the metabolic characteristics of Crabtree negative S. cerevisiae sZJD-28, its transcriptional profile was compared with that of Crabtree positive S. cerevisiae CEN.PK113-11C. The reporter GO term analysis showed that, in sZJD-28, genes associated with translational processes were down-regulated, while those related to carbon metabolism were significantly up-regulated. To verify a potential increase in carbon metabolism for the Crabtree negative strain, the production of non-ethanol chemicals, derived from different metabolic nodes, was then undertaken for both sZJD-28 and CEN.PK113-11C. At the pyruvate node, production of 2,3-butanediol and lactate in sZJD-28-based strains was remarkably higher than that of CEN.PK113-11C-based ones, representing 16.8- and 1.65-fold increase in titer, as well as 4.5-fold and 0.65-fold increase in specific titer (mg/L/OD), respectively. Similarly, for shikimate derived p-coumaric acid, the titer of sZJD-28-based strain was 0.68-fold higher than for CEN.PK113-11C-based one, with a 0.98-fold increase in specific titer. While farnesene and lycopene, two acetoacetyl-CoA derivatives, showed 0.21- and 1.88-fold increases in titer, respectively. From malonyl-CoA, the titer of 3-hydroxypropionate and fatty acids in sZJD-28-based strains were 0.19- and 0.76-fold higher than that of CEN.PK113-11C-based ones, respectively. In fact, yields of products also improved by the same fold due to the absence of residual glucose. Fed-batch fermentation further showed that the titer of free fatty acids in sZJD-28-based strain 28-FFA-E reached 6295.6 mg/L with a highest reported specific titer of 247.7 mg/L/OD in S. cerevisiae. CONCLUSIONS: Compared with CEN.PK113-11C, the Crabtree negative sZJD-28 strain displayed a significantly different transcriptional profile and obvious advantages in the biosynthesis of non-ethanol chemicals due to redirected carbon and energy sources towards metabolite biosynthesis. The findings, therefore, suggest that a Crabtree negative S. cerevisiae strain could be a promising chassis cell for the biosynthesis of various chemicals.

Microbial Hyaluronic Acid Production: A Review.

Microbial production of hyaluronic acid (HA) is an area of research that has been gaining attention in recent years due to the increasing demand for this biopolymer for several industrial applications. Hyaluronic acid is a linear, non-sulfated glycosaminoglycan that is widely distributed in nature and is mainly composed of repeating units of N-acetylglucosamine and glucuronic acid. It has a wide and unique range of properties such as viscoelasticity, lubrication, and hydration, which makes it an attractive material for several industrial applications such as cosmetics, pharmaceuticals, and medical devices. This review presents and discusses the available fermentation strategies to produce hyaluronic acid.

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.

Flavonoid Production: Current Trends in Plant Metabolic Engineering and De Novo Microbial Production.

Flavonoids are secondary metabolites that represent a heterogeneous family of plant polyphenolic compounds. Recent research has determined that the health benefits of fruits and vegetables, as well as the therapeutic potential of medicinal plants, are based on the presence of various bioactive natural products, including a high proportion of flavonoids. With current trends in plant metabolite research, flavonoids have become the center of attention due to their significant bioactivity associated with anti-cancer, antioxidant, anti-inflammatory, and anti-microbial activities. However, the use of traditional approaches, widely associated with the production of flavonoids, including plant extraction and chemical synthesis, has not been able to establish a scalable route for large-scale production on an industrial level. The renovation of biosynthetic pathways in plants and industrially significant microbes using advanced genetic engineering tools offers substantial promise for the exploration and scalable production of flavonoids. Recently, the co-culture engineering approach has emerged to prevail over the constraints and limitations of the conventional monoculture approach by harnessing the power of two or more strains of engineered microbes to reconstruct the target biosynthetic pathway. In this review, current perspectives on the biosynthesis and metabolic engineering of flavonoids in plants have been summarized. Special emphasis is placed on the most recent developments in the microbial production of major classes of flavonoids. Finally, we describe the recent achievements in genetic engineering for the combinatorial biosynthesis of flavonoids by reconstructing synthesis pathways in microorganisms via a co-culture strategy to obtain high amounts of specific bioactive compounds.

High-throughput genetic engineering tools for regulating gene expression in a microbial cell factory.

With the rapid advances in biotechnological tools and strategies, microbial cell factory-constructing strategies have been established for the production of value-added compounds. However, optimizing the tradeoff between the biomass, yield, and titer remains a challenge in microbial production. Gene regulation is necessary to optimize and control metabolic fluxes in microorganisms for high-production performance. Various high-throughput genetic engineering tools have been developed for achieving rational gene regulation and genetic perturbation, diversifying the cellular phenotype and enhancing bioproduction performance. In this paper, we review the current high-throughput genetic engineering tools for gene regulation. In particular, technological approaches used in a diverse range of genetic tools for constructing microbial cell factories are introduced, and representative applications of these tools are presented. Finally, the prospects for high-throughput genetic engineering tools for gene regulation are discussed.

Production of the antifungal biopesticide physcion through the combination of microbial fermentation and chemical post-treatment.

Physcion is an anthraquinone compound observed dominantly in medicinal herbs. This anthraquinone possesses a variety of pharmaceutically important activities and has been developed to be a widely used antifungal biopesticide. Herein, we report on the effective preparation of 3R-torosachrysone (4), a tetrahydroanthracene precursor of physcion, in Aspergillus oryzae NSAR1 by heterologous expression of related genes mined from the phlegmacins-producing ascomycete Talaromyces sp. F08Z-0631. Conditions for converting 4 into physcion were studied and optimized, leading to the development of a concise approach for extracting high-purity physcion from the alkali-treated fermentation broth of the 4-producing A. oryzae strain.

Development of Streptococcus equisimilis Group G Mutant Strains with Ability to Produce Low Polydisperse and Low-Molecular-Weight Hyaluronic Acid

Background: Background: Hyaluronic acid (HA), a natural polymer with wide applications in biomedicine and cosmetics, is mainly produced by Streptococcal fermentation at industrial scale. In the present study, chemical random mutagenesis was used for development of Streptococcus equisimilis group G mutant strains with high HA productivity. Methods: Methods: The optimum of the pH of culture condition and cultivation time for HA production by wild strain group G were assessed. At first, two rounds of mutation at different concentrations of NTG was used for mutagenesis. Then, the nonhemolytic and hyaluronidase-negative mutants were screened on the blood and HA agar. HA productivity and molecular weight were determined by carbazole assay, agarose gel electrophoresis and specific staining. Moreover, stability of the high producer mutants was evaluated within 10 generations. Results: Results: The results showed that the wild-type strain produced 1241 ± 2.1 µg/ml of HA at pH 5.5 and 4 hours of cultivation, while the screened mutants showed a 16.1-45.5% increase in HA production. Two mutant strains, named Gm2-120-21-3 (2470 ± 8.1 µg/ml) and Gm2-120-21-4 (2856 ± 4.2 µg/ml), indicated the highest titer and a consistent production. The molecular weight (Mw) of HA for the mutants was less than 160 kDa, considering as a low Mw HA. Conclusion: Conclusion: The mutant strains producing a low polydisperse, as well as low Mw of HA with high titer might be regarded as potential industrial strains for HA production after further safety investigations.

Application of quorum sensing system in microbial synthesis of valuable chemicals: a mini-review.

With advantages of low substrates cost, high optical purity of end products and environmentally friendly fermentation process, microbial production of valuable chemicals grow rapidly. Compared with static microbial strain engineering strategies, such as gene deletion, overexpression and mutation, dynamic pathway regulation is a new approach that balances cellular growth and chemical production. Quorum sensing is a natural microbial communication system responsible for cell-density-related cell behaviors. Accordingly, quorum sensing systems can be employed to achieve dynamic regulation in microorganisms without the need for manual intervention or the use of chemical inducers. In this review, natural quorum sensing systems are firstly summarized. Then, recent progress in using quorum sensing circuits in the field of metabolic engineering is highlighted. The current application challenges of quorum sensing systems and future perspectives in microbial synthesis of chemicals are also discussed.