Advances in ε-Poly-Lysine Biosynthesis, Selection of High-Yielding Strains and Regulatory Mechanisms.

British chef and food activist Jamie Oliver ignited a firestorm in January 2011 when he mentioned on the Late Show with David Letterman that castoreum, a substance used to augment some strawberry and vanilla flavorings, comes from what he described as “rendered beaver anal gland.”1 The next year, vegans were outraged to learn that Starbucks used cochineal extract, a color additive derived from insect shells, to dye their strawberry Frappuccino® drinks2 (eventually, the company decided to transition to lycopene, a pigment found in tomatoes3). Although substances like castoreum and cochineal extract may be long on the “yuck factor,”4 research has shown them to be perfectly safe for most people; strident opposition arose not from safety issues but from the ingredients’ origins. But these examples demonstrate that the public often lacks significant knowledge about the ingredients in foods and where they come from. This is not a new development; the public relationship to food additives has a long history of trust lost, regained, and in some cases lost again. The Federal Food, Drug, and Cosmetic (FD&C) Act of 19385 was passed shortly after the deaths of 100 people who took an untested new form of a popular drug, which contained what turned out to be a deadly additive.6 The new law was consumer oriented and intended to ensure that people knew what was in the products they bought, and that those products were safe. The law has been amended over the years in attempts to streamline and bring order to the sprawling task of assessing and categorizing the thousands of substances used in foods, drugs, and cosmetics. One result of this streamlining is that under current U.S. law, companies can add certain types of ingredients to foods without premarket approval from the thin-stretched Food and Drug Administration (FDA). In other words, there are substances in the food supply that are unknown to the FDA. In 2010 the Government Accountability Office (GAO) concluded that a “growing number of substances … may effectively be excluded from federal oversight.”7 Is this a problem? The answer depends on whom you ask.

PikD is a widely known pathway-specific regulator for controlling pikromycin production in Streptomyces venezuelae ATCC 15439, which is a representative of the large ATP-binding regulator of the LuxR family (LAL) in Streptomyces sp. RapH and FkbN also belong to the LAL family of transcriptional regulators, which show greatest homology with the ATP-binding motif and helix-turn-helix DNA-binding motif of PikD. Overexpression of pikD and heterologous expression of rapH and fkbN led to enhanced production of pikromycin by approximately 1.8-, 1.6-, and 1.6-fold in S. venezuelae, respectively. Cross-complementation of rapH and fkbN in the pikD deletion mutant (ΔpikD) restored pikromycin and derived macrolactone production. Overall, these results show that heterologous expression of rapH and fkbN leads to the overproduction of pikromycin and its congeners from the pikromycin biosynthetic pathway in S. venezuelae, and they have the same functionality as the pathwayspecific transcriptional activator for the pikromycin biosynthetic pathway in the ΔpikD strain. These results also show extensive "cross-communication" between pathway-specific regulators of streptomycetes and suggest revision of the current paradigm for pathwayspecific versus global regulation of secondary metabolism in Streptomyces species.

Post Brexit trade with the US

Streptomyces is taken as an important resource for producing the most abundant antibiotics and other bio-active natural products, which have been widely used in pharmaceutical and agricultural areas. Usually they are biosynthesized through secondary metabolic pathways encoded by cluster situated genes. And these gene clusters are stringently regulated by interweaved transcriptional regulatory cascades. In the past decades, great advances have been made to elucidate the regulatory mechanisms involved in antibiotic production in Streptomyces. In this review, we summarized the recent advances on the regulatory cascades of antibiotic production in Streptomyces from the following four levels: the signals triggering the biosynthesis, the global regulators, the pathway-specific regulators and the feedback regulation. The production of antibiotic can be largely enhanced by rewiring the regulatory networks, such as overexpression of positive regulators, inactivation of repressors, fine-tuning of the feedback and ribosomal engineering in Streptomyces. The enormous amount of genomic sequencing data implies that the Streptomyces has potential to produce much more antibiotics for the great diversities and wide distributions of biosynthetic gene clusters in Streptomyces genomes. Most of these gene clusters are defined cryptic for unknown or undetectable natural products. In the synthetic biology era, activation of the cryptic gene clusters has been successfully achieved by manipulation of the regulatory genes. Chemical elicitors, rewiring regulatory gene and ribosomal engineering have been employed to crack the potential of cryptic gene clusters. These have been proposed as the most promising strategy to discover new antibiotics. For the complex of regulatory network in Streptomyces, we proposed that the discovery of new antibiotics and the optimization of industrial strains would be greatly promoted by further understanding the regulatory mechanism of antibiotic production.

Background: To satisfy the need for thorough yet flexible food safety guidelines, the GRAS (Generally Recognized as Safe) certification process was introduced in 1958, establishing a process for food products and additives to be considered safe for consumption. It involves a multitude of steps from multiple parties: the author advocating for their new food product, a group of panelists who will evaluate the safety of the product, and the FDA (Food and Drug Administration), who has the final say in the GRAS status of the product. The first step involves the author submitting documents with their basic information and product details, including lab trials and experimentation. Next, the panelists follow thorough guidelines to carefully evaluate the product and its safety in the context of its intended usage. The final step involves the FDA reviewing evidence from both previous groups and reaches a singular conclusion that will determine the GRAS status of the product at the end. Overall, the GRAS process established a concrete set of guidelines to determine the safety of food products. Different conclusions and safety levels were produced to set a general standard for companies and other organizations to abide by. These different groups and multiple safety procedures are all required to maintain the highly renowned status of GRAS certification and overall maintain a standardized level of quality for all food products that are exported to the United States. This article will also evaluate the candidacy of functional food products, comparing both processes with regards to sending a functional food product to market and the GRAS notification process overall. Functional foods proved to be an excellent candidate for the GRAS certification process, passing every component of the requirements and serving as a great example of exactly what authors should be expecting when putting their food products through the GRAS certification procedure. Keywords: GRAS, GRAS notification, functional food product, FDA

SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona.

Drug induced nephrotoxicity is the current concern of research due to its awful worldwide occurrences. Generally recognized as safe (GRAS) grade food preservatives e.g. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), L-ascorbic acid (Vit.C) and gamma-tocopherol (Vit. E) exhibits potent antioxidant, anti-inflammatory properties against severe oxidative stress. The aim of this study was to evaluate the efficacy of food preservatives on carbon tetrachloride (CCl4)-induced (230 mg/ kg b wt/ rat/day) nephritic damage in rats. Nephritic markers like serum urea, blood urea nitrogen, serum creatinine; antioxidant markers such as GSH, SOD, CAT, GPx, and lipid peroxidation end product, MDA were measured to establish anti-oxidant properties of said food preservatives and vitamins. The results had shown an elevated level of serum urea (387.30%), blood urea nitrogen (376%), serum creatinine (646.82%) and marked decreased activity of antioxidant markers like SOD (81.03%), CAT (72.24%), GSH (63.04%), GPx (50.34%) as well. CCl4 induced nephrotoxicity also caused 48.14% and 59.47% increase in sodium and potassium concentration. Histological studies also confirmed that antioxidant status in renal cells was restored as BHA, BHT, L-ascorbic acid, and gamma-tocopherol successfully ameliorated certain degenerative changes caused due to CCl4 intoxication. Therefore, it can be concluded that supplementation of certain food preservatives like BHA, BHT and like Vitamins L-ascorbic acid, gamma-tocopherol may be potentially beneficial to the community affected by severe renal dysfunction. Keywords: butylated hydroxyanisole, butylated hydroxytoluene, L-ascorbic acid, gamma-tocopherol, CCl4 intoxication.

Making life look and taste better

Ascomycin (FK520) is a 23-membered macrolide antibiotic primarily produced by the Streptomyces hygroscopicus subsp. ascomycetoides. Structurally similar to tacrolimus and rapamycin, it serves as an effective immunosuppressant widely used in the treatment of rejection reactions following organ transplantation and certain autoimmune diseases. Currently, FK520 is mainly produced through microbial fermentation, but its yield remains low. Since the gene fkbR2 is a regulatory gene within the FK520 biosynthetic gene cluster that has not been studied, this paper focuses on the overexpression of the regulatory gene fkbR2 in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (WT). By constructing a strain with overexpressed fkbR2 gene, we initially obtained a high-yielding strain R2-17 through shake flask fermentation, with a 28% increase in yield compared to WT. In the process of further improving the stability of the high-yielding strain, this paper defines two indices: high-yield index and stability index. After two consecutive rounds of natural selection, strain R2-17 achieved a high-yield index of 100% and a stability index of 80%, ultimately leading to the selection of a stable high-yielding strain R2-17-3-10 with a 34% increase in yield compared to WT, reaching 686.47 mg/L. A comparative analysis between the high-yielding strain R2-17-3-10 and the original strain WT revealed differences in fermentation process parameters such as FK520 synthesis rate, pH, bacterial concentration, glycerol consumption rate, amino nitrogen level, and ammonium ion concentration. Additionally, the transcription levels of genes involved in the synthesis of precursors DHCHC (fkbO), ethylmalonyl-CoA (fkbE, fkbU, fkbS), and piperidine acid (fkbL), as well as pathway-specific regulatory genes (fkbN, fkbR1), were significantly increased at different time points in the high-yielding strain R2-17-3-10. This suggests that the gene fkbR2may enhance the supply of FK520 synthetic precursors by regulating the transcription of these genes, thereby promoting an increase in FK520 production. These results demonstrate that modifying genes within the biosynthetic gene clusters of natural products can be successfully applied to increase the yields of industrially and clinically important compounds. However it is found that fkbR2 gene may be a regulatory gene that has not been fully studied, and it is worth further studying its regulatory mechanism.

Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Stilbenes are natural phenolic compounds which function as antimicrobial phytoalexins in plants and affect human health as cardioprotective, antibaceteria, antioxidative and antineoplastic agents . In this review, the progresses of study on relevant enzymes, genes and regulation mechanism in biosynthesis pathway of stilbenes are described. Here we introduce a holistic and systematic method of researching relevant enzymes, genes and other regulatory factors in biosynthesis pathway of stilbenes - Systems biology. The application of knowledge of relative enzymes, genes and regulation mechanisms in stilbenes biosynthesis in metabolic engineering which is used as a tool of improving the disease resistance of plants and health caring quality of crops is also discussed.

Stilbenes are natural phenolic compounds which function as antimicrobial phytoalexins in plants and affect human health as cardioprotective, antibaceteria, antioxidative and antineoplastic agents. In this review, the progresses of study on relevant enzymes, genes, and regulation mechanism in biosynthesis pathway of stilbenes are described. Here we introduce a holistic and systematic method of researching relevant enzymes, genes and other regulatory factors in biosynthesis pathway of stilbenes—Systems biology. The application of knowledge of relative enzymes, genes and regulation mechanisms in stilbenes biosynthesis in metabolic engineering which is used as a tool of improving the disease resistance of plants and health caring quality of crops is also discussed.

BackgroundNorvancomycin has been widely used in clinic to treat against MRSA (Methicillin-resistant Staphylococcus aureus) and MRSE (Methicillin-resistant Staphylococcus epidermidis) infections in China. Amycolatopsis orientalis NCPC 2-48, a high yield strain derived from A. orientalis CPCC 200066, has been applied in industrial large-scale production of norvancomycin by North China Pharmaceutical Group. However, the potential high-yield and regulatory mechanism involved in norvancomycin biosynthetic pathway has not yet been addressed.ResultsHere we sequenced and compared the genomes and transcriptomes of A. orientalis CPCC 200066 and NCPC 2-48. These two genomes are extremely similar with an identity of more than 99.9%, and no duplication and structural variation was found in the norvancomycin biosynthetic gene cluster. Comparative transcriptomic analysis indicated that biosynthetic genes of norvancomycin, as well as some primary metabolite pathways for the biosynthetic precursors of norvancomycin were generally upregulated. AoStrR1 and AoLuxR1, two cluster-situated regulatory genes in norvancomycin cluster, were 23.3-fold and 5.8-fold upregulated in the high yield strain at 48 h, respectively. Over-expression of AoStrR1 and AoLuxR1 in CPCC 200066 resulted in an increase of norvancomycin production, indicating their positive roles in norvancomycin biosynthesis. Furthermore, AoStrR1 can regulate the production of norvancomycin by directly interacting with at least 8 promoters of norvancomycin biosynthetic genes or operons.ConclusionOur results suggested that the high yield of NCPC 2-48 can be ascribed to increased expression level of norvancomycin biosynthetic genes in its cluster as well as the genes responsible for the supply of its precursors. The norvancomycin biosynthetic genes are presumably regulated by AoStrR1 and AoLuxR1, of them AoStrR1 is possibly the ultimate pathway-specific regulator for the norvancomycin production. These results are helpful for further clarification of the holistic and pathway-specific regulatory mechanism of norvancomycin biosynthesis in the industrial production strain.

Two-component system AfrQ1Q2 involved in oxytetracycline biosynthesis of Streptomyces rimosus M4018 in a medium-dependent manner

Many early studies on penicillin and cephamycin biosynthesis were carried out in Streptomyces clavuligerus, but because of the commercial importance of clavulanic acid, most recent studies have focused on that compound. In particular, genetic engineering to generate improved clavulanic acid producer strains has been a main interest. S. clavuligerus grows and produces secondary metabolites optimally at 28 to 30°C, but it is unusually sensitive to elevated temperature and will not grow above 31 to 32°C. Liquid media employed by research groups studying the production of the various types of β-lactam metabolites produced by S. clavuligerus vary widely. Numerous bioprocess-type studies examining growth medium optimization as a means of improving production of clavulanic acid have appeared in recent years, typically reinforcing the association of soy-based growth medium constituents with high productivity. In S. clavuligerus, biosynthesis begins with the amino acids L-lysine, L-cysteine, and L-valine and proceeds through the synthesis of penicillin and cephalosporin intermediates to give the final cephamycin product. While improvements in productivity can be achieved by increasing production of pathway enzymes, another approach to achieving this is to identify genes encoding critical regulatory proteins and increase their expression levels. More global approaches to improvement of antibiotic production could also be coupled with strategies aimed at the pathway-specific regulators. Effects of greater magnitude are seen when pathway-specific regulators are targeted for overproduction, and combination approaches targeting both global and specific regulators along with individual pathway enzymes for overproduction, together with elimination of competing pathways, will likely yield even greater results.