Multiple copies of the oxytetracycline gene cluster in selected Streptomyces rimosus strains can provide significantly increased titers.

BACKGROUND: Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. RESULTS: In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. CONCLUSIONS: This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.

Antiviral Activities of Halogenated Emodin Derivatives against Human Coronavirus NL63.

The current COVID-19 outbreak has highlighted the need for the development of new vaccines and drugs to combat Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). Recently, various drugs have been proposed as potentially effective against COVID-19, such as remdesivir, infliximab and imatinib. Natural plants have been used as an alternative source of drugs for thousands of years, and some of them are effective for the treatment of various viral diseases. Emodin (1,3,8-trihydroxy-6-methylanthracene-9,10-dione) is a biologically active anthraquinone with antiviral activity that is found in various plants. We studied the selectivity of electrophilic aromatic substitution reactions on an emodin core (halogenation, nitration and sulfonation), which resulted in a library of emodin derivatives. The main aim of this work was to carry out an initial evaluation of the potential to improve the activity of emodin against human coronavirus NL63 (HCoV-NL63) and also to generate a set of initial SAR guidelines. We have prepared emodin derivatives which displayed significant anti-HCoV-NL63 activity. We observed that halogenation of emodin can improve its antiviral activity. The most active compound in this study was the iodinated emodin analogue E_3I, whose anti-HCoV-NL63 activity was comparable to that of remdesivir. Evaluation of the emodin analogues also revealed some unwanted toxicity to Vero cells. Since new synthetic routes are now available that allow modification of the emodin structure, it is reasonable to expect that analogues with significantly improved anti-HCoV-NL63 activity and lowered toxicity may thus be generated.

Quorum-sensing in yeast and its potential in wine making.

This mini-review synthesises the present knowledge of microbial quorum-sensing, with a specific focus on quorum-sensing in yeast, and especially in wine yeast. In vine and wine ecosystems, yeast co-interact with a large variety of microorganisms, thereby affecting the fermentation process and, consequently, the flavour of the wine. The precise connections between microbial interactions and quorum-sensing remain unclear, but we describe here how and when some species start to produce quorum-sensing molecules to synchronously adapt their collective behaviour to new conditions. In Saccharomyces cerevisiae, the quorum-sensing molecules were identified as 2-phenylethanol and tryptophol. However, it was recently shown that also a quorum-sensing molecule formerly identified only in Candida albicans, tyrosol, appears to be regulated in S. cerevisiae according to cell density. This review describes the methods for detection and quantification of those quorum-sensing molecules, their underlying mechanisms of action, and their genetic background. It also examines the external stimuli that evoke the quorum-sensing mechanism in the wine-processing environment. The review closes with insight into the biotechnological applications that are already making use of the advantages of quorum-sensing systems and indicates the important questions that still need to be addressed in future research into quorum-sensing.

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus.

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

Molecular Biology Methods in Streptomyces rimosus, a Producer of Oxytetracycline.

Streptomyces rimosus is used for production of the broad-spectrum antibiotic oxytetracycline (OTC). S. rimosus belongs to Actinomyces species, a large group of microorganisms that produce diverse set of natural metabolites of high importance in many aspects of our life. In this chapter, we describe specific molecular biology methods and a classical homologous recombination approach for targeted in-frame deletion of a target gene or entire operon in S. rimosus genome. The presented protocols will guide you through the design of experiment and construction of homology arms and their cloning into appropriate vectors, which are suitable for gene-engineering work with S. rimosus. Furthermore, two different protocols for S. rimosus transformation are described including detailed procedure for targeted gene replacement via double crossover recombination event. Gene deletion is confirmed by colony PCR, and colonies are further characterized by cultivation and metabolite analysis. As the final step, we present in trans complementation of the deleted gene, to confirm functionality of the engineering approach achieved by gene disruption. A number of methodological steps and protocols are optimized for S. rimosus strains including the use of the selected reporter genes. Protocols described in this chapter can be applied for studying function of any individual gene product in diverse OTC-producing Streptomyces rimosus strains.

Synthetic biology approaches to actinomycete strain improvement.

Their biochemical versatility and biotechnological importance make actinomycete bacteria attractive targets for ambitious genetic engineering using the toolkit of synthetic biology. But their complex biology also poses unique challenges. This mini review discusses some of the recent advances in synthetic biology approaches from an actinomycete perspective and presents examples of their application to the rational improvement of industrially relevant strains.

Quality characteristics of wheat flour dough and bread containing grape pomace flour.

Wheat bread was enriched with 6%, 10% and 15% dried and milled grape pomace flour from two grape cultivars: 'Merlot' and 'Zelen'. Rheological, textural, sensory and antioxidant properties of the enriched dough and bread were evaluated, and compared to control samples. Grape cultivar had significant impact on the rheological characteristics of the dough, and on the sensory and antioxidant properties of the final bread. Development time and dough stability were longer when 'Merlot' grape pomace flour was added compared to 'Zelen' grape pomace flour and the control. Grape pomace flour addition affected bread volume, firmness, crumb and crust colour, and odour and taste intensity. Moreover, grape pomace flour addition resulted in a stickier and less springy crumb texture, and some negative sensorial properties, such as increased intensity of aftertaste and sand feeling in the mouth. The phenolic content and antioxidant activity of bread were positively correlated with grape pomace flour addition ( r = 0.987, p = 0.01 and r = 0.941, p = 0.01 between phenolic content and ferric reducing antioxidant power and phenolic content and 2,2-diphenyl-1-picrylhydrazyl, respectively). The highest total phenolic contents were 5.92 mg gallic acid equivalents (GAE)/g dw for 'Merlot' and 3.65 mg gallic acid equivalents /g dw for 'Zelen', which were seen for the bread prepared with the highest grape pomace flour addition (15%). The highest antioxidant activity determined by the 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays were seen for the bread prepared with the highest 'Merlot' grape pomace flour addition (15%). Dough characteristic and sensory profile are strongly influenced by cultivar of grape pomace flour. Based on results of sensory profiling, the variety 'Zelen' is suggested for use.

Quorum-Sensing Kinetics in Saccharomyces cerevisiae: A Symphony of ARO Genes and Aromatic Alcohols.

The kinetics of quorum sensing in Saccharomyces cerevisiae were studied using a mini-fermentation platform. The quorum-sensing molecules were monitored using our previous HPLC approach that is here supported by quantitative real-time PCR analysis of the quorum-sensing genes. We thus initially confirm correlations between peak production rates of the monitored quorum-sensing molecules 2-phenylethanol, tryptophol, and tyrosol and peak expression of the genes responsible for their synthesis: ARO8, ARO9, and ARO10. This confirms the accuracy of our previously implemented kinetic model, thus favoring its use in further studies in this field. We also show that the quorum-sensing kinetics are precisely dependent on the population growth phase and that tyrosol production is also regulated by cell concentration, which has not been reported previously. Additionally, we show that during wine fermentation, ethanol stress reduces the production of 2-phenylethanol, tryptophol, and tyrosol, which opens new challenges in the control of wine fermentation.

Monitoring of quorum-sensing molecules during minifermentation studies in wine yeast.

At high cell density or under low nutrient conditions, yeasts collectively adapt their metabolism by secreting aromatic alcohols in what is known as quorum sensing. However, the mechanisms and role of quorum sensing in yeast are poorly understood, and the methodology behind this process is not well established. This paper describes an effective approach to study quorum sensing in yeast fermentations. The separation, detection, and quantification of the putative quorum-sensing molecules 2-phenylethanol, tryptophol, and tyrosol have been optimized on a simple HPLC-based system. With the use of a phenyl HPLC column and a fluorescence detector, the sensitivity of the system was significantly increased. This allowed extraction and concentration procedures to be eliminated and the process to be scaled down to 2 mL minifermentations. Additionally, an innovative method for rapid viable-cell counting is presented. This study forms the basis for detailed studies in kinetics and regulation of quorum sensing in yeast fermentation.