Substantiation of the development of technology for industrial production of B vitamins

Проведен анализ информационных источников по существующим технологиям получения витаминов B и B. Кратко описаны преимущества и недостатки известных технологий с использованием различных штаммов-продуцентов, а также технологий с использованием химического синтеза. Предложена принципиальная схема разрабатываемых технологий синтеза данных витаминов, определены наиболее продуктивные штаммы-продуценты и необходимые компоненты питательных сред. Обоснован и представлен перечень продуктов, предназначенных для обогащения витаминами группы В, которые будут получены по разрабатываемым технологиям. The analysis of information sources on existing technologies for obtaining vitamins B and B has been carried out. The advantages and disadvantages of known technologies using various producer strains, as well as technologies using chemical synthesis, are briefly described. A schematic diagram of the developed technologies for the synthesis of these vitamins is proposed, the most productive strains - producers and necessary components of nutrient media are identified. The list of products intended for enrichment with B vitamins, which will be obtained by the developed technologies, has been substantiated and presented.

Pyrazines Biosynthesis by Bacillus Strains Isolated from Natto Fermented Soybean

Pyrazines are organic compounds with a varied, intense aroma of roasted nuts, occasionally with hints of baked potatoes, almonds, and others. As a result, they are used in the food industry as food flavorings. Biosynthesis of pyrazines using microorganisms in environmentally friendly conditions is an alternative to chemical synthesis. However, screening is required to isolate efficient producer strains for efficient biosynthesis of this compound. The study’s goal was to assess the ability of Bacillus subtilis cultures isolated from natto (fermented soybeans) to biosynthesize a broad range of alkylpyrazines. B. subtilis isolated cultures were found to be capable of producing 2-methylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, and 2,3,5,6-tetramethylpyrazine. As a result of the screening, two cultures of B. subtilis capable of producing alkylpyrazines were isolated. At a total concentration of 3261 µg/L, the BcP4 strain primarily produced 2-methylpyrazine (690 µg/L), 2,3-dimethylpyrazine (680 µg/L), and 2,6-dimethylpyrazine (1891 µg/L). At a total concentration of 558 mg/L, the BcP21 strain produced 2,5-dimethylpyrazine (4.5 mg/L), 2,3,5-trimethylpyrazine (52.6 mg/L), and 2,3,5,6-tetramethylpyrazine (501.1 mg/L). The results show that different B. subtilis strains are predisposed to produce different alkylpyrazines.

ULTRASONIC DISINTEGRATION OF LIGNOCELLULOSE RAW MATERIALS AS A PRE-TREATMENT OF A SUBSTRATE FOR MICROBIOLOGICAL PRODUCTION OF BIOBUTANOL

Aim. The purpose of the study was to investigate the effect of ultrasonic disintegration on the lignocellulosic raw materials (biomass of the non-cereal part of rape) with its subsequent use as a substrate for the production of biobutanol. Methods. Butanol-producing strains and the biomass of the non-cereal part of rape Brassica napus were used in the present study. Ultrasonic disintegration of lignocellulosic raw materials was performed on the specially designed equipment. Results. The effect of ultrasonic disintegration on lignocellulosic raw materials was investigated for further application in biofuel production based on microbiological conversion. The possibility of using the obtained components after the pre-treatment of lignocellulose by ultrasonic disintegration as a substrate for the microbiological synthesis of butanol was shown. The highest accumulation of butanol (2.4 g/l) was obtained with the use of 5% dry matter content in the medium, 5 min treatment and the specific power of ultrasonic disintegration of 0.72 W/ml. Conclusions. The possibility of producer strains of the genus Clostridium to use cellulose in the fermentation process has been shown. When using ultrasonic disintegration for pretreatment of the non-cereal part of the biomass of rape, the accumulation of butanol increased by 3 folds.

Targeted accumulation of selective anticancer depsipeptides by reconstructing the precursor supply in the neoantimycin biosynthetic pathway

Background Neoantimycins are a group of 15-membered ring depsipeptides isolated from Streptomycetes with a broad-spectrum of anticancer activities. Neoantimycin biosynthesis is directed by the hybrid multimodular megaenzymes of non-ribosomal peptide synthetase and polyketide synthase. We previously discovered a new neoantimycin analogue unantimycin B, which was demonstrated to have selective anticancer activities and was produced from the neoantimycin biosynthetic pathway with a starter unit of 3-hydroxybenzoate, instead of the 3-formamidosalicylate unit that is common for neoantimycins. However, the low fermentation titre and tough isolation procedure have hindered in-depth pharmacological investigation of unantimycin B as an anticancer agent. Results In this work, we genetically constructed two unantimycin B producer strains and inhibited neoantimycins production by removing natO and natJ-L genes essential for 3-formamidosalicylate biosynthesis, therefore facilitating chromatographic separation of unantimycin B from the complex fermentation extract. Based on the ΔnatO mutant, we improved unantimycin B production twofold, reaching approximately 12.8 mg/L, by feeding 3-hydroxybenzoate during fermentation. Furthermore, the production was improved more than sixfold, reaching approximately 40.0 mg/L, in the ΔnatO strain introduced with a chorismatase gene highly expressed under a strong promoter for endogenously over-producing 3-hydroxybenzoate. Conclusion This work provides a case of targeting accumulation and significant production improvement of medicinally interesting natural products via genetic manipulation of precursor biosynthesis in Streptomycetes, the talented producers of pharmaceutical molecules.

Target Accumulation of Selective Anticancer Depsipeptides by Reconstructing Precursor Supply in Neoantimycins Biosynthetic Pathway

BackgroundNeoantimycins are a group of 15-membered ring depsipeptides isolated from streptomycetes with a broad-spectrum of anticancer activities. Their biosynthesis is directed by the hybrid multimodular megaenzymes of non-ribosomal peptide synthetase and polyketide synthase. We have previously discovered a new neoantimycin analogue unantimycin B, which was demonstrated with selective anticancer activities and was produced from neoantimycins biosynthetic pathway with a starter unit of 3-hydroxybenzoate, instead of the 3-formamidosalicylate for neoantimycins. However, the low fermentation yield and tough isolation procedure have been hindering in-depth pharmacology investigation of unantimycin B as anticancer agents.ResultsIn the work, we genetically constructed two unantimycin B producer strains with neoantimycins production destroyed by removing natO and natJ-L genes essential for 3-formamidosalicylate biosynthesis and therefore facilitated chromatographic separation of unantimycin B from the complex fermentation extract. Based on the △natO mutant, we improved unantimycin Bproduction by two times, reaching to approximate 12.8 mg/L, by feeding 3-hydroxybenzoate in fermentation. Further, the production was improved by more than six times, reaching to approximate 40.0 mg/L, in the △natO strain introduced with a chorismatase gene highly expressed under a strong promoter for over-producing 3-hydroxybenzoate endogenously.ConclusionThe work gives a case of targeting accumulation and significant production improvement of medicinally interesting natural products via genetically manipulation of precursor biosynthesis in streptomycetes, the talented producers of pharmaceutical molecules.

Development and implementation of strategies to improve octanoic acid production in Saccharomyces cerevisiae

Octanoic acid (C8 FA) is a medium-chain fatty acid which, in nature, mainly occurs in palm kernel oil and coconuts. It is used in various products including cleaning agents, cosmetics, pesticides and herbicides as well as in foods for preservation or flavoring. Furthermore, it is investigated for medical treatments, for instance, of high cholesterol levels. The cultivation of palm oil plants has surged in the last years to satisfy an increasing market demand. However, concerns about extensive monocultures, which often come along with deforestation of rainforest, have driven the search for more environmentally friendly production methods. A biotechnological production with microbial organisms presents an attractive, more sustainable alternative. Traditionally, the yeast Saccharomyces cerevisiae has been utilized by mankind in bread, wine, and beer making. Based on comprehensive knowledge about its metabolism and genetics, it can nowadays be metabolically engineered to produce a plethora of compounds of industrial interest. To produce octanoic acid, the cytosolic fatty acid synthase (FAS) of S. cerevisiae was utilized and engineered. Naturally, the yeast produces mostly long-chain fatty acids with chain lengths of C16 and C18, and only trace amounts of medium-chain fatty acids, i.e. C8-C14 fatty acids. To generate an S. cerevisiae strain that produces primarily octanoic acid, a mutated version of the FAS was generated (Gajewski et al., 2017) and the resulting S. cerevisiae FASR1834K strain was utilized in this work as a starting strain. The goal of this thesis was to develop and implement strategies to improve the production level of this strain. The current mode of quantification of octanoic acid includes labor-intensive, low-throughput sample preparation and measurement – a main obstacle in generating and screening for improved strain variants. To this end, a main objective of this thesis was the development of a biosensor. The biosensor was based on the pPDR12 promotor, which is regulated by the transcription factor War1. Coupling pPDR12 to GFP as the reporter gene on a multicopy plasmid allowed in vivo detection via fluorescence intensity. The developed biosensor enabled rapid and facile quantification of the short- and medium-chain fatty acids C6, C7 and C8 fatty acids (Baumann et al., 2018). This is the first biosensor that can quantify externally supplied octanoic acid as well as octanoic acid present in the culture supernatant of producer strains with a high linear and dynamic range. Its reliability was validated by correlation of the biosensor signal to the octanoic acid concentrations extracted from culture supernatants as determined by gas chromatography. The biosensor’s ability to detect octanoic acid in a linear range of 0.01-0.75 mM (≈1-110 mg/L), which is within the production range of the starting strain, and a response of up to 10-fold increase in fluorescence after activation was demonstrated. A high-throughput FACS (fluorescence-activated cell sorting) screening of an octanoic acid producer strain library was performed with the biosensor to detect improved strain variants (Baumann et al., 2020a). For this purpose, the biosensor was genomically integrated into an octanoic acid producer strain, resulting in drastically reduced single cell noise. The additional knockout of FAA2 successfully prevented medium-chain fatty acid degradation. A high-throughput screening protocol was designed to include iterative enrichment rounds which decreased false positives. The functionality of the biosensor on single cell level was validated by adding octanoic acid in the range of 0-80 mg/L and subsequent flow cytometric analysis. The biosensor-assisted FACS screening of a plasmid overexpression library of the yeast genome led to the detection of two genetic targets, FSH2 and KCS1, that in combined overexpression enhanced octanoic acid titers by 55 % compared to the parental strain. This was the first report of an effect of FSH2 and KCS1 on fatty acid titers. The presented method can also be utilized to screen other genetic libraries and is a means to facilitate future engineering efforts. In growth tests, the previously reported toxicity of octanoic acid on S. cerevisiae was confirmed. Different strategies were harnessed to create more robust strains. An adaptive laboratory evolution (ALE) experiment was conducted and several rational targets including transporter- (PDR12, TPO1) and transcription factor-encoding genes (PDR1, PDR3, WAR1) as well as the mutated acetyl-CoA carboxylase encoding gene ACC1S1157A were overexpressed or knocked out in producer or non-producer strains, respectively. Despite contrary previous reports for other strain backgrounds, an enhanced robustness was not observable. Suspecting that the utilized laboratory strains have a natively low tolerance level, four industrial S. cerevisiae strains were evaluated in growth assays with octanoic acid and inherently more robust strains were detected, which are suitable future production hosts. ...

Testing of laboratory samples of microbiological preparations based on promising producer strains for the control of apple scab

To prevent phytosanitary destabilization in apple agrocenoses, it is necessary to include ecologically safe microbiological preparations in the protection systems. In the Krasnodar region in 2016 and 2019 in the field, laboratory samples of microbiological preparations were tested in the formulation “wettable powder” from the collection of the laboratory of the biological method of the FGBNU VNIIMK against Venturia inaequalis (Cooke) G. Winter. Of the 9 laboratory samples tested in 2016, the highest efficiency, approaching the effectiveness of a chemical fungicide, was shown by samples based on fungal producer strains: RK-1, XK-1 and T-2 - 76.9-92.5%. In 2019, all variants (in the amount of 4) using laboratory samples of microbiological preparations were more effective in comparison with the active substance of chemical origin. The best scab control was provided by a laboratory sample of a three-component mixed preparation based on two fungal and one bacterial producer strain RK-1 + XK-1 + B-12 - 83.0-84.9%. The isolated samples are promising for research on the creation of microbiological preparations against apple scab.

DEVELOPMENT OF A PRODUCER OF RECOMBINANT MARAL CHYMOSIN BASED ON THE YEAST KLUYVEROMYCES LACTIS

A plasmid vector pSVB was developed, which allows integrating an expression cassette containing the maral prochymosin gene into the genome of the yeast Kluyveromyces lactis. Producer strains have been obtained that ensure the accumulation of recombinant maral chymosin in the culture medium with a milk-clotting activity of up to 145 AU / ml.

Scaling of the promising producer strains cultivation process of fungicidal metabolites

The optimal conditions in pilot bioreactors for deep cultivation of the genus Bacillus bacteria were selected according to a number of biotechnological parameters, and the dynamics of antifungal compounds synthesis in the process of the culture maintaining was monitored.