Development of a strategy induction AOX1 promoter methylotrophic yeast Komagataella phaffii

Currently, there is a significant increase in interest in the industrial production of enzyme preparations (and other recombinant proteins) using various microorganisms, including methylotrophic yeasts such as Komagataella phaffii. At the same time, the most significant productivity of the target proteins is achieved by methanol induction of heterologous genes cloned under the control of the AOX1 promoter. Thus, the efficiency of biosynthesis is largely determined by the metabolism of methanol. In this connection, the aim of the work is to develop an optimal strategy for methanol induction of the AOX1 promoter of Komagataella phaffii. The object of the study is the culture of the recombinant phospholipase A2 producing strain Komagataella phaffii. The studies were carried out in a laboratory fermenter Infors Minifors (Switzerland) on a liquid nutrient medium BSM (Basal Salt Medium) We used the generally accepted methods of studying the characteristics of metabolic activity, including the calculation of specific characteristics and productivity of the strain. The result of the study is the determination of the specific rate of consumption of methanol used as a carbon source, which was 19.2±1.8 mg/g*h. Also, the specific growth rate of Komagataella phaffii was determined and amounted to 0.24 h-1.Based on the data obtained during the research, a strategy for the induction of the AOX1 promoter in the cultivation of the methylotrophic yeast Komagataella phaffii was developed by maintaining the methanol concentration in the range of 0.6 to 2% based on the concentration of dissolved oxygen in the medium. The developed strategy of induction of the AOX1 promoter made it possible to obtain at least 1.87 g / l of recombinant protein (phospholipase A2) during cultivation of Komagataella phaffii for 96 h, which is 3.7 times higher than the known results.

Biodegradation of alicyclic amines by a newly isolated hypersaline tolerant strain Paenarthrobacter sp. TYUT067

Alicyclic amines are widely used in several types of industries, and considerable attention has been devoted to possible environmental pollution by alicyclic amines in hypersaline industrial wastewater. In this study, a new hypersaline tolerant bacterial TYUT067 capable of growing in liquid basal salt medium with cyclohexylamine (CHAM) as the sole carbon source and energy source, was isolated from soil, and discovered with highly efficient CHAM degrading ability. The strain TYUT067 was identified as Paenarthrobacter sp. based on 16S rDNA gene sequence, and its degradation characteristic was examined. The results revealed that the isolated TYUT067 could grow well under pH range of 6.5–10.0, temperature from 20 °C to 30 °C. For degradation of 60 mM of cyclohexylamine, 100% degradation could be finished within 120 h. The TYUT067 could degrade 10 mM CHAM under hypersaline conditions (3–5% NaCl, w/v), revealed the hypersaline tolerance of TYUT067. Different type of amines was also tested with TYUT067, the degradations of >90% were achieved toward several alicyclic amines. The current results suggested that TYUT067 was a potential species could be efficiently used for the degradation of alicyclic amines and might be applicable to a hypersaline wastewater treatment system for the removal of alicyclic amines.

Decomposition of Rice Chaff Using a Cocultivation System of Thermobifida fusca and Ureibacillus thermosphaericus

Lignocellulosic biomass comprises cellulose, hemicellulose, and lignin and is a potential source of fuels and chemicals. Although this complex biomass is persistent, it can be cooperatively decomposed by a microbial consortium in nature. In this study, a coculture of the moderately thermophilic bacteria Thermobifida fusca and Ureibacillus thermosphaericus was used for biodegradation of rice chaff. The bacterial strains were incubated in modified Brock’s basal salt medium (pH 8.0) supplemented with yeast extract and rice chaff at 50 °C for 7 days. The concentration of reducing sugars and the enzymatic activities of laccase, lignin peroxidase, cellulase, and xylanase in the supernatant of the culture medium were measured every day. The concentrations of reducing sugars in solo cultures of T. fusca and U. thermosphaericus and a mixed culture of the two strains after 7 days of incubation were 0.047, 0.040, and 0.195 mg/mL, respectively, indicating that the decomposition of rice chaff was enhanced in the coculture. Based on the results, it is thought that the lignin surrounding the cellulose was decomposed by laccase and lignin peroxidase secreted from U. thermosphaericus, resulting in cellulose and hemicellulose in the rice chaff being easily decomposed by enzymes from T. fusca.

PRIMARY AND SECONDARY SOMATIC EMBRYOGENESIS OF CACAO: THE EFFECT OF EXPLANT TYPES AND PLANT GROWTH REGULATORS

<p class="abstrakinggris">The success of cacao somatic embryogenesis is affected by many factors, including the basal salt medium, the genotype, the explant type, and the concentration and composition of plant growth regulators (PGRs). The study aimed to evaluate the effects of PGRs composition on the primary somatic embryo (PSE) response and the effect of explant type and PGRs composition used in inducing PSE on the secondary somatic embryogenesis (SSE) response. PSEs were induced from basal petal and staminoid explants of MCC 01 and MCC 02 clones on DKW medium containing 2,4-D 2 mg l^-1+ kinetin 0.5 mg l^-1 or 2,4-D 2 mg l^-1 + kinetin 0.125 – 0.250 mg l^-1 + thidiazuron (TDZ) 2.5 – 5 µg l^-1 or 2,4-D 2 mg l^-1 + TDZ 10 µg l^-1. Genotype, explant type, and PGR composition dependently affected PSE response. The best PSE response was obtained from staminoid explant of MCC 02 clone on medium containing 2,4-D 2 mg l^-1 + kinetin 0.5 mg l^-1 (20%, 9 embryos). The explant type and PGR composition used in inducing PSEs affect the SSE response. The highest SSE response of MCC 01 clone was obtained from petal explant with medium containing 2,4-D 2 mg l^-1 + kinetin 0.5 mg l^-1. The formation of SSEs could increase the multiplication rate of MCC 01 clone by 7 times.</p>

Optimization of cultivated strawberry micropropagation using а biogenic silica and green-tea-flavonoids-based mechanocomposite

For the first time, organogenesis and physiological characteristics of Fragaria ananassa microclones (cvs. ‘Alpha’ and ‘Solnechnaya polyanka’) under the influence of mechanocomposite (MC) based on rice husks amorphous silica and flavonoids of green tea during the multiplication stage in in vitro conditions were studied. The addition of the MC (0.0, 2.5, 5.0 and 10.0 mg·L-1) to the Gamborg- Eveleg’s basal salt medium supplemented with 0.75 mg·L-1 6-benzylaminopurine has shown beneficial action on processes of organogenesis followed by enzymatic, photosynthetic, and hormonal activities of in vitro cultured strawberry plantlets. In both cultivars, the high frequency of proliferation (100 %) and maximum number of axillary shoots increased by 1.8–2.0 times on medium supplemented with 5.0 mg·L-1 MC. The concentrations of 2.5 and 5.0 mg·L-1 MC were optimal for obtaining plantlets with high physiological state in in vitro conditions. The results may be used for the development of production systems for a healthy planting material using biotechnological approaches and recommended for commercial strawberry micropropagation.

Pengaruh Komposisi Media Dasar dan Jenis Eksplan terhadap Pembentukan Embrio Somatik Kakao

<em>The composition of basal medium determines the regeneration success of in vitro culture. The study aimed to evaluate the effect of basal medium in the primary callus induction medium and explant type on the formation of cacao somatic embryo. The research was conducted at the Tissue Culture Laboratory of IAARD, Bogor, from June 2014 to December 2015. Primary callus induction derived from staminoid and petal explants of ICCRI 4 clone used two types of basal medium, i.e. DKW+ 9 µM 2.4-D + 1.16 µM kinetin or WPM + 9 µM 2.4-D + 1.16 µM kinetin.</em> <em>After 14 days, callus was subcultured onto secondary callus induction medium (WPM + 2.4-D 9 </em><em>μM + kinetin 0.58 μM), and then onto DKW medium without growth regulators to induce the formation of somatic embryo. The research was designed in two-factor factorial design with five replications. The first factor was the type of basal medium on the primary callus induction medium (DKW and WPM) and the second factor was the type of explants (petal and staminoid). The results showed significant interaction effect between basal medium type and explant type on the formation of callus and somatic embryo of cacao. The highest percentage of callus formation was derived from staminoid explants on the DKW basal salt medium (92.5%). However, the highest percentage of somatic embryo formation and the number of somatic embryo per explant were obtained from DKW basal salt medium with petal explants (36.5% and 2.3).</em> <em>Therefore, t</em><em>he use of DKW basal salt medium and petal explant were recommended for the induction of somatic embryo of the ICCRI 4 clone.</em>

Production of xylanase under solid state fermentation using different agricultural and horticultural residue by Myceliopthora thermophila SH1 (Ascomycota: Chaetomiaceae)

An abundant amount of agricultural/horticultural waste were generated from agricultural and industrial processing. However, such wastes usually have a composition rich in sugars, minerals and proteins, and therefore, they should not be considered "wastes" but raw materials for other industrial processes. The purpose of the present work was to optimize the xylanase production by Myceliopthora thermophila SH1 (Ascomycota: Chaetomiaceae) an alkalothermophilic strain isolated from hot spring of Himachal Pradesh under solid state fermentation using different agricultural wastes/horticultural waste as a substrate by enrichment with basal salt medium at temperature 45 oC after incubating it for 7 days. The highest level of enzymes was produced using rice bran, orange pulp, sugarcane bagasse, wheat straw, wheat bran, apple pomace as a substrate. The secreted extracellular enzyme presented a property that matches the requirement in industrial environment.

Influence Of Iron Sources In The Nutrient Medium On In Vitro Shoot Multiplication And Rooting Of Magnolia And Cherry Plum

In this study, the effects of compounds providing Fe in chelated (NaFeEDTA and Fe(III)AC) and non-chelated (FeSO4·7H2O) forms as components of culture media, onin vitroshoot multiplication and rooting ofMagnolia soulangeana‘Alexandrina’,Magnolia grandifloraandPrunus cerasifera‘Nigra’ were comparatively evaluated. Each of the tested chemicals was used as a single Fe source in the basal salt medium. In the stages of shoot multiplication and rooting plant response was scored by biometrical indices (number of shoots, leaves and roots, shoot and root length, percent of rooted plants and root hairs). The occurrence of physiological disorders was estimated by visual observations. In presence of FeSO4, symptoms of chlorosis, hyperhy-dricity, early senescence and specific morphology of roots, suggesting Fe deficiency, were observed. These deteriorations were entirely prevented at the application of Fe chelates of which, in this experimental systems, Fe(III)AC was tested for the first time. The addition of Fe(III)AC positively affected the plant quality to extent comparable to that of NaFeEDTA. The obtained data suggest that both applied Fe chelates are more appropriate than non-chelated Fe form and can be alternatively used in the optimization of nutrient media for micropropagation ofMagnoliaandPrunus cerasiferagenotypes.

Biodegradation of PAHs byBurkholderiasp. VITRSB1 Isolated from Marine Sediments

The polycyclic aromatic hydrocarbons (PAHs) pollution to the environment is a major threat to the living organisms, and hence the degradation of these PAHs is necessary. Studies on PAHs degrading bacteria have focussed on terrestrial microbes and the potential of marine derived microbes is undermined. Herein we report the isolation and characterization of PAHs degradingBurkholderiasp. from lagoon sediments collected at the Southern coast of India. The strain was Gram negative, rod-shaped, motile, and ∼2–5 μm in length. Based on the phylogenetic data the strain was identified asBurkholderiaand designated as VITRSB1. Initial PAHs degradation ability of the strain was assessed using basal salt medium supplemented with diesel, kerosene, toluene, aniline, naphthalene, and phenol. The strain was found to be effectively degrading kerosene, diesel, toluene, and aniline even at higher concentration (1%). However, naphthalene and aniline were degraded only at lower concentration (0.1%) and phenol, camphor, and DAP inhibited the growth of the strain. Furthermore, the degraded end products of the PAHs were determined using FTIR. Notably, none of the end products were found to be toxic to the biosphere. Our results indicate that the isolatedBurkholderiasp. could be a prospective candidate for the effective degradation of selective PAHs.