Enhancing Saccharomyces cerevisiae Taxane Biosynthesis and Overcoming Nutritional Stress-Induced Pseudohyphal Growth

The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2×YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2×YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.

Optimization of dextran production from Leuconostoc strains isolated from different dairy products from Ouargla region Algeria

Leuconostoc (Ln) sp. belongs to a group of lactic acid bacteria, which has the capacity to produce dextran (an exopolysaccharides) in the presence of su-crose. dextran is industrially important, it was the first microbial exopolysac-charide affirmed for commercial use. This study aimed to optimize the pro-duction of the synthesized dextran by Ln strains species isolated from differ-ent dairy products. Morphological, cultural, physiological and biochemical characteristics were employed to identify 23 isolated strains. We have identi-fied the species: Ln. gelidum, Ln. carnosum, Ln. citreum, Ln. fallax, Ln. mesen-teroides subsp mesenteroides, Ln. mesenteroides subsp dextranicum, Ln. mesenteroides subsp cremoris. 20 strains had the capacity to produce dex-tran from sucrose. The precipitation and quantification of EPS on MRSs (Mark rogosa et sharpe sucrose) medium revealed a difference between the strains, by the total sugars assay method, the amount of EPS varied between 0.63 ± 0.19 and 2.41 ± 0.17 g / L of strains LnF70 and LnC1 (isolated from goat's milk), respectively. The dextran production from MRSs medium was better than from liquid MSE. The optimization of production on MRSs medi-um with different concentration of glucose, yeast extract and sucrose showed that the strains had good production with a concentration of 2% glucose, 0.3% yeast extract and 10% sucrose.

Select streptococci can degrade Candida mannan to facilitate growth

Background: Multiple studies have found that streptococci have a synergistic relationship with Candida species, but the details of these interactions are still being discovered. Candida species are covered by mannan, a polymer of mannose, which could serve as a carbon source for certain microbes. We hypothesized that streptococci that possess mannan-degrading glycosyl hydrolases would also be able to enzymatically cleave mannose residues, which could serve as a primary carbohydrate source to support growth. Methods & Results: We analyzed 90 streptococci genomes to predict the capability of streptococci to transport and utilize mannose and to degrade diverse mannose-linkages found on mannan. The genome analysis revealed mannose transporters and downstream pathways in most streptococci, but only <50% of streptococci harbored the glycosyl hydrolases required for mannan degradation. To confirm the ability of streptococci to use mannose or mannan, we grew 6 representative streptococci in a chemically defined media lacking glucose supplemented with mannose, yeast extract or purified mannan isolated from Candida and Saccharomyces strains. Although all tested Streptococcus strains could use mannose, S. salivarius and S. agalactiae , which did not possess mannan-degrading glycosyl hydrolases, could not use yeast extract or mannan to enhance their growth. In contrast, we found that S. mitis , S. parasanguinis, S. sanguinis , and S. pyogenes possessed the necessary glycosyl hydrolases to use yeast extract and isolated mannan, which promoted robust growth. Conclusions : Our data indicate that several streptococci are capable of degrading fungal mannans and harvesting mannose for energy. Importance: This work highlights a previously undescribed aspect of streptococcal- Candida interactions. Our work identifies that certain streptococci possess the enzymes required to degrade mannan and through this mechanism, they can release mannose residues from the cell wall of fungal species and use them as a nutrient source. We speculate that streptococci that can degrade fungal mannan may have a competitive advantage for colonization. This finding has broad implications for human health as streptococci and Candida are found at multiple body sites.

Probiotic powder: Optimization of the process parameters and influence of the drying method

This research paper aimed to optimize the process parameters and to select the most suitable drying method in order to obtain probiotic powder at reduced cost. The influence of the addition of growth promoters (glucose, sucrose, cheese whey, peptone, yeast extract or ammonium sulfate), incubation parameters (time and temperature) and drying method (freeze-drying or oven-drying) on the viability of the probiotic cultures Lactobacillus casei or Lactobacillus acidophilus was evaluated. The effect of the growth promoters was evaluated using a fractional factorial experimental design 26-4 and the concentration of the growth promoters and the incubation temperature were optimized through Box-Behnken experimental matrix (33). Cheese whey (16% w/v) plus ammonium sulfate (2.5% w/v) and cheese whey (12% w/v) plus yeast extract (7% w/v) promoted a higher multiplication of L. casei and L. acidophilus, respectively. The best temperature for L. casei was 35 ºC and for L. acidophilus 39 ºC, without influence of the incubation time (24 or 48 h). The oven-drying resulted in the highest populations of the probiotic cultures (above 9 log cfu/mL). This study proved that cheese whey can be a suitable growth promoter for both probiotic cultures and oven-drying could be the drying method, which can decrease the production costs. The influence of the temperature and growth promoters is strain specific, demonstrating that the growth conditions should be evaluated for each probiotic strain.

Statistical Optimization of Media Components for Xylanase Production by Aspergillus spp. Using Solid State Fermentation and its Application in Fruit Juice Clarification

Xylanases are enzymes that convert xylan into xylose, xylobiose, and xylotriose. The present study deals with the production and optimization of xylanase through Solid-State Fermentation (SSF) using different agricultural wastes by Aspergillus spp. The Plackett Burman (PB) design was used to screen significant media components affecting the xylanase production. The carbon sources screened were wheat bran, rice bran, sugarcane bagasse, corn cob, and orange peel. The nitrogen sources screened were yeast extract, peptone, (NH4)2SO4, Na2NO3, and urea. Also, nine different salts such as KCl, MgSO4, Na2HPO4, CaCl2, FeSO4, ZnSO4, Na2CO3, KH2PO4, and NaH2PO4 which act as trace elements were screened. The results showed that wheat bran, yeast extract, Na2NO3 and KCl are the significant factors that affect xylanase production. A 33 Full Factorial Design (FFD) was performed to optimize the significant media components (wheat bran, KCl, yeast extract) obtained from PB design using Response Surface Methodology (RSM). Statistical analysis of results showed that wheat bran, KCl, yeast extract, and interaction between wheat bran and yeast extract were found to be significant. The optimum concentration of wheat bran, KCl, yeast extract was 8 g/L, 0.1 g/L and 3 g/L. The Partial purification of xylanase was carried out using ammonium salt precipitation and dialysis. Gel filtration chromatography was performed to optimize the elution time, which was found to be 6 minutes. Application of xylanase in orange juice clarification was studied at 40 °C, 50 °C, and 60 °C. The optimum temperature obtained was 60 ºC.

Antibiotic Activity of Actinomycetes Isolated from Young Tectona Grandis (L.) Wood and Pith

Actinomycetes are a source for novel bioactive compounds and justify obtaining new species from various sources. Hardwoods such as Tectona grandis (L.) have not been studied for actinomycete isolation. We aim to isolate endophytic actinomycetes from young Tectona grandis wood and pith and screen for antibiotic activity. Five young wood were cut and surface sterilized using ethanol and hypochlorous acid. The wood and pith of each sample are placed in eight plates of Humic acid-vitamin B (HV), Tap water Yeast extract (TWYE), and Yeast Extract Casein Digest (YECD) medium and incubated at 27°C for four weeks. Actinomycetes were isolated from such medium, observed every week, and transferred to an International Streptomyces Project-2 (ISP-2) medium for identification and antibiotic production tested against Staphylococcus aureus, Helicobacter pylori, and Escherichia coli using the Kirby-Bauer method. Seven actinomycetes were isolated from the wood, primarily from YECD and TWYE media, with varying morphological characteristics. One isolate having maroon-colored aerial and vegetative mycelium with grey spores showed moderate antibacterial activity against S. aureus and H. pylori (13.49±1.03 mm and 14.9±0.7 mm, respectively), while two other actinomycetes showed weak activity against these bacteria. However, none of the actinomycetes show any activity against E. coli. Tectona grandis (L.) is a potential source for novel actinomycetes with an antibiotic activity which warrants further exploration

Overexpression of chalcone isomerase A gene in Astragalus trigonus for stimulating apigenin

Apigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

Effect of different yeast extract concentration in membrane-less microbial fuel cell (ML-MFC) for electricity generation using food waste as carbon sources

Electricity constitutes one of the basic energies of our everyday life and approximately 14 % of the global population does not have the access to electricity. An abundance of waste is generated daily wherein food waste constitutes 45 % of the composition. A mediator-less and membrane-less single-chambered microbial fuel cell (ML-MFC) has the potential to serve as a cost-effective solution for food waste treatment and electricity power generation at no additional cost for the substrate. Food waste from E-Idaman Sdn. Bhd. Kedah was utilised in this study to generate electrical energy while focusing on the effect of different yeast extract concentrations on the performance of the ML-MFC. Electrogenic bacterial (EB) culture employed in this study acted as a catalyst for the power generation and was isolated from a previous ML-MFC study. The proximate analysis of food waste observed carbon constituting the greatest composition at 30.02 %. From the conducted preliminary study which compared three different strains of EB to be introduced in the ML-MFC, Bacillus subtillis sp. exhibited the highest specific growth rate, μ, (0.117 g. L−1/h) and shortest doubling time, Td, (5.93 h). One-factor-at-a-time (OFAT) method was utilised to evaluate the performance of the ML-MFC. 15 g/L yeast extract concentration obtained the greatest power density (628.05 × 106 mW/m2), substrate degradation efficiency (12.3 %), COD removal (99 mg/L) and biomass (44.32 mg/L). This showcased that the addition of extra yeast extract concentration into the food waste had boosted the efficiency of EB’s growth resulting in greater consumption of carbon source (removed COD value; bioremediation) in the food waste.