Flask Fermentation Research Articles

Obtaining and Characterizing Thermostable α-Amylases Secreted by Bacillus subtilis, Originating from Bacillus amyloliquefaciens and Bacillus subtilis

The production of recombinant enzymes, primarily used for obtaining pure and functional target molecules, holds significant importance in modern biotechnology. This study aimed to obtain and characterize recombinant, extracellularly expressed α-amylases (Amy3500 and Amy1974), derived from B. amyloliquefaciens MDC1974 and B. subtilis MDC3500, respectively, using the pBE-S shuttle vector. Both α-amylase genes were molecularly cloned into the E. coli/B. subtilis pBE-S shuttle vector, both with (Amy1974sig and Amy3500sig) and without their signal peptides (Amy1974 and Amy3500), along with a signal peptide originating from the plasmid, and tested in flask fermentations. For recombinant Amy3500, the amylase variants resulted in similar levels of volumetric activity (700–750 U/mL). In contrast, the expression of Amy1974 nearly doubled compared to Amy1974sig with double signal peptides, reaching 2000 U/mL. SDS-PAGE estimated the molecular weight of Amy1974 α-amylase to be 54.6 kDa, which is consistent with the theoretical molecular mass calculations. However, the estimated molecular weight of Amy3500 α-amylase was significantly lower upon exiting the producer cells. Ca2⁺ ions exhibit a modest activating effect on the activities of Amy1974 and Amy3500 amylases, likely due to their tight binding to the protein scaffold. Both enzymes exhibited broad activity peaks between 45 and 70 °C, with a maximum at 65 °C. The Amy1974 and Amy3500 α-amylases demonstrated broad pH optima and pH-dependent thermostability, with optimum pH values at 6.5 and 5.8, and thermal stability peaks at pH 7.6 and 5.9, respectively. Both α-amylases displayed high relative activity against various starches, including corn amylopectin and potato amylose, while showing comparatively lower activity towards α-, β-, and γ-cyclodextrins. The Amy1974 amylase is effective in converting starch into dextrins of varying lengths, while Amy3500 primarily converts starch into glucose. These characteristics make them promising candidate enzymes for industrial applications.

Engineering Escherichia coli via introduction of the isopentenol utilization pathway to effectively produce geranyllinalool

BackgroundGeranyllinalool, a natural diterpenoid found in plants, has a floral and woody aroma, making it valuable in flavors and fragrances. Currently, its synthesis primarily depends on chemical methods, which are environmentally harmful and economically unsustainable. Microbial synthesis through metabolic engineering has shown potential for producing geranyllinalool. However, achieving efficient synthesis remains challenging owing to the limited availability of terpenoid precursors in microorganisms. Thus, an artificial isopentenol utilization pathway (IUP) was constructed and introduced in Escherichia coli to enhance precursor availability and further improve terpenoid synthesis.ResultsWe first constructed an artificial IUP in E. coli to enhance the supply of precursor geranylgeranyl diphosphate (GGPP) and then screened geranyllinalool synthases from plants to achieve efficient synthesis of geranyllinalool (274.78 ± 2.48 mg/L). To further improve geranyllinalool synthesis, we optimized various cultivation factors, including carbon source, IPTG concentration, and prenol addition and obtained 447.51 ± 6.92 mg/L of geranyllinalool after 72 h of shaken flask fermentation. Moreover, a scaled-up production in a 5-L fermenter was investigated to give 2.06 g/L of geranyllinalool through fed-batch fermentation. To the best of our knowledge, this is the highest reported titer so far.ConclusionsEfficient synthesis of geranyllinalool in E. coli can be achieved through a two-step pathway and optimization of culture conditions. The findings of this study provide valuable insights into the production of other terpenoids in E. coli.

Reconfiguring the Escherichia coli Electron Transport Chain to Enhance trans-2-Decenoic Acid Production.

trans-2-Decenoic acid is a pivotal α,β-medium-chain unsaturated fatty acid that serves as an essential intermediary in the synthesis of 10-hydroxy-2-decenoic acid and various pharmaceutical compounds. Biosynthesis yield of trans-2-decenoic acid by decanoic acid has significantly improved in recent years; however, the oxidative stress of Escherichia coli at high fatty acid concentrations restricts the conversion rate. Here, we introduced a combination of rational design and metabolic rewiring of the E. coli electron transport chain (ETC) to improve trans-2-decenoic acid production. Overexpressing ubiquinone (UbQ) biosynthesis genes enhanced the expression of ETC complex III: UbQ to reduce reactive oxygen species (ROS) accumulation. Furthermore, applying rotenone to inhibit ETC complex I improved the electron transfer efficiency of complex II. The integration of Vitamin B5 and B2 into the fermentation process increased the activities of fatty acyl-CoA synthetase (MaMACS) and fatty acyl-CoA dehydrogenase (PpfadE). Finally, the constructed E. coli BL21(DE3)(ΔfadBJR/pCDFDuet-1-PpfadE-MaMACS/pRSFDuet-1-sumo-CtydiI-ubiI) strain exhibited a 51.50% decrease in ROS and a 93.33% enhancement in trans-2-decenoic acid yield, reaching 1.45 g/L after 66 h, which is the highest yield reported for flask fermentation. This study reports the feasibility of rewiring the ETC regulation and energy metabolism to improve α,β-UCA biosynthesis efficiency.

Highly efficient synthesis of the chiral ACE inhibitor intermediate (R)-2-hydroxy-4-phenylbutyrate ethyl ester via engineered bi-enzyme coupled systems

(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester ((R)-HPBE) is an essential chiral intermediate in the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Its production involves the highly selective asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), catalyzed by carbonyl reductase (CpCR), with efficient cofactor regeneration playing a crucial role. In this study, an in-situ coenzyme regeneration system was developed by coupling carbonyl reductase (CpCR) with glucose dehydrogenase (GDH), resulting in the construction of five recombinant strains capable of NADPH regeneration. Among these, the recombinant strain E. coli BL21-pETDuet-1-GDH-L-CpCR, where CpCR is fused to the C-terminus of GDH, demonstrated the highest catalytic activity. This strain exhibited an enzyme activity of 69.78 U/mg and achieved a conversion rate of 98.3%, with an enantiomeric excess (ee) of 99.9% during the conversion of 30 mM OPBE to (R)-HPBE. High-density fermentation further enhanced enzyme yield, achieving an enzyme activity of 1960 U/mL in the fermentation broth, which is 16.2 times higher than the volumetric activity obtained from shake flask fermentation. Additionally, the implementation of a substrate feeding strategy enabled continuous processing, allowing the strain to efficiently convert a final OPBE concentration of 920 mM, producing 912 mM of (R)-HPBE. These findings highlight the system’s improved catalytic efficiency, stability, and scalability, making it highly suitable for industrial-scale biocatalytic production.

Engineering a novel pathway for efficient biosynthesis of salicin in Escherichia coli

Salicin is a natural glycoside compound widely used to treat fever, inflammation, and analgesia. Currently, salicin is primarily extracted from willow bark, which is not only cumbersome in terms of extraction and separate steps, but also subject to seasonal and geographic limitations. In this study, a highly efficient biosynthetic pathway for salicin synthesis was designed and constructed in E. coli. The most important precursor in the synthetic pathway of salicin designed in this study is salicyl alcohol. Building on a previously constructed biosynthetic salicylic acid metabolic pathway, the production of salicyl alcohol in shake flask fermentation reached 1.7 g/L by increasing the supply of shikimic acid pathway precursor PEP and salicyl alcohol precursor chorismate. According to the principle of substrate similarity, this study identified the key enzyme OsSGT1 from Oryza sativa, which uses E. coli endogenous UDP-glucose as a glycosyl donor to glycosylate salicyl alcohol into salicin. By redefining the optimal substrate of OsSGT1, and balancing metabolic flux along with increasing the supply of UDP-glucose, salicin production in shake flasks reached 4 g/L. Finally, culturing the high-yield strain in a 3-L fermenter resulted in the synthesis of 14.62 g/L of salicin. To the best of our knowledge, this achievement marks the highest salicin production through microbial fermentation to date.

Enhanced S-adenosyl-L-methionine synthesis in Saccharomyces cerevisiae using metabolic engineering strategies

S-adenosyl-L-methionine (SAM) plays pivotal roles in various physiological processes. With its increasing application in the treatment of diseases such as liver disease, depression, osteoarthritis and Alzheimer's, interest in SAM production aroused. Currently, Saccharomyces cerevisiae is the main industrial producer of SAM. With the surge in demand for SAM, improving the SAM biosynthesis is of importance. In this study, a multimodule engineering strategy was employed to improving SAM production: 1) Enhancing the gene expression of the sulfur assimilation pathway; 2) Strengthening the metabolic flux of the SAM synthesis pathway; 3) Weakening the SAM degradation pathway; 4) Increasing ATP supply. The resulting engineered mutant SC06 (S. cerevisiae CEN.PK2–1CΔgal80∷Tcyc1-sam2-Pgal1-Pgal10-met14-Tadh1, Δlsc2∷Tcyc1-hom6-Pgal1-Pgal10-met6-Tadh1, Δsah1Δmls1) displayed the highest SAM titer of 240.86 mg/L, which was 10.22-fold increase compared with the original strain. With optimized conditions, the SAM titer of mutant SC06 in shake flask fermentation reached 473.02 mg/L with a specific yield of 127.18 mg/g dry cell weight (DCW). In a 5 L fermenter with fed-batch fermentation, the maximal SAM yield of mutant SC06 reached 1.25 g/L with a specific yield of 166.67 mg/g DCW after 58 h cultivation. Therefore, the established metabolic engineering strategies displayed promising efficiency in improving the SAM productivity of S. cerevisiae CEN.PK2–1C, which may provide a useful tool for the improvement of SAM-producing strains.

A novel high-level phenyllactic acid fungal producer, Kodamaea ohmeri w5 screened from fermented broad bean-chili-paste

Phenyllactic acid (PLA) is a broad-spectrum and efficient antimicrobial phenolic acid with potential applications in the food industry. Previous studies have demonstrated that fungi may be ideal producers of PLA. In this study, 15 fungi screened from Doubanjiang with the ability to produce PLA were first reported, including Wickerhamomyces anomalus, Candida etchellsii, Candida parasitosis, Pichia kudriavzevii, Pichia membranifaciens and Kodamaea ohmeri. Among them, K. ohmeri w5 had the highest PLA yield, producing up to 7160 mg/L PLA in shake flask fermentation with phenylalanine as substrate, which was more than ten times higher than the PLA produced by wild-type LAB under the similar conditions. In addition, K. ohmeri w5 was able to grow under extreme hypertonic conditions of 20 % NaCl (w/v) and 50 % glucose (w/v) as well as produce 57.12 ± 0.42 and 1609.22 ± 36.26 mg/L of PLA, respectively. Furthermore, the fermentation supernatant of K. ohmeri w5 demonstrated direct inhibitory effects against foodborne pathogenic microorganisms, Aspergillus flavus and Bacillus cereus. However, the inhibitory effect was weaker than that of the PLA standard at the same concentration. Further, 12,497,932 bp of w5 genome-wide information was obtained by sequencing and assembling. And its gene model was predicted based on transcriptomic evidence, which showed that a total of 7 genes related to PLA synthesis were identified in the w5 genome. Based on qRT-PCR, structure prediction, and molecular docking, a potentially key genetic resource from K. ohmeri w5 for PLA production was uncovered. The results will provide novel producers of PLA and its potential genetic resources.

Moderately halophilic bacterium Halomonas alkalicola strain Ext as a platform for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production with fruit peels residues as sole carbon source

Polyhydroxyalkanoates (PHAs) are synthesized by a variety of microorganisms as intracellular storage granules under imbalanced nutrition and excess carbon. Carbon sources are key contributors to the high cost of PHA production, hence the need for exploration of cheap and sustainable raw materials for bacteria fermentation. In the present study, Halomonas alkalicola strain Ext isolated from a hypersaline lake in Kenya was assessed for its ability to utilize fruit peels hydrolysates (FPH) as sole carbon sources for PHA production. Sugars were extracted from dried peels of banana, mango, orange, and pineapple fruits through mechanical pretreatment and dilute acid hydrolysis. Fruit peels pretreated with 3 % H2SO4 at 121℃ were utilized for shake flask fermentation to produce PHAs by Halomonas alkalicola Ext. At optimal C:N ratios of between 20:1 and 30:1, the bacterium could produce up to 0.45±0.03, 0.394±0.12, 0.39±0.05, and 0.28±0.0 g/L of PHAs from hydrolysates of orange peels, mango peels, banana peels, and pineapple peels, respectively. A maximum PHA content of 16.92 % was achieved on orange peels hydrolysates with 4 % substrate loading. Monomer analysis with gas chromatography-mass spectrometry (GC-MS) revealed that the bacterium produced a copolymer Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 3-hydroxyvalerate (3HV) contents of 5.77 %, 6.08 %, 6.79 %, 6.845 %, for orange peels, pineapple peels, mango peels and banana peels substrates, respectively. The findings of this study suggest that fruit peels waste is a potential feedstock for sustainable production of PHAs by Halomonas alkalicola.

Systematic metabolic engineering enables highly efficient production of vitamin A in Saccharomyces cerevisiae

Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food, cosmetics, pharmaceutical, and nutraceutical industries. Synthetic biology and metabolic engineering enable microbes, especially the model organism Saccharomyces cerevisiae (generally recognised as safe) to possess great potential for the production of vitamin A. Herein, we first generated a vitamin A-producing strain by mining β-carotene 15,15′-mono(di)oxygenase from different sources and identified two isoenzymes Mbblh and Ssbco with comparable catalytic properties but different catalytic mechanisms. Combinational expression of isoenzymes increased the flux from β-carotene to vitamin A metabolism. To modulate the vitamin A components, retinol dehydrogenase 12 from Homo sapiens was introduced to achieve more than 90 % retinol purity using shake flask fermentation. Overexpressing POS5Δ17 enhanced the reduced nicotinamide adenine dinucleotide phosphate pool, and the titer of vitamin A was elevated by almost 46 %. Multi-copy integration of the key rate-limiting step gene Mbblh further improved the synthesis of vitamin A. Consequently, the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level. Eventually, the highest reported titer of 5.21 g/L vitamin A in S. cerevisiae was achieved in a 1-L bioreactor. This study unlocked the potential of S. cerevisiae for synthesising vitamin A in a sustainable and economical way, laying the foundation for the commercial-scale production of bio-based vitamin A.

High-yield magnetosome production of Magnetospirillum magneticum strain AMB-1 in flask fermentation through simplified processing and optimized iron supplementation.

Developing a simplified flask fermentation strategy utilizing magnetotactic bacterium AMB-1 and optimized iron supplementation for high-yield magnetosome production to address the challenges associated with magnetosome acquisition. A reliable processing for the pure culture of AMB-1 was established using standard laboratory consumables and equipment. Subsequently, the medium and iron supplementation were optimized to enhance the yield of AMB-1 magnetosomes. The mSLM supported higher biomass accumulation in flask fermentation, reaching an OD565 of ~ 0.7. The premixed solution of ferric quinate and EDTA-Fe (at a ratio of 0.5:0.5 and a concentration of 0.4mmol/L) stabilized Fe3+ and significantly increased the reductase activity of AMB-1. Flask fermentations with an initial volume of 15 L were then conducted employing the optimized fermentation strategy. After two rounds of iron and nutrient supplementation, the magnetosome yield reached 185.7 ± 9.5mg/batch (approximately 12mg/L), representing the highest AMB-1 flask fermentation yield to our knowledge. A flask fermentation strategy for high-yield magnetsome production was developed, eliminating the need for bioreactors and greatly simplifying the process of magnetosome acquisition.

Expression, characterization, and application of human-like recombinant gelatin

Gelatin is a product obtained through partial hydrolysis and thermal denaturation of collagen, belonging to natural biopeptides. With irreplaceable biological functions in the field of biomedical science and tissue engineering, it has been widely applied. The amino acid sequence of recombinant human-like gelatin was constructed through a newly designed hexamer composed of six protein monomer sequences in series, with the minimum repeating unit being the characteristic Gly-X-Y sequence found in type III human collagen α1 chain. The nucleotide sequence was subsequently inserted into the genome of Pichia pastoris to enable soluble secretion expression of recombinant gelatin. At the shake flask fermentation level, the yield of recombinant gelatin is up to 0.057 g/L, and its purity can rise up to 95% through affinity purification. It was confirmed in the molecular weight determination and amino acid analysis that the amino acid composition of the obtained recombinant gelatin is identical to that of the theoretically designed. Furthermore, scanning electron microscopy revealed that the freeze-dried recombinant gelatin hydrogel exhibited a porous structure. After culturing cells continuously within these gelatin microspheres for two days followed by fluorescence staining and observation through confocal laser scanning microscopy, it was observed that cells clustered together within the gelatin matrix, exhibiting three-dimensional growth characteristics while maintaining good viability. This research presents promising prospects for developing recombinant gelatin as a biomedical material.

DEVELOPMENT OF TECHNOLOGY FOR PILOT-SCALE PRODUCTION, DRYING, AND STORAGE OF EXTRACELLULARLY EXPRESSED RECOMBINANT AMY1974 ALPHA-AMYLASE

The α-amylase gene from Bacillus amyloliquefaciens MDC1974 was cloned into the pBE-S shuttle vector and expressed extracellularly using Bacillus subtilis RIK1285. Following the optimization of fermentation conditions, a pilot-scale production of 100 liters was conducted, achieving a maximum volumetric activity of 1969 U/ml, which is 1.4 times higher than the maximum activity obtained in flask fermentation. Additionally, enzyme concentration, drying, and preservation conditions were studied and optimized.

Engineering strategies for enhanced 1′, 4′-trans-ABA diol production by Botrytis cinerea

BackgroundCurrently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.ResultsIn this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1′,4′-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1′,4′-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1′,4′-trans-ABA-diol synthesis. To further increase the yield of 1′,4′-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1′,4′-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1′,4′-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.ConclusionsWe utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1′,4′-trans-ABA-diol. By overexpressing the crucial genes involved in the 1′,4′-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1′,4′-trans-ABA-diol.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica.

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

Diffusion-driven fed-batch fermentation in perforated ring flasks

PurposeSimultaneous membrane-based feeding and monitoring of the oxygen transfer rate shall be introduced to the newly established perforated ring flask, which consists of a cylindrical glass flask with an additional perforated inner glass ring, for rapid bioprocess development.Methods A 3D-printed adapter was constructed to enable monitoring of the oxygen transfer rate in the perforated ring flasks. Escherichia coli experiments in batch were performed to validate the adapter. Fed-batch experiments with different diffusion rates and feed solutions were performed.ResultsThe adapter and the performed experiments allowed a direct comparison of the perforated ring flasks with Erlenmeyer flasks. In batch cultivations, maximum oxygen transfer capacities of 80 mmol L−1 h−1 were reached with perforated ring flasks, corresponding to a 3.5 times higher capacity than in Erlenmeyer flasks. Fed-batch experiments with a feed reservoir concentration of 500 g glucose L−1 were successfully conducted. Based on the oxygen transfer rate, an ammonium limitation could be observed. By adding 40 g ammonium sulfate L−1 to the feed reservoir, the limitation could be prevented.Conclusion The membrane-based feeding, an online monitoring technique, and the perforated ring flask were successfully combined and offer a new and promising tool for screening and process development in biotechnology.

Saccharomyces cerevisiae cellular engineering for the production of FAME biodiesel

The unsustainable and widespread utilization of fossil fuels continues to drive the rapid depletion of global supplies. Biodiesel has emerged as one of the most promising alternatives to conventional diesel, leading to growing research interest in its production. Microbes can facilitate the de novo synthesis of a type of biodiesel in the form of fatty acid methyl esters (FAMEs). In this study, Saccharomyces cerevisiae metabolic activity was engineered to facilitate enhanced FAME production. Initially, free fatty acid concentrations were increased by deleting two acetyl-CoA synthetase genes (FAA1, FAA4) and an acyl-CoA oxidase gene (POX1). Intracellular S-adenosylmethionine (SAM) levels were then enhanced via the deletion of an adenosine kinase gene (ADO1) and the overexpression of a SAM synthetase gene (SAM2). Lastly, the S. cerevisiae strain overproducing free fatty acids and SAM were manipulated to express a plasmid encoding the Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). Using this combination of engineering approaches, a FAME concentration of 5.79 ± 0.56 mg/L was achieved using these cells in the context of shaking flask fermentation. To the best of our knowledge, this is the first detailed study of FAME production in S. cerevisiae. These results will provide a valuable basis for future efforts to engineer S. cerevisiae strains for highly efficient production of biodiesel.

Endophytic fungi of Panax sokpayensis produce bioactive ginsenoside Compound K in flask fermentation

Endophytes of Panax have the potential to produce their host plant secondary metabolites, ginsenosides. Panax sokpayensis, an endemic traditional medicinal plant of the Sikkim Himalayas was explored for the isolation of endophytic fungi. In the present study, we have isolated 35 endophytic fungal cultures from the rhizome of P. sokpayensis and screened for ginsenosides production by HPLC by comparing the peak retention time with that of standard ginsenosides. The HPLC analysis revealed that out of 35 isolates, the mycelial extracts of four fungal endophytes (PSRF52, PSRF53, PSRF49 and PSRF58) exhibited peaks with a similar retention time of the standard ginsenoside, Compound K (CK). LC–ESI–MS/MS analysis led to the confirmation of ginsenoside CK production by the four fungal endophytes which showed a compound with m/z 639.6278, similar to that of standard ginsenoside CK with yield in potato dextrose broth flask fermentation ranging from 0.0019 to 0.0386 mg/g of mycelial mass in dry weight basis. The four prospective fungal endophyte isolates were identified as Thermothielavioides terrestris PSRF52, Aspergillus sp. PSRF49, Rutstroemiaceae sp. strain PSRF53, and Phaeosphaeriaceae sp. strain PSRF58 based on ITS sequencing. The present finding highlights the need for further study on growth optimization and other culture parameters to exploit the endophytes as an alternative source for ginsenoside CK production.

Efficient expression of an alkaline pectin lyase from Bacillus licheniformis in Pichia pastoris

AbstractPectin lyase (PMGL) is an industrially important enzyme with widespread applications in the food, paper, and textile industries, owing to its capacity for direct degradation of highly esterified pectin. In this study, PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P. pastoris. Furthermore, diverse strategies, encompassing the optimization of expression cassette components, elevation of gene dosage, and co-expression of chaperone factors, were employed to augment PMGL-Ba production in P. pastoris. The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements. By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup, a strain yielding high PMGL-Ba production was achieved. In shake flask fermentation lasting 144 h, the total protein concentration reached 1.81 g/L, and the enzyme activity reached 1821.36 U/mL. For further scale up production, high-density fermentation transpired in a 5 L fermenter for 72 h. Remarkably, the total protein concentration increased to 12.49 g/L, and the enzyme activity reached an impressive 12668.12 U/mL. The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.

Achieving robust synthetic tolerance in industrial E. coli through negative auto-regulation of a DsrA-Hfq module

In industrial fermentation processes, microorganisms often encounter acid stress, which significantly impact their productivity. This study focused on the acid-resistant module composed of small RNA (sRNA) DsrA and the sRNA chaperone Hfq. Our previous study had shown that this module improved the cell growth of Escherichia coli MG1655 at low pH, but failed to obtain this desired phenotype in industrial strains. Here, we performed a quantitative analysis of DsrA-Hfq module to determine the optimal expression mode. We then assessed the potential of the CymR-based negative auto-regulation (NAR) circuit for industrial application, under different media, strains and pH levels. Growth assay at pH 4.5 revealed that NAR-05D04H circuit was the best acid-resistant circuit to improve the cell growth of E. coli MG1655. This circuit was robust and worked well in the industrial lysine-producing strain E. coli SCEcL3 at a starting pH of 6.8 and without pH control, resulting in a 250 % increase in lysine titer and comparable biomass in shaking flask fermentation compared to the parent strain. This study showed the practical application of NAR circuit in regulating DsrA-Hfq module, effectively and robustly improving the acid tolerance of industrial strains, which provides a new approach for breeding industrial strains with tolerance phenotype.

Metabolic Comparison and Molecular Networking of Antimicrobials in Streptomyces Species.

Streptomyces are well-known for producing bioactive secondary metabolites, with numerous antimicrobials essential to fight against infectious diseases. Globally, multidrug-resistant (MDR) microorganisms significantly challenge human and veterinary diseases. To tackle this issue, there is an urgent need for alternative antimicrobials. In the search for potent agents, we have isolated four Streptomyces species PC1, BT1, BT2, and BT3 from soils collected from various geographical regions of the Himalayan country Nepal, which were then identified based on morphology and 16S rRNA gene sequencing. The relationship of soil microbes with different Streptomyces species has been shown in phylogenetic trees. Antimicrobial potency of isolates was carried out against Staphylococcus aureus American Type Culture Collection (ATCC) 43300, Shigella sonnei ATCC 25931, Salmonella typhi ATCC 14028, Klebsiella pneumoniae ATCC 700603, and Escherichia coli ATCC 25922. Among them, Streptomyces species PC1 showed the highest zone of inhibition against tested pathogens. Furthermore, ethyl acetate extracts of shake flask fermentation of these Streptomyces strains were subjected to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis for their metabolic comparison and Global Natural Products Social Molecular Networking (GNPS) web-based molecular networking. We found very similar metabolite composition in four strains, despite their geographical variation. In addition, we have identified thirty-seven metabolites using LC-MS/MS analysis, with the majority belonging to the diketopiperazine class. Among these, to the best of our knowledge, four metabolites, namely cyclo-(Ile-Ser), 2-n-hexyl-5-n-propylresorcinol, 3-[(6-methylpyrazin-2-yl) methyl]-1H-indole, and cyclo-(d-Leu-l-Trp), were detected for the first time in Streptomyces species. Besides these, other 23 metabolites including surfactin B, surfactin C, surfactin D, and valinomycin were identified with the help of GNPS-based molecular networking.