High Amylase Production by a Novel Strain of Bacillus amyloliquefaciens M37 Isolated from Can Gio Mangrove Forest, Vietnam

Amylases are one of the most important industrial enzymes and find applications in many areas such as textiles, chemicals, food, and pharmaceuticals. Most of the amylases are derived from microbes. The objective of the present study was to evaluate amylase production by a bacterium isolated from the Can Gio mangrove forest. The bacterium was identified as a species of genus Bacillus based on morphological and biochemical characteristics. The analysis of 16S rRNA sequences was then confirmed that this strain belonged to Bacillus amyloliquefaciens species (100% similarity). The effect of culture conditions such as temperature, pH, and carbon sources on amylase production through shake-flask culture was investigated. Maximum amylase activity of 904 IU/mL was obtained after 24 h of cultivation in LB medium containing 1% soluble starch at 35oC and pH 7.0. The highest enzyme activity of 1279 IU/mL was achieved in the bioreactor after 30 h of cultivation at optimum conditions. In addition, B. amyloliquefaciens M37 can grow on soybean meal medium. The high bacterial cell number of 456 × 109 CFU/g and amylase activity of 1039 IU/g were obtained after 36 h of cultivation. This newly isolated B. amyloliquefaciens M37 could be a potential producer for industrial amylase production and probiotics with commercial implications.

Improvement of FK506 production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis

Background FK506, a macrolide mainly with immunosuppressive activity, can be produced by various Streptomyces strains. However, one of the major challenges in the fermentation of FK506 is its insufficient production, resulting in high fermentation costs and environmental burdens. Herein, we tried to improve its production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis. Results First, basing on the genome sequencing and analysis, putative competitive pathways were deleted. A better parental strain L19-2 with increased FK506 production from 140.3 to 170.3 mg/L and a cleaner metabolic background was constructed. Subsequently, the FK506 biosynthetic gene cluster was refactored by in-situ promoter-substitution strategy basing on the regulatory circuits. This strategy enhanced transcription levels of the entire FK506 biosynthetic gene cluster in a fine-tuning manner and dramatically increased the FK506 production to 410.3 mg/mL, 1.41-fold higher than the parental strain L19-2 (170.3 mg/L). Finally, the FK506 production was further increased from 410.3 to 603 mg/L in shake-flask culture by adding L-isoleucine at a final concentration of 6 g/L. Moreover, the potential of FK506 production capacity was also evaluated in a 15-L fermenter, resulting in the FK506 production of 830.3 mg/L. Conclusion From the aspects of competitive pathways, refactoring of the FK506 biosynthetic gene cluster and nutrients-addition, a strategy for hyper-production and potentially industrial application of FK506 was developed and a hyper-production strain L19-9 was constructed. The strategy presented here can be generally applicable to other Streptomyces for improvement of FK506 production and streamline hyper-production of other valuable secondary metabolites.

Anthocyanin extract from Lycium ruthenicum enhanced production of biomass and polysaccharides during submerged fermentation of Agaricus bitorquis (Quél.) Sacc. Chaidam

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC) is a wild edible fungus uniquely found in the Tibet Plateau. ABSC is rich in polysaccharides that are considered biologically active. This study aimed to determine the feasibility of enhancing exopolysaccharide (EPS) production by ABSC in shake flask culture by supplementing the fermentation medium with anthocyanin extract. Different concentrations of Lycium ruthenicum Murr. (LRM) anthocyanin crude extract were tested on ABSC fermentation. The activity of phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM), and phosphomannose isomerase (PMI), enzymes presumably involved in EPS synthesis by ABSC, was determined. ABSC transcriptomic profile in response to the presence of anthocyanins during fermentation was also investigated. LRM anthocyanin crude extract (0.06 mg/mL) was most effective in increasing EPS content and mycelial biomass (by 208.10% and 105.30%, respectively, P < 0.01). The activity of PGI, PGM, and PMI was increased in a medium where LRM anthocyanin extract and its main components (proanthocyanidins and petunia anthocyanin) were added. RNA-Seq analysis showed that 349 genes of ABSC were differentially expressed during fermentation in the medium containing anthocyanin extract of LRM; 93 genes were up-regulated and 256 genes down-regulated. From gene ontology enrichment analysis, differentially expressed genes were mostly assigned to carbohydrate metabolism and signal transduction categories. Collectively, LRM anthocyanins extract positively affected EPS production and mycelial biomass during ABSC fermentation. Our study provides a novel strategy for improving EPS production and mycelial growth during ABSC liquid submerged fermentation.

Large scale production of indole-3-acetic acid and evaluation of the inhibitory effect of indole-3-acetic acid on weed growth

Indole-3-acetic acid (IAA) is the most common plant hormone of the auxin class and regulates various plant growth processes. The present study investigated IAA production by the basidiomycetous yeast Rhodosporidiobolus fluvialis DMKU-CP293 using the one-factor-at-a-time (OFAT) method and response surface methodology (RSM). IAA production was optimized in shake-flask culture using a cost-effective medium containing 4.5% crude glycerol, 2% CSL and 0.55% feed-grade l-tryptophan. The optimized medium resulted in a 3.3-fold improvement in IAA production and a 3.6-fold reduction in cost compared with those obtained with a non-optimized medium. Production was then scaled up to a 15-L bioreactor and to a pilot-scale (100-L) bioreactor based on the constant impeller tip speed (Vtip) strategy. By doing so, IAA was successfully produced at a concentration of 3569.32 mg/L at the pilot scale. To the best of our knowledge, this is the first report of pilot-scale IAA production by microorganisms. In addition, we evaluated the effect of crude IAA on weed growth. The results showed that weed (Cyperus rotundus L.) growth could be inhibited by 50 mg/L of crude IAA. IAA therefore has the potential to be developed as a herbicidal bioproduct to replace the chemical herbicides that have been banned in various countries, including Thailand.

Integration of Proteomics and Metabolomics Into the Design, Build, Test, Learn Cycle to Improve 3-Hydroxypropionic Acid Production in Aspergillus pseudoterreus

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4 × after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

Production of Indole-3-Acetic Acid: A White Biotechnology for Weed Biocontrol

Indole-3-acetic acid (IAA) is the most common plant hormone of the auxin class and regulates various plant growth processes. The present study investigated IAA production by the basidiomycetous yeast Rhodosporidiobolus fluvialis DMKU-CP293 using the one-factor-at-a-time (OFAT) method and response surface methodology (RSM). IAA production was optimized in shake-flask culture using a cost-effective medium containing 4.5% crude glycerol, 2% CSL and 0.55% feed-grade L-tryptophan. The optimized medium resulted in a 3.3-fold improvement in IAA production and a 3.6-fold reduction in cost compared with those obtained with a non-optimized medium. Production was then scaled up to a 15-L bioreactor and to a pilot-scale (100-L) bioreactor based on the constant impeller tip speed (Vtip) strategy. By doing so, IAA was successfully produced at a concentration of 3,569.32 mg/L at the pilot scale. To the best of our knowledge, this is the first report of pilot-scale IAA production by microorganisms. In addition, we evaluated the effect of crude IAA on weed growth. The results showed that weed (Cyperus rotundus L.) growth could be inhibited by 50 mg/L of crude IAA. IAA therefore has the potential to be developed as a herbicidal bioproduct to replace the chemical herbicides that have been banned in various countries, including Thailand.

Production of chemotherapeutic enzyme L-asparaginase from fungal source

Background: L-Asparaginase is an antineoplastic agent used in the treatment of acute myeloid and acute lymphoblastic leukemia. The present study deals with the production of this chemotherapeutic enzyme drug from Aspergillus flavus NCIM 526. The production of enzymes was carried out using oil-extracted cakes in shake flask culture. Process parameters like carbon and nitrogen sources were taken into account. Methods: A total of six isolates were used to screen out efficient microorganisms for enzyme production. Aspergillus flavus NCIM 526 exhibited 138 IU/ml of enzyme activity in oil extracted mix cake after 96 hours of the incubation period. Molasses and l-asparagine were proved the best carbon and nitrogen sources for enzyme production. The enzyme was purified by column chromatography and the finest enzyme exhibited specific activity of 28 IU/mg. Results and Discussion: The fungal enzyme exhibited low Km values as compared with standard E. coli L-asparaginase, proving more substrate affinity of fungal enzyme than bacterial enzymes. Conclusion: The study explored the Aspergillus flavus NCIM 526 as a potential fungal source for high yield production of antileukemic enzyme drugs.

Offloading Role of a Discrete Thioesterase in Type II Polyketide Biosynthesis

ABSTRACT Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KSα), chain length factor (KSβ), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KSα or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

Optimization of L-asparaginase production from Escherichia coli using response surface methodology

Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. In previous study, the L-asparaginase from Erwinia chrysanthermy was expressed in Escherichia coli BL21(DE3). The recombinant L-asparaginase was produced from recombinant E.coli BL21(DE3) under different cultivation conditions (inducer concentration, inoculum concentration and KH2PO4 concentration). The optimized conditions by response surface methodology using face centered central composite design. The analysis of variance coupled with larger value of R2 (0.9) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced L-asparaginase activity of 123.74 U/ml at 1.03 mM IPTG, 3% (v/v) inoculum and 0.5% (w/v) KH2PO4. Recombinant protein was purified by two step using gel filtration and DEAE chromatography. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 462 U/mg and identified by MALDI-TOF mass spectrometry. Results of MALDI-TOF analysis confirmed that recombinant protein was L-asparaginase II. Recombinant L-asparaginase has antiproliferative activity with K562 cell line. In conclusion, this study has innovatively developed cultivation conditions for better production of recombinant L-asparaginase in shake flask culture.