Journal of Industrial Microbiology & Biotechnology最新文献

Although the α-amylase (AmyS) from Geobacillus stearothermophilus exhibits high thermostability, its low enzymatic activity (44.36 ± 2.02 U/mL) severely hinders industrial applications. Given its strong protein secretion capability and GRAS (generally recognized as safe) status, this study employed Bacillus subtilis as the host to enhance AmyS production. Through error-prone PCR and high-throughput screening, a triple mutant T151A/K178E/T458A (AmySM) was generated, showing a 17.67% increase in activity (51.34 ± 1.11 U/mL). AmySM activity was further increased by 29.32% to 65.23 ± 2.33 U/mL using the signal peptide SPykwD, selected from a comprehensive library for superior secretion efficiency. The promoter PgsiB enhanced activity by 37.61% to 89.54 ± 2.95 U/mL. Optimizing the ribosome binding site (RBS) resulted in an additional 48.83% increase in activity, yielding a final activity of 134.02 ± 3.54 U/mL, which corresponds to a 3.02-fold improvement over the initial strain WBSW (44.36 ± 2.02 U/mL). Ultimately, scale-up fermentation in a 5-L bioreactor yielded a maximum extracellular activity of 1244.17 ± 48.66 U/mL at 72 hours, a 2.84-fold increase over the control. This multi-level strategy provides a rational framework for high-efficiency AmySM production, paving the way for extracellular production of high-value proteins in the GRAS host B. subtilis.

Rhodotorula toruloides is a red oleaginous yeast with growing commercial interest because of its hardiness and exceptional lipid production capacity. Because it is a basidiomycete yeast with a complex life cycle, many of the classical breeding methods used with ascomycetes are unavailable for strain improvement. However, we have been able to construct polyploid yeast by fusing protoplasts of parents with the same mating type. Fusing of Y-6985 (A2) and Y- 48 190 (A2), which had been transformed with complementary antibiotic markers, led to the recovery of two diploids and one triploid. The stability of the fusion yeasts was tested by plating them on non-selective medium after several growth cycles under antibiotics and then testing five colonies per strain for nuclear DNA contents using flow cytometry and standard cell cycle analysis: the triploid and one diploid were stable. Fusants inherited their mitochondria from a single parent, which was demonstrated using restriction fragment length polymorphism (RFLP) of mitochondrial DNA. The phenotypic properties of the parents and fusants were compared in glucose fed-batch bioreactor studies and cellulosic sugar batch cultures. The final lipid titers for the fed-batch cultures were 24.9-39.7 g/L with Y-6985 and the diploid and triploid performing the best and worst, respectively. The fusants demonstrated intermediate hardiness for growth on hydrolysate prepared with dilute-acid pretreated switchgrass and were outperformed by Y-48190. Unlike one of the haploid parents, the fusants grew in 70%v/v concentrated hydrolysate. However, they did not grow as fast as the other haploid. In this study, a modernized protoplast fusion method is resurrected a useful tool for strain development in this yeast, which is complementary with other available methods.

Epoxyquinoids are a unique class of natural products featuring an epoxide embedded within a quinone/quinol scaffold, typically as an epoxycyclohexenone (ECH). Their striking stereochemical diversity arises from multiple permutations of epoxide and hydroxyl configurations across the epoxyquinol and epoxyhydroquinone families. These highly oxygenated cores contain contiguous stereocenters and reactive functionalities, and their structural diversity correlates with broad bioactivity spanning antibacterial, antifungal, antiparasitic, anti-inflammatory, and antiproliferative effects. A shared epoxide-quinone (or keto-epoxide) pharmacophore acts as an electrophilic warhead that covalently engages protein nucleophiles via Michael addition and epoxide opening, underpinning diverse modes of action. Recent biosynthetic advances have uncovered tailoring enzymes with unusual catalytic strategies that forge the epoxide, install additional functionality, and tune oxidation states through redox chemistry. This review highlights current knowledge of experimentally characterized pathways and enzyme functions across Actinobacteria, Ascomycota, and Basidiomycota, illuminating common logic and organism-specific innovations in epoxyquinoid assembly.

Indigo is a plant-based natural blue dye that can be produced via chemical synthesis and biological pathways. However, the toxic reduction processes and intracellular production of indigo through microbial metabolism are often limited by insolubility of indigo and complex downstream processing, causing environmental issues in the dyeing processes. Additionally, indican, a precursor of indigo with a glucose moiety, is highly soluble and can be easily converted into indoxyl by β-glucosidase, forming indigo under mild conditions. We constructed an indican-producing strain Escherichia coli BL21 HI201 by introducing a UDP-glycosyltransferase (ugt) into an indoxyl production system containing tryptophanse (tnaA) and flavin-containing monooxygenase (FMO) genes, enabling conversion of tryptophan into indican. Testing of the effect by various carbon sources suggested that glucose is one of the major factors affecting the ratio of indigo to indican, and increase in glucose concentration to more than 1.5% could produce sole indican without indigo. Under optimal conditions, E. coli BL21 HI201 biosynthesized 5.65 mM indican from tryptophan. Additionally, after deletion of various β-glucosidase genes, the bglA knockout strain E. coli BL21 HI204 produced more indican, achieving 6.79 mM after 24 hr of cultivation. This study demonstrated the strategic production of indican through the installation of a production system, deletion of a byproduct pathway, and control of glucose concentration.

One-sentence summary: This paper demonstrates the strategic enhancement of indican production in genetically engineered Escherichia coli BL21 by optimizing metabolic pathways and controlling glucose concentrations.

According to the Food and Agricultural Organisation 2024 statement, developing single-cell protein technology is important to reduce the burden on conventional feed protein production sectors. In this regard, improved commercial strains rich in amino acids, especially Lys and Met, may provide a sustainable alternative source of protein in aquaculture diets. The developed and laboratory-validated methodology for creating protein-synthesizing mutants will strengthen the competitiveness of SCP production technology. The present work provides unique results on improving the protein-producing properties of wild-type Phaffia rhodozyma DSM 5626 by mutagenesis and screening on herbicide-containing medium as a selective agent for amino acid biosynthesis inhibition. Inhibitory concentrations of pure herbicide actives were determined for S-(2-aminoethyl)-L-cysteine (AEC) hydrochloride and glufosinate-ammonium (GA) for complete inhibition and strong inhibition of the DSM 5626 strain. GA at a concentration of 50 mM and 100 mM and AEC at 0.5 mM and 2.5 mM were chosen for mutant selection after chemical mutagenesis. The use of herbicides resulted in the selection of mutants with significantly improved synthesis of Met and Lys. Specifically, mutants GA6/4 and GA7/5 exhibited 37% and 26% higher Met levels, respectively, while GA6/3 had a 14% increase in Lys compared to the wild-type strain. The AEC3/9 mutant demonstrated a 35% increase in Met, 24% in Lys, 8% in Ile, and 6% in Phe, underscoring the efficacy of this screening approach in enhancing essential amino acid content. The protein quality parameters essential amino acid index and amino acid score of these mutants became higher compared with commercial strains of SCP yeast such as C. utilis, S. cerevisiae, K. marxianus, etc.

One-sentence summary: Mutagenesis combined with selective screening using herbicides is an effective approach to enhancing amino acid biosynthesis in yeast.

L-Arginase is a therapeutic enzyme that hydrolyzes L-arginine to ornithine and urea. The L-arginase extracted from bacteria has an anticancer activity by causing starvation of nutrients for cancer cells. This study aimed to screen and characterize L-arginase-producing bacteria and to optimize different factors influencing L-arginase production. Isolation and primary screening were carried out by using mineral arginine agar media using phenol red as an indicator. Molecular identification of the isolates was employed by using 16S ribosomal RNA sequencing and phylogenetic tree construction. L-Arginase assay by colorimetric method was carried out to measure the amount of urea liberated from the hydrolysis of L-arginine for quantitative screening. From 31 water samples, 102 colonies were isolated, and those colonies that convert the media to pink were selected as arginase-producing bacteria. 7 isolates were screened from qualitative screening method. Based on quantitative screening, the highest L-arginase was produced from bacteria Alcaligenes aquatilis BC2 (92.46 ± 0.19 U/ml) followed by Paenalcaligenes suwonensis BCW8 (59.29 ± 0.66 U/ml). Following their mean difference, isolate BC2 was selected for further optimization process of 8 parameters. After optimization, the isolate shows the maximum (163.85 U/ml) enzyme activity. The result of this study implies that novel bacteria were isolated from soda lakes that produce a considerable amount of L-arginase, which can be used as a promising anticancer activity. One-Sentence Summary: This study successfully isolated and characterized a novel L-arginase-producing bacterium, Alcaligenes aquatilis BC2, from Ethiopian soda lakes and optimized its enzyme production parameters for potential anticancer applications.

Rubromycins are bacterial aromatic polyketides containing a hallmark spiroketal pharmacophore produced by type II polyketide synthases and accessory enzymes. They generally display cytotoxic and antimicrobial properties, frequently disrupting cellular processes and proteins associated with nucleic acids, such as DNA helicase or telomerase. Among the known rubromycin congeners, hyaluromycin stands out due to a 2-amino-3-hydroxycyclopent-2-enone (C5N) substitution that is presumably installed by an amide bond synthetase (ABS). Here, we used bioinformatic analysis to identify uncharacterized biosynthetic gene clusters and potential rubromycin producer strains encoding putative ABSs but lacking the enzymes responsible for C5N formation, suggesting potentially novel substituents. One of these strains, Lentzea tibetensis, was successfully cultivated and confirmed to produce a previously undescribed aminocoumarin-substituted rubromycin polyketide, named coumarubrin, as verified by high-resolution mass spectrometry (HRMS) and comprehensive nuclear magnetic resonance (NMR) spectroscopy. Electronic circular dichroism spectroscopy indicates an absolute configuration identical to that of previously characterized rubromycins, while the first bioactivity assays demonstrated potent inhibitory activity against Gram-positive bacteria. One-Sentence Summary: This study reports the discovery of a novel member of the rubromycins, antibiotic and cytotoxic aromatic polyketides produced by Actinobacteria, which is fused to a distinct aminocoumarin moiety.

Glucosamine (GlcN) and GlcN-based supplements, e.g. glucosamine hydrochloride, glucosamine sulfate, and N-acetyl glucosamine (GlcNAc), provide symptomatic relief to osteoarthritis patients and have been used as one of the most popular nutraceuticals. To meet the increasing demands, scientists have explored cost-effective methods for GlcN and GlcNAc production using low-cost raw materials such as seafood waste. However, the commercially available GlcN and GlcNAc production methods are environmentally harmful because of the use of toxic reagents. Moreover, the raw material used might be unsafe for consumers with shrimp allergies. On the other hand, bio-based GlcN production is gaining popularity because of its eco-friendly production approach and optimum reaction conditions. In this mini-review, we will discuss the recent developments to produce GlcN and GlcNAc through (1) the chemical and enzyme-mediated approaches of crude chitin hydrolysis, primarily obtained from shrimp and crabs; (2) the whole cell-based systems for fungal derived chitin bio-transformation and fungal fermentation; and (3) the metabolic engineering and the adaptive evolution based microbial biocatalyst for a balanced cell growth and optimal production of GlcN and GlcNAc. One-Sentence Summary: This article summarizes the mechanism of glucosamine and N-acetyl glucosamine production using bacteria, fungi, and chemical processes.

In spite of wonderful industrial applications of microbial enzymes, still most of habitats in various parts of world are unexplored for bioprospecting of industrial potent microbes. This study represents the first bioprospecting effort in Layyah district to explore indigenous bacterial diversity, enzymatic potential, and phylogenetic relationships in untapped contaminated soil habitat. Contaminated soils serve as reservoirs of industrially significant bacteria with unique enzymatic degradation capabilities, offering solutions for sustainable industrial applications and environmental remediation. An effort for comparative bioprospecting-based study for bacterial diversity exhibiting amylase potential across unaddressed contaminated soil samples [industrial, household, poultry, and animal waste (AW)] using qualitative and quantitative methods, was conducted. AW-contaminated soil exhibited the highest bacterial load (2.51 × 1010 CFUs) and amylase activity (51, amylase zones), whereas industrial waste soil showed the lowest CFUs (1.24 × 1010). Household waste soil, however, displayed the greatest Shannon diversity index (H'= 2.192262) for amylase-producing bacteria. Among isolates, Priestia flexa AW3 (OQ446563) demonstrated exceptional amylase production, forming 30 mm hydrolysis zones on starch agar and achieving optimal activity (1.76 ± 0.05 OD; 1.23 ± 0.03 AU/mL) at pH 7 and 37°C after 48 h. Notably, the strain retained enzymatic stability under extreme conditions, temperature up to 50°C, NaCl concentrations (0.5%-10%), and a broad pH range. Phylogenetic analysis via 16S rRNA sequencing confirmed its identity as P. flexa. This study underscores the untapped potential of contaminated soils in Layyah as sources of robust industrial microbes and highlights the value of bioprospecting in discovering novel bacterial strains for biotechnology and environmental sustainability. One-Sentence Summary: Bioprospecting for industrially important bacteria with unique enzymatic potential from untapped habitats is highly needed to solve sever environmental problems.

Biocatalysis provides access to synthetically challenging molecules and commercially and pharmaceutically relevant natural product analogs while adhering to principles of green chemistry. Cytochromes P450 (P450s) are among the most superlative and versatile oxidative enzymes found in nature and are desired regio- and stereoselective biocatalysts, particularly for structurally complex hydrocarbon skeletons. We used 10 genome-sequenced Streptomyces strains, selected based on their preponderance of P450s, to biotransform the bioactive diterpenoid abietic acid. We isolated and structurally characterized seven oxidized abietic acid derivatives from three different strains, including four products that are new bacterial biotransformants or enzymatic products. Oxidations (hydroxylation, dehydrogenation, and aromatization) were seen on both the B and C rings of abietic acid and five products had multiple modifications. Notable conversions observed in the study were that of abietic acid to 15-hydroxy-7-oxo-8,11,13-abietatrien-18-oic acid, 7, which involves multiple hydroxylation reactions and dehydrogenation. The findings from this study will lead to identifying P450s or other enzymes that may act as general biocatalysts to modify abietanes and other labdane-type diterpenoid skeletons.

One-sentence summary: Genome-guided biotransformation of the bioactive diterpenoid abietic acid in Streptomyces yielded seven oxidized derivatives including four that have not been previously seen from bacteria.