Journal of Industrial Microbiology & Biotechnology最新文献

Designer cellulosomes are engineered multi-enzyme complexes inspired by natural cellulosomes, designed to improve lignocellulose breakdown. Their modular architecture enables the spatial colocalization of diverse catalytic activities, potentially enhancing depolymerization efficiency compared to free enzymes. Although conceptually promising, little is known about how they perform on complex lignocellulosic substrates. In this study, we developed a tetravalent designer cellulosome using a modular VersaTile assembly approach, incorporating endoglucanase, cellobiohydrolase, β-glucosidase, and endoxylanase activities. The process involved (i) delineating catalytic modules from Cellvibrio japonicus enzymes, (ii) generating docking enzyme variants via combinatorial cloning, and (iii) selecting optimal candidates based on expression, activity, and cohesin-dockerin binding before assembling them onto a scaffoldin with four cohesins and a cellulose-binding module. The resulting designer cellulosome was tested on two industrially relevant substrates: agro-industrial wheat fibers and genome-edited low-lignin poplar biomass under controlled laboratory conditions. It achieved cellulose-to-glucose conversion yields of 24.98% (150 pmol designer cellulosome/ml) and 0.82% (200 pmol designer cellulosome/ml), respectively, under the test conditions. By comparing the saccharification efficiencies of the enzymes in their free and complexed forms, we found that colocalization on a common scaffoldin significantly enhanced synergistic activity. This effect was most pronounced under low enzyme concentrations and when acting on complex lignocellulosic substrates, increasing glucose release compared to free enzymes. These observations highlight that the benefits of colocalization are substrate-dependent and occur under conditions that mimic the natural environment of biomass degradation, conditions that differ from typical industrial settings. This work advances our understanding of designer cellulosome behavior on real-world substrates, providing essential insights for evaluating their economic viability in industrial applications.

Specialized metabolites encoded by biosynthetic gene clusters (BGCs) in the oral microbiome remain largely unexplored in the context of oral health and disease. Previous genome-centric surveys have identified hundreds of uncharacterized BGCs in the oral cavity associated with health and disease, but these studies relied on reference genomes and did not capture strain-level variation or the native distribution of BGCs. Here, we assembled three independently sourced metagenomic datasets from healthy and dental caries samples, extracted BGCs, and quantified their metagenomic abundance and transcriptional activity. We found that aryl polyene, ribosomally synthesized and post-translationally modified peptide (RiPP), and nonribosomal peptide (NRP) encoding BGCs were the most prominent BGCs identified across the three metagenomic datasets. We grouped the identified BGCs into homology-based gene cluster families (GCFs) and found that specific GCFs were consistently associated with either health or caries across diverse taxa, suggesting that some specialized metabolites may perform conserved ecological functions. Conversely, other BGCs showed more restricted taxonomic distributions and were linked to disease-associated taxa, such as Propionibacterium acidifaciens, suggesting niche-specific biosynthetic capacities within the oral environment. Applying elastic-net regression to the metatranscriptomic dataset further identified a subset of 51 BGCs out > 3 000 that distinguished healthy from caries samples, reinforcing the discriminatory power of BGC expression patterns. Together, these results demonstrate that BGCs provide functional resolution beyond taxonomic profiling and that BGC expression, rather than genomic presence alone, differentiates oral microbial community states. This underscores the relevance of specialized metabolism to oral health and supports the use of BGC-centric analyses to interrogate microbial interactions underlying community stability and disease-associated shifts.

Microbial synthesis of carotenoids has garnered significant attention as an eco-friendly alternative to conventional synthetic methods and its facile extraction for impressive yield. This study delves into the efficacy of carotenoid production from a red yeast strain, as well as the biodiversity of yeast species from Thai flowers. The research involved the collection of flower samples within Thailand, along with 12 yeast species from 10 genera of Ascomycetes and 4 genera of Basidiomycetes which were isolated by identifying the D1/D2 domain of the large subunit rRNA gene. Unexpectedly, Rhodotorula paludigena SWU-FKT03 emerged as the top performing yeast strain, boasting an impressive carotenoid production rate of 183.30 ± 5.00 mg/L among the 36 red yeast strains isolated. Subsequently, a further investigation was performed, focusing on optimized culture conditions for carotenoid production from this yeast strain. The results were promising, as carotenoid production surged to 288.27 mg/L when 20 g/L of glucose and 10 g/L of monosodium glutamate served as the carbon and nitrogen sources, respectively. These findings underscore the potential of the R. paludigena SWU-FKT03 as a high-yield carotenoid producer when cultivated in shaking flasks, exhibiting a three-fold increase in carotenoid content when under optimized conditions. These results hint at the potential of this approach for future large-scale carotenoid production. One-sentence summary A novel red yeast, Rhodotorula paludigena SWU-FKT03, isolated from Thai floral ecosystems, demonstrated high-yield carotenoid production of 288.27 mg/L after fermentation optimization, establishing a significant potential for industrial applications.

Here, we present the first complete, fully phased diploid genome of type strain Yarrowia lipolytica YB-423 (=ATCC 18942™), constructed using a combination of Oxford Nanopore long-read and Illumina short-read sequencing. Y. lipolytica is an industrially relevant yeast species known for its metabolic versatility, particularly its ability to degrade hydrophobic compounds and express useful products such as fatty acids. Despite its growing use in biotechnology, a high-quality genome assembly of the species' diploid type-strain has been lacking. The assembly and annotations presented here span six chromosomes of paired "haplotigs" and a mitochondrial genome, capturing large-scale structural variations and prominent levels of genome-wide heterozygosity. Variant analysis revealed 13,908 heterozygous alleles, of which 3,201 alleles were distributed among 1,237 protein-coding genes. Gene set enrichment analysis showed that these variants are enriched among genes involved in transmembrane transport, suggesting a role in environmental adaptability. Comparative analysis of matched haplotigs for the same chromosome uncovered multiple inversions and transpositions, as well as allele-specific insertions of retrotransposons, providing new insights into the structural complexity and evolutionary dynamics of the genome. The fully phased, finished diploid genome of Y. lipolytica YB-423 represents a crucial step toward unlocking the full genetic potential of Y. lipolytica. Our work will provide a valuable foundation for future comparative and functional genomics and strain engineering studies, particularly for industrial microbiology and biotechnology applications.

Successful design of an industrial biological product such as a vaccine requires an efficient, robust and reproducible process. In this study, we investigated conditions for process optimization of a promising recombinant factor H binding protein (fHbp)-Porin A (PorA) chimeric protein-based vaccine candidate against Meningococcal B serogroup in Escherichia coli B834 strain using the inducible T7-lac promoter system. Random screening of components of complex culture media for growth and expression using IPTG as an inducer, showed inconsistency when analyzed using Plackett-Burman design (PBD). Further analysis identified galactose present either in tryptone (as lactose) or soy-phytone, has caused strong autoinduction and is surmised as an interfering factor for IPTG induction. Synergistic combination of soy-phytone and yeast extract under conditions of controlled growth and auto-induction gave a good correlation between the cell growth and expression of the fHbp-PorA chimeric protein when evaluated using PBD and central composite design. Using response optimization conditions, an optimized media was attained. A shake flask study was performed to validate the optimized media, and later, a fed-batch fermentation at 10 L scale was established to prove the scalability, consistency, and product quality using the optimized media. One-sentence summary This study reports optimization of cell growth and expression conditions for a recombinant chimeric meningococcal protein in E. coli that assures its industrial scale production and suitability for preclinical and clinical studies as a vaccine component against Meningococcal B serogroup.

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-48190 (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.

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.