Microbial Cell Factories最新文献

Background: Friedelin is a pharmacologically valuable pentacyclic triterpenoid with anti-inflammatory, anticancer, antiviral, and antiobesity properties. Conventional methods of friedelin production rely on solvent-intensive extraction from plant biomass, which is often expensive, inefficient, and environmentally unsustainable. Phaeodactylum tricornutum, a model marine diatom with a unique chimeric sterol biosynthetic pathway and native oxidosqualene accumulation, presents a promising platform for heterologous triterpenoid biosynthesis.

Results: In this study, the TwOSC4 gene from Tripterygium wilfordii, which encodes friedelin synthase, was successfully expressed in P. tricornutum via biolistic transformation. As a result, two transgenic strains, Pt-OSC4-20 and Pt-OSC4-48, were confirmed to express TwOSC4 at both transcript and protein levels. Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) analysis validated friedelin production in these strains, with a baseline accumulation of 26 ng/mL under standard conditions. Upon treatment with Ro 48-8071, an oxidosqualene cyclase inhibitor that suppresses endogenous sterol biosynthesis, friedelin production increased up to 55 ng/mL. Nile red staining revealed increased lipid droplet formation in the transgenic strains, suggesting the possible intracellular storage of friedelin. Importantly, transgene expression did not impair cell growth, indicating the metabolic compatibility of the host with exogenous triterpenoid synthesis.

Conclusions: This is the first study to demonstrate successful biosynthesis of friedelin in a microalgal system, highlighting the potential of P. tricornutum as a sustainable phototrophic chassis for the production of plant-derived triterpenoids. Unlike yeast-based systems, which require extensive metabolic amplification, P. tricornutum enables simpler genetic engineering and simultaneous coproduction of valuable compounds, such as fucoxanthin and Omega-3 Eicosapentaenoic Acid (EPA). These findings lay the groundwork for further strain optimization aimed at increasing friedelin yield and broadening the scope of triterpenoid biosynthesis in microalgae.

Background: Foot-and-mouth disease (FMD) is a highly infectious disease that mainly affects cloven-hoofed animals. The rapid spread of this disease hinders the success of control measures. This necessitates the urgent development of a vaccine to ensure the safe rearing of affected animals. However, traditional vaccines have some drawbacks including partial protection against persistent infection and a lack of cross-protection against different strains.

Results: We developed an oral vaccine that targets VP1, the major immunogenic protein of the causative FMD virus (FMDV). We expressed this protein using the surface display method and subsequently analyzed its immune efficacy. Western blot analysis and confocal imaging confirmed that our constructs effectively expressed VP1 on the surface of yeast cells. However, the expression of surface-displayed VP1 was substantially lower than that of intracellular VP1. The immune response of mice fed with cells expressing surface-displayed VP1 was greater than that of mice fed with an equal number of cells expressing intracellular VP1.

Conclusions: Our study indicates that yeast surface expressed VP1 elicits a superior immune response against FMDV than intracellularly expressed VP1 for immune response against FMDV, which offers a potential breakthrough in the effort to provide a simple and highly effective oral vaccine.

Background: Corn bran arabinoxylan (CBAX) is one of the most structurally complex xylans in nature. The bioconversion of CBAX into value-added products remains challenging because the substrate is resistant to pure xylanases and commercial enzyme cocktails. The carbohydrate-active enzymes (CAZymes) of Penicillium parvum 4-14 have been shown to efficiently hydrolyze CBAX. This study aimed to investigate the expression patterns and functional roles of CAZymes involved in the degradation of complex arabinoxylans in the fungus using transcriptomic and proteomic technologies.

Results: P. parvum 4-14 grew on CBAX and corn cob arabinoxylan (CCAX) with different substitution patterns and produced secretomes with varied compositions. The CBAX- or CCAX-induced fungal secretomes showed similar ratios (76.2% and 75.1%) on monosaccharide release from CBAX, but the former has a 12% higher ratio on monosaccharide release from CCAX than the latter. Integrated transcriptomic and proteomic analyses revealed distinct patterns of functional gene expression and CAZyme secretion in P. parvum cells induced by the two types of arabinoxylans, implying that the fungus has a complex regulatory system for CAZyme synthesis. A total of 26 CAZymes were inferred to be involved in the degradation of CBAX on the basis of multi-omics data and substrate structures. At the same fungal growth stage (48 h), 24 of these 26 CAZyme showed 0.42- to 5.74-fold higher gene transcription levels under CBAX culture than under CCAX culture.

Conclusions: Different sources of arabinoxylans significantly affect the production of extracellular CAZymes in P. parvum. These findings are valuable for understanding the key CBAX-degrading enzymes and engineering tailored enzyme systems to valorize complex hemicelluloses.

Despite the ability of mega synthases to construct complex structures with high precision, the main product is often accompanied by minor congeners because of relaxed selectivity of biosynthetic steps. In the case of polyketides, these may arise from promiscuity of acyltransferase domains while selecting the extender building blocks. This yields compounds which can differ only slightly from the main product and are therefore notoriously difficult to separate. For compounds in medicinal use, such congeners are considered impurities and must be precisely quantified in commercial production through use of reference standards. The immunosuppressants FK506, FK520, and its chlorinated derivate pimecrolimus, are well known for accumulation of minor congeners in the fermentation, which demands costly and lengthy purification with industrial HPLC to obtain material of sufficient purity. The relaxed selectivity of the AT domain in module 4 of FK506/520 PKS results in production of FK506, FK506D, FK520, and FK523 from the same system. Here, we investigate additional minor impurities, specifically the 11-, 17-, and 19-ethyl FK520 derivatives. Because the PKS can process these analogues to completion, it is reasonable to expect that strategic replacement of AT domains in modules 9, 6, and 5 (respectively), would enhance their production. Indeed, we show a direct, exclusive and high titer biosynthesis of these analogues through PKS engineering, replacing tedious and costly isolation by preparative HPLC and significantly improving access to these minor congeners. In addition, by using the AT4 as the donor domain, an interesting system is obtained, containing identical promiscuous AT domains at two different positions, and consequently distinct intra- and intermodular context. Media-dependent extender pool modulation toward methylmalonyl-CoA showed unexpected but consistent distribution of the four predicted analogues for each of the engineered systems. Despite the use of identical ATs, extender incorporation preference is different, depending on the modular context. It appears that the selectivity of the downstream AT4-containing module depends on the structural features of the incoming acyl chain; features installed by a module well upstream in the assembly line. Therefore, the global context of the PKS may be more impactful to the outcome of AT-engineering experiments than is generally considered.

Background: Talaromyces sp., as powerful cellulolytic fungi, display a nearly complete enzymatic system, which exhibits a complex regulatory system involving multiple transcription factors to control the expression of cellulase genes. However, there are fewer studies on the transcriptional regulatory factors of cellulase expression in Talaromyces sp. This study provides a basis for in-depth analysis of the transcription regulatory mechanism of Talaromyces endophyticus for cellulase expression, offering new ways for the preparation of fermented sugars using straw resources to produce high value-added products and biofuels.

Results: In this study, a novel transcription factor was investigated for T. endophyticus NEAU-6 which was a high cellulase-producing strain. Results revealed that TeSrdA was nuclear protein, which was stably expressed in the nucleus. TeSrdA deletion caused a remarkable increase of the activities of FPase (121.1%), CMCase (36.6%), pNPCase (97.0%), β-glucosidase (75.1%) and Xylanase (98.4%). Microscopic analysis showed that ΔTeSrdA strains increased the length of the hyphae and the number of branching. Notably, TeSrdA deletion could accelerated the microbial growth, which was unlike most studies in which deletion of transcription factor led to the reduction of cell growth. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses revealed that the expression of eg7A, cbh6A, bgl3A, xyl11A genes was up-regulated obviously in the ΔTeSrdA strain. Through Electrophoretic mobility shift assays (EMSA), we further discovered that the transcription factor TeSrdA regulated the transcription of eg7A, cbh6A, cbh6B, bgl3A, xyl11A and xyl11B by directly binding to the promoters of these enzyme genes, thereby repressing the cellulase production.

Conclusions: TeSrdA plays an important role by increasing hyphal branching, accelerating strain growth, and promoting cellulase and hemicellulase production. The novel transcription factor TeSrdA is of great significance for studying the transcriptional regulatory network related to enzyme production and growth of strains in Talaromyces sp.

Background: Feather waste, a byproduct of the poultry industry, remains underutilized due to its recalcitrant nature. While microbial conversion holds substantial potential, the scarcity of high-efficiency degrading strains hampers industrial application.

Results: A novel feather-degrading actinobacterium, designated KK^T, exhibited highly efficient decomposition of feather waste. When cultured with 10% (w/v) chicken feathers as sole nutrient source, it achieved over 50% degradation within 8 days. Taxonomic characterization identified strain KK^T as a novel species of the genus Streptomyces, with the proposed name Streptomyces shaoguanensis sp. nov.. Genomic analysis of strain KK^T revealed an abundance of functionally uncharacterized genetic elements and 26 predicted biosynthetic gene clusters (BGCs) for secondary metabolites. Integrated transcriptomic and biochemical analyses suggested that feather degradation by S. shaoguanensis KK^T represents an adaptive physiological response. This process was found to sustain an alkaline fermentation environment through continuous ammonia release and to efficiently disrupt disulfide bonds via a non-sulfite-dependent mechanism mediated by cysteine, H₂S and reductases. Simultaneously, highly efficient degradation was achieved through the temporally coordinated action of multiple proteases. Furthermore, when applied as a biofertilizer, the feather hydrolysate significantly promoted the growth of Brassica rapa subsp. chinensis (Pak Choi) compared to commercial amino acid fertilizers, achieving 13.1% higher fresh weight, 14.4% greater leaf area, 16.3% increased chlorophyll content, and 45.3% elevated soluble protein levels.

Conclusions: Here, a novel Streptomyces species strain KK^T with superior feather-degrading efficiency was reported. A wealth of functionally uncharacterized genes and significant biosynthetic potential in the genome of strain KK^T laid a genetic groundwork for the exploration of its novel physiological functions and the discovery of uncharacterized metabolites. Integrative analyses of genomics, transcriptomics, and biochemical profiles of the degradation metabolites, together, uncovered the underlying mechanism of superior feather-degrading capacity. Additionally, the feather hydrolysate demonstrated a significant growth-promoting effect on Pak Choi. This finding provides a solid foundation for the sustainable valorization of feather waste and the development of novel biofertilizers.

Background: Lactic acid is a highly versatile molecule whose increasing demand across the polymer, food, pharmaceutical, chemical, and cosmetics industries underscores its industrial and economic significance. Currently, lactic acid is predominantly produced via microbial fermentation using lactic acid bacteria facing limitations such as sensitivity to low pH, complex nutritional requirement and waste product generation during downstream processing.

Results: To address these challenges, we employed a genetically modified Saccharomyces cerevisiae strain capable of producing lactic acid and subjected it to long-term adaptive laboratory evolution. The strain was cultured in serial shake flask cultivations over a period of 35 months under elevating lactic acid concentrations and increasing stress to low pH. The evolved populations showed improved production of up to 250% in final lactic acid titers compared to the parental strain. The best-performing strains reached 67 g L⁻¹ at a final pH of 2.4 without pH control or 165 g L⁻¹ lactic acid at pH 3.0 with the addition of pH neutralizers, representing - to our knowledge - the highest LA titer reported in shake flask cultivations for S. cerevisiae.

Conclusion: Overall, our results prove the great potential of long-term adaptive laboratory evolution in developing robust yeast cell factories for industrial organic acid production.

Salinity stress is a major environmental problem affecting agricultural productivity worldwide. Bioagents such as plant growth-promoting fungi (PGPF) are gained increasing attention to improve plant growth and resilience to this problem. This study addresses the isolation and screening of endophytic fungal isolates from Atriplex nummularia as well as soil fungi for salinity tolerance. Screening revealed two fungal isolates AS1 and B4, exhibiting exceptional salt tolerance at different concentrations of NaCl from 2 to 10%. Morphological and molecular identification confirmed AS1 was identified as Alternaria sp. and B4 as Aspergillus terreus. Results revealed that, both fungal strains are plant growth promoters under normal and saline conditions in vitro. In normal conditions, endophytic Alternaria sp. AS1 produced indole acetic acid (IAA) and solubilized phosphate with quantities 39.0 and 58.438 µg/ml; and A. terreus B4 with quantities 52.90 and 63.07 µg/ml respectively. In saline conditions, IAA production by both fungal strains was decreased gradually with increasing salt concentration. On the other hand, phosphates solubilization was increased with increasing salt concentration up to 8% where the quantity was 81.917 and 85.677 in the case of endophytic Alternaria sp. AS1 and A. terreus B4, respectively. Furthermore, both fungi produced siderophores and hydrogen cyanide, with A. terreus exhibiting high production under both normal and saline conditions compared to the endophytic Alternaria sp. AS1. Antagonistic assays revealed that both AS1 and B4 effectively inhibited the growth of fungal plant pathogens Alternaria alternata and Fusarium oxysporum using dual culture technique. Antimicrobial assay demonstrated significant efficacy of ethyl acetate extracts of both fungi against A. alternata, F. oxysporum and Ralstonia solanacearum using the agar well diffusion method. Furthermore, seed treatment with both fungal strains and their consortia alleviated the harmful effect of salinity stress and improved seedling growth parameters compared to untreated wheat seeds. Our findings suggest that endophytic Alternaria sp. and soil fungus Aspergillus terreus have potential as bio-inoculants to improve plant growth and its resilience in saline environments.

Background: ArsR-based whole-cell biosensors offer sensitive colorimetric detection of arsenite [As(III)], yet their broad reactivity toward Group 15 metalloids-especially antimonite [Sb(III)]-limits field specificity. The recently identified ant operon from Comamonas testosteroni JL40 confers Sb(III)-selective resistance via the efflux ATPase AntA, the metallochaperone AntC, and the regulator AntR, providing genetic parts to suppress Sb(III) cross-talk.

Results: We systematically introduced antA, antC, and three antR homologs into an ArsR regulator coupled with a deoxyviolacein reporter chassis (pJ23119-K12). Co-expression of AntA and AntC under a moderate constitutive promoter (PceuR) shifted the Sb(III) limit of detection (LOD) from 0.073 µM to 0.586 µM, with a modest increase in the As(III) LOD to 0.018 µM. Subsequent integration of AntR1 not only maintained the As(III) LOD at 0.018 µM but also unexpectedly amplified the As(III) signal, extending the linear range to 36 nM-37.5 µM (R² = 0.991). It suggests that AntR1 may modulate the transcriptional circuitry via cross-regulation, warranting further mechanistic inquiry. The modified biosensor TOP10/pJ23119-antACR1 exhibited high selectivity for As(III) over divalent metals (Cd, Pb, Cu, Hg, Mn, Mg) and tolerated Sb(III) up to 1 µM. Performance was retained in 90% freshwater and 50% seawater matrices, enabling accurate quantification of 0-2.5 µM As(III) in deionized, tap, surface, and marine samples.

Conclusion: By coupling Sb(III)-specific ant efflux/sequestration components with an ArsR-based sensing module, we developed a portable, low-cost biosensor that overcomes longstanding As(III)/Sb(III) cross-reactivity and performs robustly in complex environmental waters.

Backgroud: Succinic acid (SA) is a significant C4-dicarboxylic acid with broad applications in the food, chemical, and pharmaceutical industries. In microbial SA production, Yarrowia lipolytica shows great potential. Polysulfides are vital for maintaining redox balance and cellular health in yeast.

Results: In this study, we changed the polysulfides metabolism of Y. lipolytica to enhance SA production. The 3-mercaptopyruvate sulfurtransferase (3-MST) and rhodanese (RHOD) encoding genes were disrupted in Y. lipolytica PGC01003, which led to increased biomass and SA production. In a 3-L scale bioreactor, the mutant strain produced 64.5 g/L SA, representing a 37.8% increase compared with PGC01003. Further investigations indicated that the number of mitochondria was decreased, but the ATP production and oxygen consumption rate were increased in the mutant strain. Transcriptomic analysis indicated that apoptosis genes were downregulated, and cell cycle related genes were upregulated.

Conclusions: This study demonstrated that polysulfides affected the overall growth and metabolism of Y. lipolytica. The same strategy may have the potential to be applied in improving other cell factories.