Microbial Cell Factories最新文献

Programmed autolytic bacteria, also termed controlled self-disruptive or self-destructive bacteria, are bacterial systems that express certain lytic genes and undergo cell lysis at a predetermined time point to release the intracellular contents or to commit suicide. Such systems have wide applications in high-throughput screening of protein libraries, synthesis and recovery of bio-products, population control of heterogeneous cultures or synthetic co-cultures, drug delivery, and food fermentation. Recently, great achievements have been reported regarding on-demand control of cell autolysis for different purposes, highlighting the potential of autolytic strains in biomanufacturing and biomedicine. In this review article, we first introduce the various applications of such bacteria, followed by a summarization of the approaches used in the establishment of autolytic bacterial systems, including cell autolysis mediated by cell wall hydrolases with or without facilitating proteins and by membrane-disturbing proteins. Next, we describe in detail the methodologies adopted to control and initiate cell lysis, including induction by chemical inducers, stimulation by physical signals, auto-induction by metabolic status or nutrient limitation, and constitutive expression of the lytic genes. This article is ended with discussions on the remaining problems and possible future directions. This review provides comprehensive information on autolytic bacteria and insightful guidance to the development of highly efficient, robust, and smart autolytic bacterial platforms.

Background: Geranyllinalool, 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.

Results: We 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.

Conclusions: Efficient 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.

Background: Succinic acid (SA) is an important bio-based C4 platform chemical with versatile applications, including the production of 1,4-butanediol, tetrahydrofuran, and γ-butyrolactone. The non-conventional yeast Yarrowia lipolytica has garnered substantial interest as a robust cell factory for SA production at low pH. However, the high concentrations of SA, especially under acidic conditions, can impose significant stress on microbial cells, leading to reduced glucose metabolism viability and compromised production performance. Therefore, it is important to develop Y. lipolytica strains with enhanced SA tolerance for industrial-scale SA production.

Results: An SA-tolerant Y. lipolytica strain E501 with improved SA production was obtained through adaptive laboratory evolution (ALE). In a 5-L bioreactor, the evolved strain E501 produced 89.62 g/L SA, representing a 7.2% increase over the starting strain Hi-SA2. Genome resequencing and transcriptome analysis identified a mutation in the 26S proteasome regulatory subunit Rpn1, as well as genes involved in transmembrane transport, which may be associated with enhanced SA tolerance. By further fine-tuning the glycolytic pathway flux, the highest SA titer of 112.54 g/L to date at low pH was achieved, with a yield of 0.67 g/g glucose and a productivity of 2.08 g/L/h.

Conclusion: This study provided a robust engineered Y. lipolytica strain capable of efficiently producing SA at low pH, thereby reducing the cost of industrial SA fermentation.

Background: Microalgae are potential sustainable resources for the production of value-added chemicals that can be used as biofuels, pharmaceuticals, and nutritional supplements. Arachidonic acid (ARA), a omega-6 fatty acid, plays a crucial role in infant development and immune response, and can be used in cosmetics and pharmaceuticals. Demand for industrial-scale ARA production is continuously increasing because of its broad applicability. To address this demand, there has been a significant shift towards microorganism-based ARA production. To accelerate large-scale ARA production, it is crucial to select suitable strains and establish optimal culture conditions.

Results: Here, we isolated a novel microalga Lobosphaera incisa CFRC-1, a valuable strain that holds promise as a feedstock for ARA production. Optimal cultivation conditions were investigated using a high-throughput screening method to enhance ARA production in this novel strain. Out of 71 candidates, four organic carbon substrates were identified that could be utilized by L. incisa CFRC-1. Through flask-scale verification, fructose was confirmed as the optimal organic carbon substrate for promoting microalgal growth, total lipid accumulation, and ARA production. Subsequently, we investigated appropriate substrate concentration and cultivation temperature, confirming that the optimal conditions were 30 g L^- 1 of fructose and 27 ℃ of temperature. Under these optimized conditions, biomass and ARA production reached 13.05 ± 0.40 g L^- 1 and 97.98 ± 7.33 mg L^- 1, respectively, representing 9.6-fold and 5.3-fold increases compared to the conditions before optimization conditions. These results achieved the highest biomass and ARA production in flask-scale cultivation, indicating that our approach effectively improved both production titer and productivity.

Conclusions: This study presents a novel microalgae and optimized conditions for enhancing biomass and ARA production, suggesting that this approach is a practical way to accelerate the production of valuable microalgae-based chemicals. These findings provide a basis for large-scale production of ARA-utilizing microalgae for industrial applications.

Background: The high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use.

Results: In this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells.

Conclusions: For efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.

Background: L-Tyrosine (L-Tyr) is a significant aromatic amino acid that is experiencing an increasing demand in the market due to its distinctive characteristics. Traditional production methods exhibit various limitations, prompting researchers to place greater emphasis on microbial synthesis as an alternative approach.

Results: Here, we developed a metabolic engineering-based method for efficient production of L-Tyr from Corynebacterium crenatum, including the elimination of competing pathways, the overexpression of aroB, aroD, and aroE, and the introduction of the mutated E. coli tyrA^fbr gene for elevating L-Tyr generation. Moreover, the mtlR gene was knocked out, and the mtlD and pfkB genes were overexpressed, allowing C. crenatum to produce L-Tyr from mannitol. The L-Tyr production achieved 6.42 g/L at a glucose-to-mannitol ratio of 3:1 in a shake flask, which was 16.9% higher than that of glucose alone. Notably, the L-Tyr production of the fed-batch fermentation was elevated to 34.6 g/L, exhibiting the highest titers among those of C. glutamicum previously reported.

Conclusion: The importance of this research is underscored by its pioneering application of mannitol as a carbon source for the biosynthesis of L-Tyr, as well as its examination of the influence of mannitol-associated genes in microbial metabolism. A promising platform is provided for the production of target compounds that does not compete with human food source.

This study explores the potential of strain selection and adaptation for developing a fungi-yeast-microalgae consortium capable of integrated bioethanol production and livestock wastewater treatment. We employed a multi-stage approach involving isolation and strain selection/adaptation of these consortiums. The study started with screening some isolated fungi to grow on the cellulosic biomass of the livestock wastewater (saccharification) followed by a fermentation process using yeast for bioethanol production. The results revealed that Penicillium chrysogenum (Cla) and Saccharomyces cerevisiae (Sc) produced a remarkable 99.32 ppm of bioethanol and a concentration of glucose measuring 0.56 mg ml^- 1. Following the impact of fungi and yeast, we diluted the livestock wastewater using distilled water and subsequently inoculated Nile River microalgae into the wastewater. The findings demonstrated that Chlorella vulgaris emerged as the dominant species in the microalgal community. Particularly, the growth rate reached its peak at a 5% organic load (0.105385), indicating that this concentration provided the most favorable conditions for the flourishing of microalgae. The results demonstrated the effectiveness of the microalgal treatment in removing the remaining nutrients and organic load, achieving a 92.5% reduction in ammonia, a 94.1% reduction in nitrate, and complete removal of phosphate (100%). The algal treatment also showed remarkable reductions in COD (96.5%) and BOD (96.1%). These findings underscore the potential of fungi, yeast, and Nile River microalgae in the growth and impact on livestock wastewater, with the additional benefit of bioethanol production.

Background: Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom.

Results: Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system.

Conclusions: Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications.

Background: Marine actinomycetes, especially Streptomyces, are recognized as excellent producers of diverse and bioactive secondary metabolites on account of the multiplicity of marine habitations and unique ecological conditions, which are yet to be explored in terms of taxonomy, ecology, and functional activity. Isolation, culture and genome analysis of novel species of Streptomyces to explore their potential for discovering bioactive compounds is an important approach in natural product research.

Results: A marine actinobacteria, designated strain SCSIO 75703 ^T, was isolated, and the potential for bioactive natural product discovery was evaluated based on genome mining, compound detection, and antimicrobial activity assays. The phylogenetic, phenotypic and chemotaxonomic analyses indicate that strain SCSIO 75703 ^T represents a novel species in genus Streptomyces, for which the name Streptomyces sediminicola sp. nov. is proposed. Genome analysis revealed the presence of 25 secondary metabolite biosynthetic gene clusters. The screening for antibacterial activity reveals the potential to produce bioactive metabolites, highlighting its value for in-depth exploration of chemical constituents. Seven compounds (1-7) were separated from the fractions guided by antibacterial activities, including three indole alkaloids (1-3), three polyketide derivatives (4-6), and 4-(dimethylamino)benzoic acid (7). These primarily antibacterial components were identified as anthracimycin (4), 2-epi-anthracimycin (5) and β-rubromycin (6), presenting strong antibacterial activities against Gram-positive bacteria with the MIC value ranged from 0.125 to 16 μg/mL. Additionally,, monaprenylindole A (1) and 3-cyanomethyl-6-prenylindole (2) displayed moderate inhibitory activities against α-glucosidase with the IC₅₀ values of 83.27 and 86.21 μg/mL, respectively.

Conclusion: Strain SCSIO 75703 ^T was isolated from marine sediment and identified as a novel species within the genus Streptomyces. Based on genomic analysis, compounds isolation and bioactivity studies, seven compounds were identified, with anthracimycin and β-rubromycin showing significant biological activity and promising potential for further applications.

Background: Milk clotting enzymes, essential for milk coagulation in cheese production, are obtained from the stomach of young ruminants, an expensive and limited source. This study was accomplished by finding a suitable alternative. Bacterial isolates recovered from honey were screened for milk clotting enzyme activity. and further, by immobilization of the microorganisms to enhance stability and facilitate their repeated use.

Result: The most effective enzyme was produced by a microbe identified as Bacillus amyloliquefaciens based on 16 S rRNA sequencing. The cells were encapsulated in Ca²⁺ alginate beads. These beads retained complete enzyme production after being used five times. Glucose and Soybean were selected as the most favorable carbon and nitrogen sources, respectively. The optimum temperature for activity was 35 ℃ for both free and immobilized cells but as the temperature was increased to 55 °C and above, the encapsulated form retained more activity than the free cells. The pH optimum shifted from 6.5 to 7 for the free cells to 7-7.5 for the immobilized cells. The immobilization process decreased the activation energy for enzyme production and activity, prolonged the enzyme half-life, and increased the deactivation energy. Enzyme produced by immobilized cells generated a more compact cheese.

Conclusions: The finding of this study was to identify a less expensive source of milk-clotting enzymes and confirm the success of cell immobilization in improving cell rigidity and stability. Also, immobilization of this B. amyloliquefaciens strain offers an enzyme source of value for industrial production of cheese.