As the increasing demand from both chemical and fuel markets, the interest in producing n-butanol using biological route has been rejuvenated to engineer an economical fermentation process, competing with the currently-dominant chemical synthesis. n-Butanol has been traditionally produced from the ABE fermentation of Clostridium acetobutylicum. This system, however, is not economically feasible due to its limited efficiency and the lack of genetic modification tools for further improvements. Alternatively, n-butanol synthesis pathway was successfully transferred into Escherichia coli and rapidly improved to reach a level of production comparable to the native producer. Nevertheless, the toxicity of n-butanol has become a common issue that either approach has to deal with. Previously, we reported our success in improving n-butanol tolerance in E. coli by engineering an Artificial Transcription Factor (ATF) that can modify the expression level of multiple targets simultaneously and improved the n-butanol tolerance of MG1655 strain to 1.5% (vol/vol) n-butanol. However, it was observed that some possible n-butanol tolerance mechanisms did not occurred upon the ATF expression, especially the membrane-related functions such as the homeoviscous adaptation, iron uptaking system, and efflux pump system. In this work, we attempted to enhance the n-butanol tolerance associated with the ATF by combining it with the membrane-related functions in E. coli, including the overexpression of fatty acid synthesis genes, iron-uptaking protein FeoA, and introducing a SrpABC efflux pump from Pseudomonas putida into E. coli. The synergistic effect of this combinatorial approach led to 4, 5, and 9-fold improved growths in the cultures containing 1, 1.5, and 2% (vol/vol) n-butanol, respectively, of an MG1655 knockout strain engineered for n-butanol production, and expanded the tolerance limit to 2% (vol/vol) n-butanol.
Minor ginsenodes are of great interest due to their diverse pharmacological activities such as their anti-cancer, anti-diabetic, neuroprotective, immunomodulator, and anti-inflammatory effects. The miniscule amount of minor ginsenosides in ginseng plants has driven the development of their mass production methods. Among the various production methods for minor ginsenosides, the utilization of microorganisms and their enzymes are considered as highly specific, safe, and environmentally friendly. In this review, various minor ginsenosides production strategies, namely utilizing microorganisms and recombinant microbial enzymes, for biotransforming major ginsenosides into minor ginsenoside, as well as constructing synthetic minor ginsenosides production pathways in yeast cell factories, are described and discussed. Furthermore, the present challenges and future research direction for producing minor ginsenosides using those approaches are discussed.
Early diagnosis of infectious diseases represents powerful means to increase patient survival rate, avoid disease spreading, and decrease healthcare costs. Current Polymerase Chain Reaction (PCR)-and antibody-based diagnostic methods for detecting pathogens offer rapid analysis with highly accurate and specific results. However, those methods are still hampered by the need of sophisticated infrastructures and highly-skilled technicians, which limit the deployment in developing area. Synthetic biology with its rational and short design-to-production cycles has the potential to overcome those limitations. Here, we discuss two promising efforts for pathogen nucleic acids detection using synthetic biology approaches: Synthetic RNA-based and Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated (CRISPR/Cas)-based biosensors. The two systems were reported to show remarkable specificity and sensitivity on detecting and reporting the presence of pathogen via pathogen nucleic acid recognition with lower development and operational costs when compared to current PCR-and antibody-based diagnostic tools. Moreover, both systems can be applied to paper-based platforms which simplify the distribution and utilization in low resource-settings.
Microbes have been the preferred hosts for producing high-value chemicals from cheap raw materials. However, metabolic flux imbalance, the presence of competing pathways, and toxic intermediates often lead to low production efficiency. The spatial organization of the substrates, intermediates, and enzymes is critical to ensuring efficient metabolic activity by microorganisms. One of the most common approaches for bringing the key components of biosynthetic pathways together is through molecular scaffolds, which involves the clustering of pathway enzymes on engineered molecules via different interacting mechanisms. In particular, synthetic scaffold systems have been applied to improve the efficiency of various heterologous and synthetic pathways in Escherichia coli and Saccharomyces cerevisiae, with varying degrees of success. Herein, we review the recent developments and applications of protein-based and nucleic acid-based scaffold systems and discuss current challenges and future directions in the use of such approaches.
Biodiesel, or fatty acid ethyl ester (FAEE), is an environmentally safe, next-generation biofuel. Conventionally, FAEE is produced by the conversion of oil/fats, obtained from plants, animals, and microorganisms, by transesterification. Recently, metabolic engineering of bacteria for ready-to-use biodiesel was developed. In Escherichia coli, it is produced by fatty acyl-carrier proteins and ethanol, with the help of thioesterase (TesB) and wax synthase (WS) enzymes. One of the foremost barriers in microbial FAEE production is the feedback inhibition of the fatty acid (FA) operon (fabHDG). Here, we studied the effect of biodiesel biosynthesis in E. coli with an engineered fabHDG operon. With a basic FAEE producing BD1 strain harboring tes and ws genes, biodiesel of 32 mg/L were produced. Optimal FAEE biosynthesis was achieved in the BD2 strain that carries an overexpressed operon (fabH, fabD, and fabG genes) and achieved up to 1291 mg/L of biodiesel, a 40-fold rise compared to the BD1 strain. The composition of FAEE obtained from the BD2 strain was 65% (C10:C2, decanoic acid ethyl ester) and 35% (C12:C2, dodecanoic acid ethyl ester). Our findings indicate that overexpression of the native FA operon, along with FAEE biosynthesis enzymes, improved biodiesel biosynthesis in E. coli.
Soil salinity and mineral deficiency are major problems in agriculture. Many studies have reported that plant-associated microbiota, particularly rhizosphere and root microbiota, play a crucial role in tolerance against salinity and mineral deficiency. Nevertheless, there are still many unknown parts of plant–microbe interaction, especially regarding their role in halophyte adaptation to coastal ecosystems. Here, we report the bacterial community associated with the roots of coastal sand dune halophytes Spinifex littoreus and Calotropis gigantea, and the soil properties that affect their composition. Strong correlations were observed between root bacterial diversity and soil mineral composition, especially with soil Calcium (Ca), Titanium (Ti), Cuprum (Cu), and Zinc (Zn) content. Soil Ti and Zn content showed a positive correlation with bacterial diversity, while soil Ca and Cu had a negative effect on bacterial diversity. A strong correlation was also found between the abundance of several bacterial species with soil salinity and mineral content, suggesting that some bacteria are responsive to changes in soil salinity and mineral content. Some of the identified bacteria, such as Bacillus idriensis and Kibdelosporangium aridum, are known to have growth-promoting effects on plants. Together, the findings of this work provided valuable information regarding bacterial communities associated with the roots of sand dune halophytes and their interactions with soil properties. Furthermore, we also identified several bacterial species that might be involved in tolerance against stresses. Further work will be focused on isolation and transplantation of these potential microbes, to validate their role in plant tolerance against stresses, not only in their native hosts but also in crops.
The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3ʹ untranslated region of a target mRNA by fusing with an RNA-binding domain in the chaperone-recruiting mRNA scaffold (CRAS) system, or coupled the expression of DnaJ and a target recombinant protein using the overlapping stop-start codons 5ʹ-TAATG-3ʹ between the two genes in a chaperone-substrate co-localized expression (CLEX) system. By engineering the untranslated and intergenic sequences of the mRNA transcript, bacterial molecular chaperones are spatially constrained to the location of protein translation, expressing selected aggregation-prone proteins in their functionally active, soluble form. Our mRNA engineering methods surpassed the in-vivo solubilization efficiency of the simple DnaJ chaperone co-overexpression method, thus providing more effective tools for producing soluble therapeutic proteins and enzymes.
Background: Candida albicans is responsible for a wide range of medical ailments, from harmless cutaneous to life-threatening bloodstream infections. Growing cases of antifungal-drug resistance strains of C. albicans become a rationale to explore and develop novel anti-candida agents. In this paper, we assessed the anti-candida activity of the methanolic extracts of various tropical medicinal plants from Myrtaceae, Poaceae, and Zingiberaceae, commonly used in Indonesia to treat fungal infections. Methods: Candida albicans strain ATCC 10231 was used as a subject to assess the anti-Candida activities of plant methanolic extracts through disc diffusion assay. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) were observed. Results: All plant extracts in this study showed antifungal activities against C. albicans. Among them, Cymbopogon citratus, Curcuma xanthorrhiza, Curcuma aeruginosa, and Zingiber officinale var. rubrum showed the lowest MIC and MFC value of 3.8 mg/mL. Conclusions: The growth inhibition of C. albicans on disc diffusion assay was demonstrated by Z. officinale var. rubrum and C. longa, which were comparable to antifungal nystatin. Further investigation of the chemical constituents of the extracts and the cytotoxicity test is needed to further develop plant-derived anti-candida agents.
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