Betulinic acid (BA), a penta-cyclic triterpenoid found as a ubiquitous secondary metabolite throughout the plant kingdom, has aroused tremendous interests due to its different pharmacological properties, which lead to large market demand. However, the content of BA in plant is low for phytoextraction. Although chemical semi-synthesis or biotransformation of BA from betulin with high conversion efficiency is achieved, it still relies on phytoextraction from the bark of medicinal trees. To circumvent this issue, the biotechnological synthesis of BA in engineered yeasts has been developed. In this review, the pharmacological properties of BA are first summarized, including antitumor, anti-HIV, antiprotozoal, anti-inflammatory, apoptosis activator and anti-metabolic syndrome. Then, the traditional phytoextraction, semi-synthesis and biotechnological synthesis of BA are discussed. Particularly, current advances in its biotechnological synthesis and strategies to improve BA production are focused. Moreover, potential strategies for further promotion of BA yield, including the introduction of artificial isopentenol utilization pathway, semi-rational mutagenesis of lupeol synthase and cytochrome P450, and subcellular morphology and compartmentalization, are discussed.
Due to the increasing demand for microbially manufactured products in various industries, it has become important to find optimal designs for microbial cell factories by changing the direction of metabolic flow and its flux size by means of metabolic engineering such as knocking out competing pathways and introducing exogenous pathways to increase the yield of desired products. Recently, with the gradual cross-fertilization between computer science and bioinformatics fields, machine learning and intelligent optimization-based approaches have received much attention in Genome-scale metabolic network models (GSMMs) based on constrained optimization methods, and many high-quality related works have been published. Therefore, this paper focuses on the advances and applications of machine learning and intelligent optimization algorithms in metabolic engineering, with special emphasis on GSMMs. Specifically, the development history of GSMMs is first reviewed. Then, the analysis methods of GSMMs based on constraint optimization are presented. Next, this paper mainly reviews the development and application of machine learning and intelligent optimization algorithms in genome-scale metabolic models. In addition, the research gaps and future research potential in machine learning and intelligent optimization methods applied in GSMMs are discussed.
Genome-scale metabolic models (GEMs) have been widely used to design cell factories in silico. However, initial flux balance analysis only considers stoichiometry and reaction direction constraints, so it cannot accurately describe the distribution of metabolic flux under the control of various regulatory mechanisms. In the recent years, by introducing enzymology, thermodynamics, and other multiomics-based constraints into GEMs, the metabolic state of cells under different conditions was more accurately simulated and a series of algorithms have been presented for microbial phenotypic analysis. Herein, the development of multiconstrained GEMs was reviewed by taking the constraints of enzyme kinetics, thermodynamics, and transcriptional regulatory mechanisms as examples. This review focused on introducing and summarizing GEMs application tools and cases in cell factory design. The challenges and prospects of GEMs development were also discussed.
Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets. Compared to antibodies, aptamers are easy to in vitro synthesize with low cost, and exhibit excellent thermal stability and programmability. With these features, aptamers have been widely used in biology and medicine-related fields. In the meantime, a variety of systematic evolution of ligands by exponential enrichment (SELEX) technologies have been developed to screen aptamers for various targets. According to the characteristics of targets, customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity. In this review, we first summarize the latest research on the systematic bio-fabrication of aptamers, including various SELEX technologies, post-SELEX optimization, and aptamer modification technology. These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers. The latter provides a new perspective for the systems bio-fabrication of aptamers. Furthermore, on this basis, we review the applications of aptamers, particularly in the fields of engineering biology, including industrial biotechnology, medical and health engineering, and environmental and food safety monitoring. And the encountered challenges and prospects are discussed, providing an outlook for the future development of aptamers.
Microbial biorefineries to produce chemicals from renewable feedstock provides attractive advantages, including mild reaction conditions and sustainable manufacturing. However, low-efficiency biorefineries always result in an uncompetitive biological process compared to the current petrochemical process. Thus, improving microbial capacity to maximize product yield, productivity, and titer has been recognized as a central goal for bioengineers and biochemists. The knowledge of cellular biochemistry has enabled the regulation of microbial physiology to couple with chemical production. The rapid development in metabolic engineering provides diverse strategies to enhance the efficiency of chemical biosynthesis pathways. New synthetic biology tools as well as novel regulatory targets also offer the opportunity to improve biorefinery environmental adaptivity. In this review, the recent advances in building efficient biorefineries were showcased. In addition, the challenges and future perspectives of microbial host engineering for increased microbial capacity of a biorefinery were discussed.
This study aimed to reveal the effect of salt reduction on the nutritional, functional and sensory aspects of doubanjiang through preparing three sets of doubanjiang fermentation (Sample H, Sample M and Sample L) with different salt concentrations. The results showed that fermentation with lower salt concentration led to significantly higher amino acids concentrations in doubanjiang, while the organic acids concentrations were also slightly increased. For biologically active compounds, the concentrations of total flavonoids, total phenols and five monomer isoflavones in Sample L were all significantly higher than those in Sample M and Sample H. Moreover, better anti-oxidant ability was observed in doubanjiang samples fermented with lower salt concentration. Bacillus and Millerozyma genus were found to be closely related to the formation of amino acids and biologically active compounds in doubanjiang, while organic acids were highly correlated with Cronobacter, Erwinia, Trabulsiella, Enterobacter and Millerozyma genus. Through sensory evaluation and electric tongue, unsatisfactory sensory characteristics, such as lighter color, strong acid taste and off-flavor, were found in lower salt fermented doubanjiang samples. This indicated that lower salt concentration favored the nutrition and function of doubanjiang while negatively influenced doubanjiang flavor. This study deepened our understanding of the roles of salt concentration on doubanjiang fermentation and provided guidance for the further development of low-salt doubanjiang.
The trisaccharide unit in lipopolysaccharide of Bordetella pertussis is important for immune recognition, but how the rare sugar 2-acetamido-4-methylamino-fucose in the trisaccharide unit is synthesized and transferred to the core of lipopolysaccharide in B. pertussis remains unclear. In this study, we demonstrated that the genes bplF, bplG, bplL and bplI are involved in the synthesis and transfer of 2-acetamido-4-methylamino-fucose to the core of lipopolysaccharide in B. pertussis. These genes were overexpressed with various combination in a recombinant E. coli strain which can synthesize B. pertussis core-lipid A. Lipopolysaccharides were isolated from these strains and analyzed by using SDS-PAGE, ion chromatography and NMR. Based on these analyses, the mechanism of biosynthesis and transfer of 2-acetamido-4-methylamino-l-fucose to the core-lipid A in B. pertussis is proposed.
Streptomyces griseus trypsin (SGT) is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions. However, SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue. In this study, by engineering the α signal peptide in Pichia pastoris, we increased the SGT amidase activity to 67.91 U mL−1 in shake flask cultures. Afterwards, we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S, V177I and E180M with increased catalytic efficiencies. On this basis, we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL−1 in shake flasks and 2506 U mL−1 in 3-L fed-batch cultures. Moreover, single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5, which was 2 times better than the animal-sourced trypsin.
The purpose of this study was to assess the potential application of cell surface display in Candida tropicalis. Surface display gene cassettes were constructed using five anchoring proteins from Saccharomyces cerevisiae, three of which [(suppression of exponential defect protein, SED1), (cell wall protein 2, CWP2) and (delayed anaerobic protein 4, DAN4)] were reported to show higher activity of heterologous proteins than α-agglutinin (AGα1). The performance of yeast-enhanced green fluorescent protein (yeGFP) was evaluated using laser scanning confocal microscopy and flow cytometry. The results showed that the three anchoring regions (SED1, CWP2 and AGα1) successfully displayed yeGFP on the cell wall. To investigate the effect of the three anchoring proteins on the surface display of Rhizopus oryzae α-amylase (ROA1) and Aspergillus aculeatus β-glucosidase (BGL1) in C. tropicalis, we constructed surface display gene cassettes for ROA1 and BGL1, respectively. The strains containing the anchoring proteins SED1 and CWP2 showed higher activity of ROA1 and BGL1 than the strains containing the anchoring protein AGα1. The highest ROA1 and BGL1 activities of strains with SED1 were 6.37 U/g CDW and 7.93 U/g CDW, respectively, which were sixfold and eightfold higher than those of strain with AGα1. In addition, we also optimized signal peptides. The results indicated that signal peptides have an impact on enzyme activity.
The current scenario of COVID-19 makes us to think about the devastating diseases that kill so many people every year. Analysis of viral proteins contributes many things that are utterly useful in the evolution of therapeutic drugs and vaccines. In this study, sequence and structure of fusion glycoproteins and major surface glycoproteins of respiratory syncytial virus (RSV) were analysed to reveal the stability and transmission rate. RSV A has the highest abundance of aromatic residues. The Kyte–Doolittle scale indicates the hydrophilic nature of RSV A protein which leads to the higher transmission rate of this virus. Intra-protein interactions such as carbonyl interactions, cation–pi, and salt bridges were shown to be greater in RSV A compared to RSV B, which might lead to improved stability. This study discovered the presence of a network aromatic–sulphur interaction in viral proteins. Analysis of ligand binding pocket of RSV proteins indicated that drugs are performing better on RSV B than RSV A. It was also shown that increasing the number of tunnels in RSV A proteins boosts catalytic activity. This study will be helpful in drug discovery and vaccine development.
Malonate is a high-value chemical that can be used to produce value-added compounds. Due to the toxic by-products and low product yield for malonate production through hydrolysis of cyanoacetic acid, microbial production methods have attracted significant attention. Previously, the β-alanine pathway has been engineered in Escherichia coli for malonate production. In this study, the β-alanine pathway was constructed in Saccharomyces cerevisiae by introducing the heterologous genes of BcBAPAT and TcPAND to convert l-aspartate to malonic semialdehyde, combining with co-expression genes of AAT2 and UGA2 to improve precursor supply and malonate producing. Through delta sequence-based integration of the two heterologous genes, the engineered strain produced with 7.21 mg/L malonate was screened. Further, replaced the succinic semialdehyde dehydrogenase gene UGA2 with yneI from E. coli which was utilized to produce malonate in previous study, increased the malonate titer to 7.96 mg/L in flask culture. Following optimization, fermentation of the final engineered strain in shake flasks yielded a maximum malonate titer of 12.83 mg/L, and this was increased to 91.53 mg/L during fed-batch fermentation in a 5 L bioreactor which increased by two-fold compared with that of the engineered strain overexpressing UGA2.
A total of 79 bacteria and 101 actinobacteria strains were isolated from the sediment samples of the different points of Baratang mud volcano viz., point of the eruption (M), middle of the volcano (MD), and the dried part of the mud volcano (E). Based on the biochemical and molecular characterization, the isolates were categorized under the phyla Proteobacteria, Firmicutes and Proteobacteria included representatives of Classes Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria of 29 genera with 38 distinct ribotypes. Thirty-eight bacterial strains from four different regions of mud volcano revealed excellent activity for indole-3-acetic acid (IAA) production. Excellent antagonistic property, plant growth promoting properties such as IAA production, phosphate, potassium and zinc solubilization were identified in Bacillus megaterium NIOT_MV 31 strain of 38 studied isolates. In this study, we investigated the optimization of IAA production by B. megaterium NIOT_MV 31 and its formulation as a plant growth promoter to improve economic and agricultural development. Maximum IAA yield was achieved using optimal conditions (42.63 mg/mL) in the presence of optimized tryptophan after 4 days of incubation. Twofold increase in the plant growth parameters were observed to that of control plants. Optimization of culture conditions resulted in a fourfold increase in IAA production by B. megaterium NIOT_MV 31 cells. The results clearly demonstrated that, B. megaterium NIOT_MV 31 holds great potential as a source for IAA production and may be useful for commercial applications.
14α-hydroxy-androst-4-ene-3,17-dione (14α-OH-AD) is an important precursor for the synthesis of steroid drugs with anti-cancer and carcinolytic activity. Initially, 14α-OH-AD was mostly synthesized by whole-cell fermentation of mold fungi using androstenedione (AD) as a substrate, which had difficulties in product isolation and purification as well as problems of high production cost. In this study, the source of the 14α-hydroxylase gene was expanded. And 14α-hydroxylase genes were heterologously expressed in Mycolicibacterium neoaurum (MNR) M3ΔksdD, which enabled the one-step biotransformation from the cheap substrate phytosterols (PS) to 14α-OH-AD, reducing the difficulty of product purification and production cost. What is more, to alleviate the problem of poor activity of 14α-hydroxylase, the 14α-hydroxylase gene was co-expressed with the electron transport chain element genes and the coenzyme regeneration genes, and a superior engineered strain MNR M3ΔksdD/pMV261-14α-G6PDH was obtained. Finally, the transformation conditions were optimized for the transformation of PS by the engineered strain. The molar yield of 14α-OH-AD reached to 60.4 ± 2.3% (about 0.22 g/L productivity). This study investigated for the first time the effects of the tandem electron transport chain element genes and the tandem coenzyme regeneration genes on the 14α-hydroxylation reaction, providing a theoretical basis for the industrial production of 14α-OH-AD.
In this study, a highly porous chemically activated granular activated carbon (GAC) was prepared from coconut husk and tested as an adsorbent to remove nitrate from contaminated groundwater. The prepared GAC was characterized by Fourier-transform infrared spectroscopy (FTIR), thermogravimetric and differential thermal analysis (TGA/DTA), scanning electron microscopy (SEM) and the Brunauer–Emmett–Teller (BET) surface area (S BET) analysis. The effects of various process parameters such as initial nitrate concentration, contact time and adsorbent dose on nitrate removal efficiency (response) by the modified GAC were investigated using the statistically significant response surface methodology and Box–Behnken design of experiments. The experimental data were fitted to well-known adsorption isotherms and kinetic models to ascertain the mechanism of the adsorption process. Analysis of variance (ANOVA) was performed to determine the significance of the individual and the interactive effects of process variables on the response. The BET surface area (S BET) and micropore volume of the prepared GAC from coconut husk was 1120 m2/g and 0.392 cm3/g, respectively. The experimental results showed that physisorption was the main adsorption mechanism governing the process, while the rate of adsorption was limited at initial nitrate concentrations > 10 mg/L. The Langmuir mono-layer adsorption isotherm best fitted the experimental data with a maximum adsorption capacity of 6.0 ± 1.3 mg/g (~ 92.5%) with an adsorbent dose of 0.1 g/50 mL, an equilibrium time of 6 h at 28 ± 2 °C, and at pH 7.6 (± 0.2). Among the tested process variables, the adsorbent dose and initial nitrate concentration showed significant effects on the nitrate removal efficiency.
Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness, high stereoselectivity, and mild reaction conditions. However, amine dehydrogenase has low catalytic activity, which greatly limits its application in the large-scale synthesis of chiral amines. In this study, a novel amine dehydrogenase was obtained by modifying the substrate specificity of leucine dehydrogenase via computer-aided protein engineering strategy. Furthermore, conservation analysis, homology modeling and molecular docking analysis were carried out via biocomputing strategy to select the mutation sites, and the mutants L52S and T143C were obtained. The enzyme activities of the two mutants to 2-pentanone were 1.55 U/mg and 2.06 U/mg, respectively. The enzyme activity of the latter was 188% and the T m value was 2.55 °Chigher than those of the original mutant, which laid a foundation for the efficient preparation of chiral amines by using this novel enzyme.