Biotechnology Letters最新文献

Itaconic acid is an excellent polymeric precursor with a wide range of industrial applications. The efficient production of itaconate from various renewable substrates was demonstrated by engineered Escherichia coli. However, limitation in the itaconic acid precursor supply was revealed by finding out the key intermediate of the tricarboxylic acid in the itaconic acid pathway. Efforts of enhancing the cis-aconitate flux and preserving the isocitrate pool to increase itaconic acid productivity are required. In this study, we introduce a synthetic protein scaffold system between CadA and AcnA to physically combine the two enzymes. Through the introduction of a synthetic protein scaffold, 2.1 g L^-1 of itaconic acid was produced at pH 7 and 37 °C. By fermentation, 20.1 g L^-1 for 48 h of itaconic acid was produced with a yield of 0.34 g g^-1 glycerol. These results suggest that carbon flux was successfully increased itaconic acid productivity.

Methanotrophs of the genus Methylocystis are frequently found in rice paddies. Although more than ten facultative methanotrophs have been reported since 2005, none of these strains was isolated from paddy soil. Here, a facultative methane-oxidizing bacterium, Methylocystis iwaonis SD4, was isolated and characterized from rhizosphere samples of rice plants in Nanjing, China. This strain grew well on methane or methanol but was able to grow slowly using acetate or ethanol. Moreover, strain SD4 showed sustained growth at low concentrations of methane (100 and 500 ppmv). M. iwaonis SD4 could utilize diverse nitrogen sources, including nitrate, urea, ammonium as well as dinitrogen. Strain SD4 possessed genes encoding both the particulate methane monooxygenase and the soluble methane monooxygenase. Simple and rapid genetic manipulation methods were established for this strain, enabling vector transformation and unmarked genetic manipulation. Fast growth rate and efficient genetic tools make M. iwaonis SD4 an ideal model to study facultative methanotrophs, and the ability to grow on low concentration of methane implies its potential in methane removal.

Salmonella typhimurium, a pathogenic bacterium with significant implications in medicine and the food industry, poses a substantial threat by causing foodborne illnesses such as typhoid fever. Accurate diagnosis of S. typhimurium is challenging due to its overlap symptoms with various diseases. This underscores the need for a precise and efficient diagnostic approach. In this study, we developed a biosensor using the Taguchi optimization method based on aptamer lateral flow assay (LFA) for the detection of S. typhimurium. Therefore, signal probe and nanobioprobe were designed using anti-Salmonella aptamer, conjugated with gold nanoparticles (GNPs), and used in LFA. The strategy of this test is based on a competitive format between the bacteria immobilized on the membrane and the bacteria present in the tested sample. Moreovere, the optimization of various factors affecting the aptamer LFA, including the concentration of bacteria (immobilized and into the sample) and the concentration of nanobioprop, were performed using the Taguchi test designing method. The data showed that the optimal conditions for the LFA reaction was 10⁸ CFU/mL of immobilized bacteria and 1.5 μg/μL of nanobioprop concentration. Then, the visual detection limit of S. typhimurium was estimated as 10⁵ CFU/mL. The reaction results were obtained within 20 min, and there were no significant cross-reactions with other food pathogens. In conclusion, the aptamer-LFA diagnostic method, optimized using the Taguchi approach, emerges as a reliable, straightforward, and accurate tool for the detection of S. typhimurium. Overall, this method can be a portable diagnostic kit for the detection and identification of bacteria.

The effective recovery of the immobilized enzymes using magnetic carriers has led to growing interest in this technology. The objective of this research was to evaluate the efficiency of immobilized laccase on magnetized multiwall carbon nanotubes (m-MWCNTs) in terms of stability and reusability. Laccases were efficiently adsorbed onto magnetized multiwall carbon nanotubes (m-MWCNTs) synthesized using water. The concentration of 7 mg laccase/mL was found to be ideal for immobilization. The optimal activity of both free and immobilized laccases was observed at pH 5, while for the latter, the optimal temperature was shifted from 40 to 50 °C. Compared to the free laccase, the immobilized laccase exhibited a greater range of stability at more extreme temperatures. At the fourth cycle of reactions, the immobilized laccase exhibited more than 60% relative activity in terms of reusability. Based on the fourier-transform infrared spectroscopy (FTIR) peak at 2921 cm^-1, saccharification of paddy straw using immobilized laccase verified lignin degradation. The easy recovery of the immobilized laccase on m-MWCNTs lends credence to its potential use in biomass hydrolysis.

One of the most remarkable techniques recently introduced into the field of bioprocess engineering is machine learning. Bioprocess engineering has drawn much attention due to its vast application in different domains like biopharmaceuticals, fossil fuel alternatives, environmental remediation, and food and beverage industry, etc. However, due to their unpredictable mechanisms, they are very often challenging to optimize. Furthermore, biological systems are extremely complicated; hence, machine learning algorithms could potentially be utilized to improve and build new biotechnological processes. Gaining insight into the fundamental mathematical understanding of commonly used machine learning algorithms, including Support Vector Machine, Principal Component Analysis, Partial Least Squares and Reinforcement Learning, the present study aims to discuss various case studies related to the application of machine learning in bioprocess engineering. Recent advancements as well as challenges posed in this area along with their potential solutions are also presented.

The present work reports the application of novel gut strains Bacillus safensis CGK192 (Accession No. OM658336) and Bacillus australimaris CGK221 (Accession No. OM658338) in the biological degradation of synthetic polymer i.e., high-density polyethylene (HDPE). The biodegradation assay based on polymer weight loss was conducted under laboratory conditions for a period of 90 days along with regular evaluation of bacterial biomass in terms of total protein content and viable cells (CFU/cm²). Notably, both strains achieved significant weight reduction for HDPE films without any physical or chemical pretreatment in comparison to control. Hydrophobicity and biosurfactant characterization were also done in order to assess strains ability to form bacterial biofilm over the polymer surface. The post-degradation characterization of HDPE was also performed to confirm degradation using analytical techniques such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Field emission scanning electronic microscopy (FE-SEM) coupled with energy dispersive X-ray (EDX), and Gas chromatography-mass spectrometry (GC-MS). Interestingly strain CGK221 was found to be more efficient in forming biofilm over polymer surface as indicated by lower half-life (i.e., 0.00032 day^-1) and higher carbonyl index in comparison to strain CGK192. The findings reflect the ability of our strains to develop biofilm and introduce an oxygenic functional group into the polymer surface, thereby making it more susceptible to degradation.

To enhance the import of heme for the production of active hemoproteins in Escherichia coli C41 (DE3) lacking the special heme import system, heme receptor ChuA from E. coli Nissle 1917 was modified through molecular docking and the other components (ChuTUV) for heme import was overexpressed, while heme import was tested through growth assay and heme sensor HS1 detection. A ChuA mutant G360K was selected, which could import 3.91 nM heme, compared with 2.92 nM of the wild-type ChuA. In addition, it presented that the expression of heme transporters ChuTUV was not necessary for heme import. Based on the modification of ChuA (G360K), the titer of human hemoglobin and the peroxidase activity of leghemoglobin reached 1.19 μg g^-1 DCW and 24.16 10³ U g^-1 DCW, compared with 1.09 μg g^-1 DCW and 21.56 10³ U g^-1 DCW of the wild-type ChuA, respectively. Heme import can be improved through the modification of heme receptor and the engineered strain with improved heme import has a potential to efficiently produce high-active hemoproteins.

Protein FadR is known as a fatty acid metabolism global regulator that sustains cell envelope integrity by changing the profile of fatty acid. Here, we present its unique participation in the defense against reactive oxygen species (ROS) in the bacterium. FadR contributes to defending extracellular ROS by maintaining the permeability of the cell membrane. It also facilitates the ROS detoxification process by increasing the expression of ROS neutralizers (KatB, KatG, and AhpCF). FadR also represses the leakage of ROS by alleviating the respiratory action conducted by terminal cytochrome cbb3-type heme-copper oxidases (ccoNOQP). These findings suggest that FadR plays a comprehensive role in modulating the bacterial oxidative stress response, instead of merely strengthening the cellular barrier against the environment. This study sheds light on the complex mechanisms of bacterial ROS defense and offers FadR as a novel target for ROS control research.

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheC_PS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheC_PS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheC_PS, the catalytic efficiency (k_cat(S)-ECH/K_m(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM^-1 s^-1), while the catalytic efficiency (k_cat(1,3-DCP)/K_m(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM^-1 s^-1. With 40 mM 1,3-DCP as substrate, HheC_PS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.

(+)-Ambrein is the primary component of ambergris, a rare product found in sperm whales (Physeter microcephalus). Microbial production using sustainable resources is a promising way to replace animal extraction and chemical synthesis. We constructed an engineered yeast strain to produce (+)-ambrein de novo. Squalene is a substrate for the biosynthesis of (+)-ambrein. Firstly, strain LQ2, with a squalene yield of 384.4 mg/L was obtained by optimizing the mevalonate pathway. Then we engineered a method for the de novo production of (+)-ambrein using glucose as a carbon source by overexpressing codon-optimized tetraprenyl-β-curcumene cyclase (BmeTC) and its double mutant enzyme (BmeTC^Y167A/D373C), evaluating different promoters, knocking out GAL80, and fusing the protein with BmeTC and squalene synthase (AtSQS2). Nevertheless, the synthesis of (+)-ambrein is still limited, causing low catalytic activity in BmeTC. We carried out a protein surface amino acid modification of BmeTC. The dominant mutant BmeTC^{K6A/Q9E/N454A} for the first step was obtained to improve its catalytic activity. The yield of (+)-ambrein increased from 35.2 to 59.0 mg/L in the shake flask and finally reached 457.4 mg/L in the 2 L fermenter, the highest titer currently available for yeast. Efficiently engineered strains and inexpensive fermentation conditions for the industrial production of (+)-ambrein. The metabolic engineering tools provide directions for optimizing the biosynthesis of other high-value triterpenes.