Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering.

Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel "PEACES" system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

Deciphering Multidrug-Resistant Plasmids in Disinfection Residual Bacteria from a Wastewater Treatment Plant.

Current disinfection processes pose an emerging environmental risk due to the ineffective removal of antibiotic-resistant bacteria, especially disinfection residual bacteria (DRB) carrying multidrug-resistant plasmids (MRPs). However, the characteristics of DRB-carried MRPs are poorly understood. In this study, qPCR analysis reveals that the total absolute abundance of four plasmids in postdisinfection effluent decreases by 1.15 log units, while their relative abundance increases by 0.11 copies/cell compared to investigated wastewater treatment plant (WWTP) influent. We obtain three distinctive DRB-carried MRPs (pWWTP-01-03) from postdisinfection effluent, each carrying 9-11 antibiotic-resistant genes (ARGs). pWWTP-01 contains all 11 ARGs within an ∼25 Kbp chimeric genomic island showing strong patterns of recombination with MRPs from foodborne outbreaks and hospitals. Antibiotic-, disinfectant-, and heavy-metal-resistant genes on the same plasmid underscore the potential roles of disinfectants and heavy metals in the coselection of ARGs. Additionally, pWWTP-02 harbors an adhesin-type virulence operon, implying risks of both antibiotic resistance and pathogenicity upon entering environments. Furthermore, some MRPs from DRB are capable of transferring and could confer selective advantages to recipients under environmentally relevant antibiotic pressure. Overall, this study advances our understanding of DRB-carried MRPs and highlights the imminent need to monitor and control wastewater MRPs for environmental security.

Simple Genomic Traits Predict Rates of Polysaccharide Biodegradation.

Natural and chemically modified polysaccharides are extensively employed across a wide array of industries, leading to their prevalence in the waste streams of industrialized societies. With projected increasing demand, a pressing challenge is to swiftly assess and predict their biodegradability to inform the development of new sustainable materials. In this study, we developed a scalable method to evaluate polysaccharide breakdown by measuring microbial growth and analyzing microbial genomes. Our approach, applied to polysaccharides with various structures, correlates strongly with well-established regulatory methods based on oxygen demand. We show that modifications to the polysaccharide structure decreased degradability and favored the growth of microbes adapted to break down chemically modified sugars. More broadly, we discovered two main types of microbial communities associated with different polysaccharide structures─one dominated by fast-growing microbes and another by specialized degraders. Surprisingly, we were able to predict biodegradation rates based only on two genomic features that define these communities: the abundance of genes related to rRNA (indicating fast growth) and the abundance of glycoside hydrolases (enzymes that break down polysaccharides), which together predict nearly 70% of the variation in polysaccharide breakdown. This suggests a trade-off, whereby microbes are either adapted for fast growth or for degrading complex polysaccharide chains, but not both. Finally, we observe that viral elements (prophages) encoded in the genomes of degrading microbes are induced in easily degradable polysaccharides, leading to complex dynamics in biomass accumulation during degradation. In summary, our work provides a practical approach for efficiently assessing polymer degradability and offers genomic insights into how microbes break down polysaccharides.

Interval Split Covariance Intersection Filter: Theory and Its Application to Cooperative Localization in a Multi-Sensor Multi-Vehicle System.

The data incest problem causes inter-estimate correlation during data fusion processes, which yields inconsistent data fusion results. Especially in the multi-sensor multi-vehicle (MSMV) system, the data incest problem is serious due to multiple relative position estimations, which not only lead to pessimistic estimation but also cause additional computational overhead. In order to address the data incest problem, we propose a new data fusion method termed the interval split covariance intersection filter (ISCIF). The general consistency of the ISCIF is proven, serving as supplementary proof for the split covariance intersection filter (SCIF). Moreover, a decentralized MSMV localization system including absolute and relative positioning stages is designed. In the absolute positioning stage, each vehicle uses the ISCIF algorithm to update its own position based on absolute measurements. In the relative position stage, the interval constraint propagation (ICP) method is implemented to preprocess multiple relative position estimates and initially prepare input data for ISCIF. Then, the proposed ISCIF algorithm is employed to realize relative positioning. In addition, comparative simulations demonstrate that the proposed method can achieve both accurate and consistent results compared with the state-of-the-art methods.

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

[Effects of N, P, and K application rates on distribution of 13C assimilates, starch accumulation in grains and fertilizer utilization of wheat]. / 氮磷钾施用量对小麦旗叶13CåŒåŒ–ç‰©åˆ†é ã€ç±½ç²’æ·€ç²‰ç§¯ç´¯å’Œè‚¥æ–™åˆ©ç”¨çš„å½±å“.

Clarifying the appropriate application rates of N, P, and K fertilizers and the physiological mechanisms of wheat under water-saving recharge irrigation in the North China Plain would provide a theoretical basis for formulating reasonable fertilization plans for high-yield and high-efficiency wheat production. We established four treatments with different amounts of nitrogen (N), phosphorus (P2O5), and potassium (K2O) application: 0, 0, and 0 kg·hm-2 (F0), 180, 75, and 60 kg·hm-2 (F1), 225, 120, and 105 kg·hm-2 (F2), and 270, 165, and 150 kg·hm-2 (F3). During the jointing and anthesis stages of wheat, the relative water content of each treatment in the 0-40 cm soil layer was replenished to 70%, to investigate the differences in wheat flag leaf photosynthetic characteristics, distribution of 13C assimilates, grain starch accumulation, and fertilizer utilization. The results showed that the relative chlorophyll content of flag leaves, photosynthetic and chlorophyll fluorescence parameters, 13C assimilate allocation in each organ, enzyme activities involved in starch synthesis, and starch accumulation in the F1 treatment were significantly higher than that in F0 treatment, which was an important physiological basis for the 20.9% increase in grain yield. The above parameters and yield in the F2 and F3 treatments showed no significant increase compared to F1 treatment, while fertilizer productivity and agronomic efficiency of N, P, and K decreased by 17.5%-58.4% and 12.7%-50.7%, respectively. Therefore, F1 could promote flag leaf photosynthetic assimilate production and grain starch accumulation under water-saving supplementary irrigation conditions, resulting in higher grain yield and fertilizer utilization efficiency.

A 16S RNA Analysis of Yangzhou Geese with Varying Body Weights: Gut Microbial Difference and Its Correlation with Body Weight Parameters.

China is a major goose-raising country, and the geese industry plays a significant role in animal husbandry. Therefore, goose growth performance (body weight) is a critical topic. Goose gut microbiota influences weight gain by regulating its energy metabolism and digestion. Additionally, the impact of cecal microbial community structure on goose growth and development, energy metabolism, and immunity has been examined. However, most studies have used different additives or feeds as variables. Improving the understanding of the dynamic changes in gut microbial communities in geese of different body weights during their growth and development and their correlation with the host's body weight is necessary. In this study, the cecal microbiota of healthy Yangzhou geese with large (L) and small (S) body weights, all at the same age (70 days old) and under the same feeding conditions, were sequenced using 16S rRNA. The sequencing results were annotated using QIIME2 (classify-sklearn algorithm) software, and the linkET package was used to explore the correlation between intestinal microorganisms and the body weight of the Yangzhou goose (Spearman). At the phylum level, the Firmicutes/Bacteroidetes ratio in the large body weight group was approximately 20% higher than that in the small body weight group, with Bacteroidetes and Firmicutes exhibiting a highly significant negative correlation. At the genus level, Bacteroides constituted the most abundant microbial group in both groups, although the Prevotellaceae_Ga6A1_group exhibited a higher abundance in the large than the small weight group. Spearman correlation analysis and the linkET package were used to analyze the correlation between cecal microflora and production performance indicators that showed significant differences between the two groups and showed that birth weight was significantly positively correlated with Deferribacterota at the phylum level. At the genus level, leg and chest muscle weights exhibited significant positive correlations with Prevotellace-ae_Ga6A1_group, suggesting its critical role in promoting the growth and development of goose leg and chest muscles. A significant negative correlation was observed between [Ruminococ-cus]_torque and Prevotellaceae_Ga6A1_group. These findings offer a crucial theoretical foundation for the study of gastrointestinal microorganisms and provide insights into the development and formulation of poultry probiotics.

Production of pyruvic acid with Candida glabrata using self-fermenting spent yeast cell dry powder as a seed nitrogen source.

Pyruvic acid is an important organic acid and a key industrial raw material. It is widely used in the chemical, agricultural, and food fields. Candida glabrata is the preferred strain for pyruvic acid production. The waste yeast cell for pyruvic acid fermentation with C. glabrata are rich in protein, amino acid, nucleic acid, and vitamins, as potential and cost-effective nitrogen source raw material. In this study, the potential of C. glabrata to produce pyruvic acid using spent yeast cell dry powder was evaluated. When 30 g/L of spray-dried spent yeast cell powder was used as the seed nitrogen source, a high titer of pyruvic acid was obtained. The pyruvic acid production reached 63.4 g/L with a yield of 0.59 g/g in a 5 L bioreactor. After scale-up to a 50 L bioreactor using the fermented spent yeast cell dry powder as a seed nitrogen source, 65.1 g/L of pyruvic acid was harvested, with a yield of 0.61 g/g. This study proposes a promisingapproach for increasing the pyruvic acid titer and reducing the costs.

Bioproduction of quercetin using recombinant thermostable glycosidases from Dictyoglomus thermophilum.

Quercetin is an essential ingredient in functional foods and nutritional supplements, as well as a promising therapeutic reagent. Also, the green technique to produce quercetin via rutin biotransformation is attractive. Genes encoding two thermostable glycosidases from Dictyoglomus thermophilum were cloned and expressed in Escherichia coli, which were applied in rutin biotransformation to produce highly pure quercetin at a high temperature. The production of biocatalysts were scaled up in a 5-L bioreactor, yielding a several-fold increase in total enzyme activity and a quercetin production of 14.22 ± 0.26 g/L from 30 g/L of rutin. Feeding strategies were optimized to boost biomass and enzyme production, achieving an activity of 104,801.80 ± 161.99 U/L for rhamnosidase and 12,637.23 ± 17.94 U/L for glucosidase, and a quercetin yield of 20.24 ± 0.27 g/L from the complete conversion of rutin. This study proposes a promising approach for producing high-quality quercetin in an industrial setting.

Efficient 1,3-dihydroxyacetone biosynthesis in Gluconobacter oxydans using metabolic engineering and a fed-batch strategy.

1,3-Dihydroxyacetone (DHA) is a commercially important chemical and widely used in cosmetics, pharmaceuticals, and food industries as it prevents excessive water evaporation, and provides anti-ultraviolet radiation protection and antioxidant activity. Currently, the industrial production of DHA is based on a biotechnological synthetic route using Gluconobacter oxydans. However, achieving higher production requires more improvements in the synthetic process. In this study, we compared DHA synthesis levels in five industrial wild-type Gluconobacter strains, after which the G. oxydans WSH-003 strain was selected. Then, 16 dehydrogenase genes, unrelated to DHA synthesis, were individually knocked out, with one strain significantly enhancing DHA production, reaching 89.49 g L-1 and 42.27% higher than the wild-type strain. By optimizing the culture media, including seed culture and fermentation media, DHA production was further enhanced. Finally, using an established fed-batch fermentation system, DHA production reached 198.81 g L-1 in a 5 L bioreactor, with a glycerol conversion rate of 82.84%.