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The salt–tolerant yeast Zygosaccharomyces rouxii is a typical aroma–producing yeast used in food brewing, but its mechanism of high temperature tolerance is still unclear. In this study, the response mechanism of Z. rouxii to glucose under high temperature stress at 40 °C was explored, based on the total synthetic lowest–nutrient medium. The results of the growth curves and scanning electron microscopy showed that high glucose was necessary for Z. rouxii to restore growth under high temperature stress, with the biomass at 300 g/L of glucose (OD600, 120h = 2.44 ± 0.26) being 8.71 times higher than that at 20 g/L (OD600, 120h = 0.28 ± 0.08). The results of the transcriptome analysis, combined with RT–qPCR, showed that the KEGG analysis of differentially expressed genes was enriched in pathways related to glucose metabolism, and high glucose (300 g/L) could effectively stimulate the gene expression of glucose transporters, trehalose synthesis pathways, and xylitol synthesis pathways under a high temperature, especially the expression of the glucose receptor gene RGT2 (up–regulated 193.7 times at 12 h). The corresponding metabolic characteristics showed that the contents of intracellular metabolites, such as glucose (Cmax, 6h = 6.50 ± 0.12 mg/g DCW), trehalose (Cmax, 8h = 369.00 ± 17.82 μg/g DCW), xylitol (Cmax, 8h = 1.79 ± 0.27 mg/g DCW), and glycerol (Cmax, 8h = 268.10 ± 44.49 μg/g DCW), also increased with time. The accumulation of acetic acid, as the main product of overflow metabolism under high temperature stress (intracellular Cmax, 2h = 126.30 ± 10.96 μg/g DCW; extracellular Cmax, 12h = 499.63 ± 27.16 mg/L), indicated that the downstream glycolysis pathway was active. Compared with the normal physiological concentration of glucose, a high glucose concentration can effectively stimulate the gene expression and metabolism of salt–tolerant Z. rouxii under high–temperature conditions to restore growth. This study helps to deepen the current understanding of the thermoadaptive growth mechanism of salt–tolerant Z. rouxii. [ABSTRACT FROM AUTHOR]

During the production of nattokinase (NK) by Bacillus species, certain by-products such as poly-γ-glutamic acid (γ-PGA) are simultaneously synthesized. The impact of γ-PGA synthesis on NK production remains unclear. In this study, we knocked out the pgsC gene, a component of the γ-PGA synthetase cluster (pgsBCA), and constructed a novel recombinant strain, Bacillus licheniformis BL11. Next, we compared the fed-batch fermentation profiles of BL11 and its parental strain BL10, conducted transcriptional analysis, and measured intracellular ATP content. We also optimized glucose-feeding strategies under varying oxygen supply conditions. Our results indicated that the utilization rates of glucose and soybean meal were both improved in the pgsC-deficient strain BL11, and NK activity was enhanced. Furthermore, the transcriptional levels of genes involved in glycolysis and the TCA cycle were relatively upregulated in BL11. The maximal NK activity reached 2522.2 FU/mL at 54 h of fermentation using a constant glucose-feeding rate of 5.0 g/(L·h) under high oxygen supply conditions. The newly developed recombinant strain B. licheniformis BL11, along with the optimized feeding strategy, shows promise for large-scale NK production. [ABSTRACT FROM AUTHOR]

Biological CO2 activation and conversion to high-value ethanol are a feasible and green strategy to close the carbon cycle. However, naturally evolved CO2 utilization pathways involve carbon loss or ATP consumption. Herein, we report a complete anabolic pathway for direct conversion of CO2 to ethanol by constructing and assembling three functional modules including CO2 activation, formaldehyde → acetyl-CoA, and ethanol synthesis in a carbon-conserved and ATP-independent system. These artificial ethanol metabolic pathway fluxes were strengthened by screening efficient key enzymes for CO2 activation, promoting formaldehyde assimilation, and employing a two-step reaction. 13C-labeled CO2 demonstrated the feasibility of the pathway converting CO2 into ethanol in vitro by detecting the carbon flow. With this anabolic pathway, we obtained an ethanol concentration of 0.37 mM at a conversion rate of 4.33 nmol CO2 min−1 mg−1 enzyme and 0.5 mM R5P supply. This modularization strategy provides a new avenue in the construction of artificial metabolic pathways for ethanol synthesis from CO2. [ABSTRACT FROM AUTHOR]

Hydroxytyrosol (HT) is a natural antioxidant that exhibits various health-related properties. In this work, we focused on establishing and optimizing the production of HT in Bacillus licheniformis. Through protein engineering ketoacid decarboxylase, boosting the phosphoenolpyruvate (PEP) supply, releasing feedback inhibition, and blocking competing pathways, HT production increased to 2078 mg L−1 from zero. Remarkably, the tyrosol catabolic pathway was identified and deletion of the alcohol dehydrogenase gene adhA significantly reduced tyrosol degradation. Next, multiple rate-liming factors were identified, including insufficiency of PEP, erythrose-4-phosphate and 4-hydroxyphenylpyruvate supply, and imbalance of KivDV461I and HpaBC expression levels. The carbon flux toward HT was significantly improved by modulating the glucose transport system, fine-tuning the expression levels of kivDV461I and hpaBC, and enhancing the expression of tyrAfbr, tkt, and zwf, which increased the HT titer to 6299 mg L−1. Finally, the final strain DHT23 produced 9475 mg L−1 HT under fed-batch conditions, with a yield and productivity of 135.4 mg g−1 glucose and 395 mg (L h)−1, respectively, which represents the highest HT production to date. Taken together, this study establishes B. licheniformis as a framework for HT hyperproduction and paves the way for achieving the industrial production of HT in the future. [ABSTRACT FROM AUTHOR]

The current expansion of building structures has created a demand for efficient and smart surface quality evaluation at the acceptance phase. However, the conventional approach mainly relies on manual work, which is labor-intensive, time-consuming, and unrepeatable. This study presents a systematic and practical solution for surface quality evaluation of indoor building elements during the acceptance phase using point cloud. The practical indoor scanning parameters determination procedure was proposed by analyzing the project requirements, room environment, and apparatus. An improved DBSCAN algorithm was developed by introducing a plane validation and coplanar checking to facilitate the surface segmentation from the point cloud. And a revised Least Median of Square-based algorithm was proposed to identify the best-fit plane. Afterwards, the flatness, verticality, and squareness were evaluated and depicted using a color-coded map based on the segmented point cloud. The experiment on an apartment showcases how the system improves the information flow and accuracy during building acceptance, resulting in a potentially smart acceptance activity. [ABSTRACT FROM AUTHOR]

In order to investigate the seismic performance of a rubber-modified soil isolation layer, a three-dimensional finite element model was constructed using finite element analysis software, utilizing a two-story frame structure as the engineering background. Nonlinear dynamic time history analysis and comparisons were performed against the seismic performance of the structure. The evaluation was based on several parameters, including the contact area of the base, the thickness of the rubber-particle-modified soil isolation layer, ground motion records with varying amplitudes, and seismic frequency spectrum characteristics. The research findings indicate that the implementation of a rubber-modified soil isolation layer effectively mitigates the peak acceleration, horizontal displacement, and shear stress of the frame structure. This not only enhances the seismic performance of the structure but also enlarges the contact area of the base. Increasing the thickness of the rubber-modified soil isolation layer will effectively decrease the peak acceleration, horizontal displacement, and shear stress of the structure during seismic events. The effectiveness of the isolation provided by the rubber-modified soil layer improves as the intensity of the ground motion record increases. [ABSTRACT FROM AUTHOR]

Bacitracin is a kind of macrocyclic dodecapeptide that produced by Bacillus, precursor supply served as a critical role in bacitracin production, here, the aim of this study wants to improve bacitracin production by enhancing Lysine (Lys) supply via metabolic engineering of B. licheniformis DW2, an industrial strain for bacitracin production. Firstly, exogenous addition of Lys was proven to be favorable for bacitracin production, and the strain LYS2 was attained through strengthening Lys synthetic pathways via overexpressing diaminopimelate decarboxylase LysA from B. licheniformis and diaminopimelate dehydrogenase DdH from Corynebacterium glutamicum, and the bacitracin produced by LYS2 was increased to 838.53 U/mL by 10.85%, compared with that of DW2 (756.45 U/mL). Secondly, oxaloacetate, the precursor of Lys, was accumulated by overexpressing pyruvate carboxylase PycA in LYS2, and 17.06% increase of bacitracin yield was attained in LYS3 (885.53 U/mL), compared with DW2. Thirdly, lysine decarboxylase gene yaaO was deleted to weaken Lys degradation, and the attained strain LYS4 showed further increased bacitracin production from 885.53 to 923.43 U/mL. Lastly, the transporter LysE was confirmed to act as a Lys exporter; LysP and YvsH were identified as the Lys importers in B. licheniformis DW2, and bacitracin yield was increased to 975.43 U/mL by 28.95% in final strain LYS5 via engineering the Lys transporters. Taken together, this study implied that metabolic engineering of Lys supply modules is an efficient strategy for enhancement production of bacitracin, and provided a promising strain of B. licheniformis for industrial production of bacitracin. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with various applications. Teichoic acid (TA) is a special component of cell wall in gram-positive bacteria, and its D-alanylation modification can change the net negative charge of cell surface, autolysin activity and cationic binding efficiency, and might further affect metabolic production. In this research, four genes (dltA, dltB, dltC , and dltD) of dlt operon were, respectively, deleted and overexpressed in the γ-PGA producing strain Bacillus licheniformis WX-02. Our results implied that overexpression of these genes could all significantly increase γ-PGA synthetic capabilities, among these strains, the dltB overexpression strain WX-02/pHY- dltB owned the highest γ-PGA yield (2.54 g/L), which was 93.42% higher than that of the control strain WX-02/pHY300 (1.31 g/L). While, the gene deletion strains produced lower γ-PGA titers. Furthermore, 13C-Metabolic flux analysis was conducted to investigate the influence of dltB overexpression on metabolic flux redistribution during γ-PGA synthesis. The simulation data demonstrated that fluxes of pentose phosphate pathway and tricarboxylic acid cycle in WX-02/pHY- dltB were 36.41 and 19.18 mmol/g DCW/h, increased by 7.82 and 38.38% compared to WX-02/pHY300 (33.77 and 13.86 mmol/g DCW/h), respectively. The synthetic capabilities of ATP and NADPH were also increased slightly. Meanwhile, the fluxes of glycolytic and by-product synthetic pathways were all reduced in WX-02/pHY- dltB. All these above phenomenons were beneficial for γ-PGA synthesis. Collectively, this study clarified that overexpression of dltB strengthened the fluxes of PPP pathway, TCA cycle and energy metabolism for γ-PGA synthesis, and provided an effective strategy for enhanced production of γ-PGA. [ABSTRACT FROM AUTHOR]

Poly‐γ‐glutamic acid (γ‐PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ‐PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ‐PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ‐PGA yield. In the third approach, the genes purB and adK in ATP‐biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ‐PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two‐component system ResD–ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ‐PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 μmol/g of DCW, the mutant WX‐BCVAR with this enhancement produced 43.81 g/L of γ‐PGA, a 38.64% improvement compared to wild‐type strain WX‐02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ‐PGA production. The respiratory chain is optimized to reduce metabolism coefficient, and the VHB and AdK are strengthened to improve ATP supply for enhanced γ‐PGA production. The roles of NarGHIJ, NasBC and ResD∼E are identified in the BFCNA medium during γ‐PGA production, and Strengthening of NarG, NasC and ResD improve the nitrate metabolism, ATP supply and γ‐PGA yield. The γ‐PGA yield produced by WX‐BCVAR is increased by 38.64% via improvement of ATP supply [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is an important biochemical product with a variety of applications. This work reports a novel approach to improve γ-PGA through over expression of key enzymes in cofactor NADPH generating process for NADPH pool. Six genes encoding the key enzymes in NADPH generation were over-expressed in the γ-PGA producing strain B. licheniformis WX-02. Among various recombinants, the strain over-expressing zwf gene (coding for glucose-6-phosphate dehydrogenase), WX-zwf, produced the highest γ-PGA concentration (9.13 g/L), 35% improvement compared to the control strain WX-pHY300. However, the growth rates and glucose uptake rates of the mutant WX-zwf were decreased. The transcriptional levels of the genes pgsB and pgsC responsible for γ-PGA biosynthesis were increased by 8.21- and 5.26-fold, respectively. The Zwf activity of the zwf over expression strain increased by 9.28-fold, which led to the improvement of the NADPH generation, and decrease of accumulation of by-products acetoin and 2,3-butanediol. Collectively, these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the γ-PGA production in B. licheniformis. [ABSTRACT FROM AUTHOR]

2-Phenylethanol (2-PE) is a kind of bulk flavor and fragrance, widely applied in food, fragrance, and cosmetic industries. In this work, a sustainable platform for 2-PE biosynthesis was developed by engineering Bacillus licheniformis using agro-industrial wastes as feedstocks. Firstly, the endogenous sucrose catabolism pathway of B. licheniformis was replaced by introducing CscB from Escherichia coli W and SucP from Bifdobacterium adolescentis. The 2-PE titer and yield of the resulting strain PE24 increased to 6.52 ± 0.20 g/L and 0.16 g/g sucrose, respectively, which increased by 16% compared to the control strain PE23 using 40 g/L sucrose as the carbon source. Subsequently, the effects of molasses and crude glycerol as the feedstocks on 2-PE production were investigated. By optimizing the molasses pretreatment methods and medium components, the 2-PE production was increased from 2.21 g/L to 5.74 g/L using 50 g/L pretreated molasses, 15 g/L crude glycerol, and 7 g/L corn steep liquor powder as the raw materials. Finally, we characterized the PE24 strain by fed-batch fermentation in 5-L fermenter and achieved a titer of 6.43 g/L of 2-PE, representing the highest level of 2-PE production. This study provided a cost-effective process for 2-PE sustainable biosynthesis. [Display omitted] • Construction of an efficient surcose utilization pathway in B. licheniformis. • A cost-effective process was established for the sustainable production of 2-PE. • The highest titer of 2-PE (6.43 g/L) was obtained from agro-industrial wastes to date. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is a natural and multifunctional biopolymer. Glycerol, especially crude glycerol, was used as fermentation raw material for production of a variety of chemical products. Here, glycerol was used with nitrate addition for γ-PGA production by Bacillus licheniformis WX-02 at a 5 L fermenter scale. However, nitrite accumulation was discovered in batch fermentation, which resulted in low γ-PGA yield (19.58 g/L). Furthermore, based on transcription analysis and intracellular NADH measurement, nitrite accumulation was probably due to the low content of intracellular NADH in glycerol-based medium, which might be resulted from low efficiency of NADH generation rate and quick NADH consumption by nitrate reduction. Accordingly, different nitrate feeding strategies were designed and optimized in γ-PGA fermentation with glycerol, then applied in fermentation with crude glycerol. Finally, 35.63 g/L of γ-PGA was achieved with the constant-rate nitrate feeding of 0.38 g/(L h) by using crude glycerol, while 36.83 g/L of γ-PGA was achieved with pH-feedback controlling. Therefore, this study provided a novel strategy for cost-effective production of γ-PGA from crude glycerol. • Nitrite accumulation observed in glycerol medium resulted in low γ-PGA yield. • Intracellular NADH content was proven as the key factor for nitrite conversion. • 35 g/L γ-PGA was achieved by using optimized constant-rate nitrate feeding. • pH-feedback control reduced nitrate addition by half with 34 g/L γ-PGA production. • The nitrate feeding strategy was applied for γ-PGA production with crude glycerol. [ABSTRACT FROM AUTHOR]

L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SP SacC , SP AmyL , SP AprE , SP YwbN and SP WapA ) were screened, among which SP SacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, P ykzA-P43 , P Ubay and P bacA ) from Bacillus were screened, and tandem promoter P ykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts ( B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/P ykzA-P43 -SP SacC -ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.

Currently, industrial production of l-threonine (Thr) is based on direct fermentation with microorganisms such as Escherichia coli , which has the characteristics of low cost and high productivity. In order to elucidate the key metabolic features of the synthesis pathway of Thr in E. coli to provide clues for metabolic regulation or engineering of the strain, this study was carried out on an l-threonine over-producing strain, in terms of analyses of metabolic flux, enzyme control and metabonomics. Since environmental disturbance and genetic modification are considered to be two important methods of metabolic analysis, addition of phosphate in the media and comparison of strains with different genotypes were selected as the two candidates due to their significant influence in the biosynthesis of Thr. Some important targets including key nodes, enzymes and biomarkers were identified, which may provide target sites for rational design through engineering the Thrproducing strain. Finally, metabolic regulation aimed at one biomarker identified in this study was set as an example, which confirms that combined metabolic analyses may guide to improve the production of threonine in E. coli ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Cai, Chen, Chen and Luo.)

Iturin A is a biosurfactant with various applications, and its low synthesis capability limits its production and application development. Fatty acids play a critical role in cellular metabolism and target product syntheses, and the relationship between fatty acid supplies and iturin A synthesis is unclear. In this study, we attempted to increase iturin A production via strengthening fatty acid synthesis pathways in Bacillus amyloliquefaciens . First, acetyl-CoA carboxylase AccAD and ACP S-malonyltransferase fabD were overexpressed via promoter replacement, and iturin A yield was increased to 1.36 g/L by 2.78-fold in the resultant strain HZ-ADF1. Then, soluble acyl-ACP thioesterase derived from Escherichia coli showed the best performance for iturin A synthesis, as compared to those derived from B. amyloliquefaciens and Corynebacterium glutamicum , the introduction of which in HZ-ADF1 further led to a 57.35% increase of iturin A yield, reaching 2.14 g/L. Finally, long-chain fatty acid-CoA ligase LcfA was overexpressed in HZ-ADFT to attain the final strain HZ-ADFTL2, and iturin A yield reached 2.96 g/L, increasing by 6.59-fold, and the contents of fatty acids were enhanced significantly in HZ-ADFTL2, as compared to the original strain HZ-12. Taken together, our results implied that strengthening fatty acid supplies was an efficient approach for iturin A production, and this research provided a promising strain for industrial production of iturin A., Competing Interests: The author JG was employed by the Hubei Corporation of China National Tobacco Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gao, She, Shi, Cai, Wang, Xiong, Shen, Gao, Zhang, Yang and Chen.)