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• Dual promoters significantly enhanced gene expression levels both at log and stationary phases. • An efficient dual-promoter Pdual3 was attained via coupling the sigB- and sigA-type promoters and sequence optimization. • The yields of heterologous proteins and target metabolites were all significantly enhanced by means of promoter Pdual3. Promoter plays the critical role in regulating gene transcription, and dual-promoter has received the widespread attentions due to its high efficiency and continuity, here, we want to construct an efficient dual-promoter for protein production and metabolic pathway enhancement. Firstly, our results indicated that P43 promoter efficiently transcribed at logarithmic period, while the σB-type promoters (PylB, PgsiB, PykzA) were active at stationary phase. Then, several dual promoters were constructed by coupling these σB-type promoters with P43, and the attained dual-promoter PykzA-P43 showed the best performance, which led to 1.72-, 3.46- and 1.85-fold increases of green fluorescence intensity, red fluorescence intensity and α-amylase activity, compared with those of the recognized strong promoter P43, respectively. Furthermore, α-amylase activity was further increased to 389.65 U/mL by 32.20 % via optimizing sigma factor binding sites (-10 and -35 boxes) of PykzA-P43, attaining the optimized dual promoter Pdual3. Finally, Pdual3 was applied in metabolic pathway enhancement, and the yields of Poly γ-glutamic acid, acetoin and 2, 3-butanediol were respectively improved by 82.01 %, 17.09 % and 99.39 %. Our results indicated that dual-promoter significantly enhanced gene expression, and this study provided an energetic dual-promoter Pdual3 for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis. [ABSTRACT FROM AUTHOR]

• The promoter PbacA was proven as the most efficient promoter for iturin A synthesis. • Deletion of regulator gene abrB improved iturin A synthetase gene cluster transcription and iturin A production. • The titer of 2013.43 mg/L iturin A was produced by optimizing fermentation medium formulas, increased by 392.15 %. • Enhancing the synthetic capability of iturin A was beneficial for the suppression of A. alternate. Brown spot disease, caused by Alternaria alternate , is one of the most destructive leaf spot diseases that made a major impact on tobacco growing. Biocontrol has attracted increasing attentions as its features of environmental friendship, promoting plant growth, etc. Iturin A, with broad spectrum antifungal activity, is a kind of biosurfactant that mainly produced by Bacillus. In this study, the promoter of iturin A synthetase cluster of Bacillus amyloliquefaciens HZ-12 was respectively replaced by promoters P43, P bacA , P srfA and P ylb , our results implied that transcriptional level of gene ituD and iturin A titer showed consistent change trends, and P bacA was proven as the most efficient promoter, iturin A titer of which reached 950.08 ± 19.43 mg/L. Furthermore, regulator gene abrB was deleted to release the repression effect of AbrB on P bacA , ituD transcriptional level and iturin A titer were increased by 133.25 % and 20.88 %, respectively. Then, the maximum iturin A titer reached 2013.43 ± 32.86 mg/L by optimizing fermentation medium, increased by 392.15 % compared to the original (408.97 ± 21.35 mg/L). Finally, our results demonstrated that enhancing iturin A synthetic capability benefited the suppression of A. alternate. Collectively, this study provided a promising strain with an efficient fermentation medium for large-scale industrial production of iturin A. [ABSTRACT FROM AUTHOR]

Cell surface engineering was proven as the efficient strategy for enhanced production of target metabolites. In this study, we want to improve the yield of target protein by engineering cell surface in Bacillus licheniformis. First, our results confirmed that deletions of d-alanyl-lipoteichoic acid synthetase gene dltD, cardiolipin synthase gene clsA and CDP-diacylglycerol-serine O-phosphatidyltransferase gene pssA were not conducive to cell growth, and the biomass of gene deletion strains were, respectively, decreased by 10.54 ± 1.43%, 14.17 ± 1.51%, and 17.55 ± 1.28%, while the concentrations of total extracellular proteins were improved, due to the increases of cell surface net negative charge and cell membrane permeability. In addition, the activities of target proteins, nattokinase, and α-amylase were also improved significantly in gene deletion strains. Furthermore, the triplicate gene (dltD, clsA, and pssA) deletion strain was constructed, which further led to the 45.71 ± 2.43% increase of cell surface net negative charge and 26.45 ± 2.31% increase of cell membrane permeability, and the activities of nattokinase and α-amylase reached 37.15 ± 0.89 FU/mL and 305.3 ± 8.4 U/mL, increased by 46.09 ± 3.51% and 96.34 ± 7.24%, respectively. Taken together, our results confirmed that cell surface engineering via deleting dltD, clsA, and pssA is an efficient strategy for enhanced production of target proteins, and this research provided a promising host strain of B. licheniformis for efficient protein expression. [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]

Soybean meal is commonly applied as the raw material in the bio-fermentation industry, and bacitracin is a widely used feed additive in the feed industry. In this study, we investigated the influence of subtilisin enhancement on soybean meal utilization and bacitracin production in Bacillus licheniformis DW2, an industrial strain for bacitracin production. Firstly, blocking sRNA aprA expression benefited bacitracin synthesis, and the bacitracin yield produced by aprA-deficient strain DW2△PaprA reached 931.43 U/mL, 18.92% higher than that of DW2 (783.25 U/mL). The bacitracin yield was reduced by 14.27% in the aprA overexpression strain. Furthermore, our results showed that deficiency of aprA led to a 6.54-fold increase of the aprE transcriptional level and a 1.84-fold increase of subtilisin activity, respectively, which led to the increases of soybean meal utilization rate (28.86%) and precursor amino acid supplies for bacitracin synthesis. Additionally, strengthening the utilization rate of soybean meal also benefited heterologous protein production, and the α-amylase and nattokinase activities were respectively enhanced by 59.81% and 50.53% in aprA-deficient strains. Collectively, this research demonstrated that strengthening subtilisin production could improve the utilization rate of soybean meal and thereby enhance bacitracin and target protein production; also, this strategy would be useful for the improvement of protein/peptide production using soybean meal as the main nitrogen source in the fermentation process. [ABSTRACT FROM AUTHOR]

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91 g L−1, 85.22% higher than that of the wild type (7.51 g L−1). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24 μmol gDCW−1) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis. [ABSTRACT FROM AUTHOR]

Nattokinase (EC 3.4.21.62) is a profibrinolytic serine protease with a potent fibrin-degrading activity, and it has been produced by many host strains. Compared to other fibrinolytic enzymes (urokinase, t-PA and streprokinase), nattokinase shows the advantages of having no side effects, low cost and long life-time, and it has the potential to be used as a drug for treating cardiovascular disease and served as a functional food additive. In this review, we focused on screening of producing strains, genetic engineering, fermentation process optimization for microbial nattokinase production, and the extraction and purification of nattokinase were also discussed in this particular chapter. The selection of optimal nattokinase producing strain was the crucial starting element for improvement of nattokinase production. Genetic engineering, protein engineering, fermentation optimization and process control have been proved to be the effective strategies for enhancement of nattokinase production. Also, extraction and purification of nattokinase are critical for the quality evaluation of nattokinase. Finally, the prospect of microbial nattokinase production was also discussed regarding the recent progress, challenge, and trends in this field. [ABSTRACT FROM AUTHOR]

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA. [ABSTRACT FROM AUTHOR]

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5′-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. Key points: • Optimizing 5′-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far. [ABSTRACT FROM AUTHOR]

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]

As a kind of biosurfactants, iturin A has attracted people’s wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5′-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. Key points: • Optimizing 5′-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far. [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]

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]

Despite industrial bio-manufacturing progress using Bacillus licheniformis , the absence of a well-characterized toolbox allowing precise regulation of multiple genes limits its expansion for basic research and application. Here, a novel gene expression toolbox (GET) was developed for precise regulation of gene expression and high-level production of 2-phenylethanol. Firstly, we established a novel promoter core region mosaic combination model to combine, characterize and analyze different core regions. Characterization and orthogonal design of promoter ribbons allowed convenient construction of an adaptable and robust GET, gene gfp expression intensity was 0.64%–16755.77%, with a dynamic range of 2.61 × 104 times, which is the largest regulatory range of GET in Bacillus based on modification of promoter P43. Then we verified the protein and species universality of GET using different proteins expressed in B. licheniformis and Bacillus subtilis. Finally, the GET for 2-phenylethanol metabolic breeding, resulting in a plasmid-free strain producing 6.95 g/L 2-phenylethanol with a yield and productivity of 0.15 g/g glucose and 0.14 g/L/h, respectively, the highest de novo synthesis yield of 2-phenylethanol reported. Taken together, this is the first report elucidating the impact of mosaic combination and tandem of multiple core regions to initiate transcription and improve the output of proteins and metabolites, which provides strong support for gene regulation and diversified product production in Bacillus. • A novel promoter core region mosaic combination model to improve promoter performance. • An adaptable and wide-range gene expression toolbox that expands the Bacillus Synthetic biology toolbox. • The toolbox has great application value in Bacillus protein expression and metabolic engineering breeding. [ABSTRACT FROM AUTHOR]

[Display omitted] • Achieving cost-effective bioproduction from starch by engineered B. licheniformis. • Modular metabolic engineering to improve starch utilization in B. licheniformis. • Starch conversion rate was increased by over 22% in multiple products production. • The highest bacitracin titer of 1252.2 U/mL was achieved in batch culture. Starch is an attractive feedstock in biorefinery processes, while the low natural conversion rate of most microorganisms limits its applications. Herein, starch metabolic pathway was systematically investigated using Bacillus licheniformis DW2 as the host organism. Initially, the effects of overexpressing amylolytic enzymes on starch hydrolysis were evaluated. Subsequently, the transmembrane transport system and intracellular degradation module were modified to accelerate the uptake of hydrolysates and their further conversion to glucose-6-phosphate. The DW2-derived strains exhibited robust growth in starch medium, and productivity of bacitracin and subtilisin were improved by 38.5% and 32.6%, with an 32.3% and 22.9% increase of starch conversion rate, respectively. Lastly, the employment of engineering strategies enabled another B. licheniformis WX-02 to produce poly-γ-glutamic acid from starch with a 2.1-fold increase of starch conversion rate. This study not only provided excellent B. licheniformis chassis for sustainable bioproduction from starch, but shed light on researches of substrate utilization. [ABSTRACT FROM AUTHOR]

Due to its pleasant rose-like scent, 2-phenylethanol (2-PE) has been widely used in the fields of cosmetics and food. Microbial production of 2-PE offers a natural and sustainable production process. However, the current bioprocesses for de novo production of 2-PE suffer from low titer, yield, and productivity. In this work, a multilevel metabolic engineering strategy was employed for the high-level production of 2-PE. Firstly, the native alcohol dehydrogenase YugJ was identified and characterized for 2-PE production via genome mining and gene function analysis. Subsequently, the redirection of carbon flux into 2-PE biosynthesis by combining optimization of Ehrlich pathway, central metabolic pathway, and phenylpyruvate pathway enabled the production of 2-PE to a titer of 1.81 g/L. Specifically, AroK and AroD were identified as the rate-limiting enzymes of 2-PE production through transcription and metabolite analyses, and overexpression of aroK and aroD efficiently boosted 2-PE synthesis. The precursor competing pathways were blocked by eliminating byproduct formation pathways and modulating the glucose transport system. Under the optimal condition, the engineered strain PE23 produced 6.24 g/L of 2-PE with a yield and productivity of 0.14 g/g glucose and 0.13 g/L/h, respectively, using a complex medium in shake flasks. This work achieves the highest titer, yield, and productivity of 2-PE from glucose via the phenylpyruvate pathway. This study provides a promising platform that might be widely useful for improving the production of aromatic-derived chemicals. • Multilevel metabolic engineering strategy was performed to achieve production of 2-PE. • Alcohol dehydrogenase YugJ was identified and characterized for production of 2-PE. • AroK and AroD were identified as the rate-limiting enzymes of 2-PE production. • Engineered B. licheniformis produced 6.24 g/L of 2-PE with yield of 0.14 g/g glucose. • 2-PE yield achieved 41% of the maximum theoretical yield on glucose. [ABSTRACT FROM AUTHOR]

2-Phenylethanol is a valuable flavoring agent with many applications. Although the bioproduction of 2-phenylethanol has been achieved by microbial fermentation, the low titer and high cost hinder its industrial-scale production. The goal of this study is to develop an efficient process for high-level production of 2-phenylethanol from L-phenylalanine. Firstly, candidate hosts for 2-phenylethanol synthesis were screened by evaluating their tolerance to 2-phenylethanol, and Bacillus licheniformis DW2 was proven to be a promising strain for 2-phenylethanol production. Subsequently, phenylpyruvate decarboxylase and alcohol dehydrogenase from different hosts were screened, and the combination of KivD from Lactococcus lactis and YqhD from Escherichia coli owned the best performance on 2-phenylethanol synthesis, and the attained strain DE4 produced 3.04 g/L 2-phenylethanol from 5.00 g/L L-phenylalanine using glucose as carbon source. Furthermore, the fermentation process was optimized using molasses as carbon source, and 2-phenylethanol titer was increased to 4.41 g/L. In fed-batch fermentation, the maximum 2-phenylethanol titer reached 5.16 g/L, with a yield of 0.65 g/g on L-phenylalanine and productivity of 0.12 g/(L.h), which was the highest 2-phenylethnol titer reported to date when molasses was used as carbon source. Collectively, this study develops a robust strain as well as the cost-efficient process for 2-phenylethanol production, which lays a substantial foundation for industrial production of 2-phenylethanol. Key points •Bacillus licheniformis is an excellent 2-PE stress-tolerant strain. •Coexpressed kivD and yqhD is most suitable for 2-PE production in B. licheniformis. •High-level production of 2-PE (5.16 g/L) was obtained by engineered strain DE4. [ABSTRACT FROM AUTHOR]

Background: Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. Results: In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100–150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. Conclusions: This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and l-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward l-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in l-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the l-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis WX-02 is a well-studied strain to produce poly-γ-glutamic acid (γ-PGA) with numerous applications. This study is to improve WX-02 strain's capability of assimilating glycerol, a major byproduct of biofuels industries, through metabolic manipulation. Through gene knockout, the GlpK pathway was identified as the sole functional glycerol catabolism pathway, while the DhaK pathway was inactive for this strain under either aerobic or anaerobic conditions. The enhancement of glycerol utilization was attempted by substituting the native glpFK promoter with the constitutive promoter (P43), ytzE promoter (PytzE), and bacABC operon promoter (PbacA), respectively. The glycerol consumptions of the corresponding mutant strains WX02-P43glpFK, WX02-PytzEglpFK, and WX02-PbacAglpFK were 30.9, 26.42, and 18.8% higher than that of the WX-02 strain, respectively. The γ-PGA concentrations produced by the three mutant strains were 33.71, 23.39, and 30.05% higher than that of WX-02 strain, respectively. When biodiesel-derived crude glycerol was used as the carbon source, the mutant WX02-P43glpFK produced 16.63 g L of γ-PGA, with a productivity of 0.35 g L h. Collectively, this study demonstrated that glycerol can be used as an effective substrate for producing γ-PGA by metabolic engineering B. licheniformis strains. [ABSTRACT FROM AUTHOR]

Disulfide bonds in proteins have strongly influence on the folding efficiency by constraining the conformational space. The inefficient disulfide bond formation of proteins is the main limiting factor of enzyme activity and stability. This study aimed to increase the activity of disulfide-bond-containing proteins via promoting disulfide bonds formation in Bacillus licheniformis. Initially, the glutamate decarboxylase GAD from Escherichia coli was selected as the model protein and introduced into the B. licheniformis. Then, the disulfide isomerase and oxidoreductase from different sources were excavated and overexpressed successively to improve the catalytic efficiency of GAD. The final engineered B. licheniformis showed significantly improved GAD specific activity (from 10.4 U/mg to 80.0 U/mg), which also presented perfect adaptability for other disulfide-bond-containing proteins, for instance, UDP-glucosyltransferase from Arabidopsis thaliana. Taken together, our work demonstrated that the activity of GAD in B. licheniformis was regulated by the disulfide bonds formation status and provided a promising platform for the expression of disulfide-bond-containing proteins. [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]

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes ( mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • Aerobic denitrification performance was improved by Bacillus licheniformis. • B. licheniformis enhanced nitrate and nitrite removal via secreted metabolites. • Lipopeptides biosurfactant lichenysin was detected in co-culture system by LC-MS. • Membrane permeability was promoted by lichenysin. • Nitrate and glucose transport rates were accelerated by lichenysin. Aerobic denitrification efficiency is not always satisfactory and high nitrite accumulation and N 2 O emission occur frequently. Though NOx reduction during aerobic denitrification is influenced by neighboring microbes, the underlying mechanism has been seldomly studied. Here, we showed markedly improved denitrification performance of a common aerobic denitrifier in its response to co-culturing with an abundant co-existing bacterium Bacillus licheniformis , and NOx removal by B. licheniformis was marginal. Compared with that in solo culture, the growth rate of denitrifiers in co-culture varied slightly, but the cytoplasmic nitrate reductase (NAR) exhibited obvious reduction capability and its encoding genes narGHI increased to 95.5 ∼ 122.2 times. It suggested that B. licheniformis improved nitrate removal by enhancing NAR activity instead of denitrifiers density. Transwell assay demonstrated non-contacting B. licheniformis could also promote nitrate and nitrite removal, and following LC-MS showed noticeable difference in the concentration of lichenysin, a lipopeptides biosurfactant metabolite from B. licheniformis , in its solo-culture and co-culture with denitrifiers. Following observation that exogenous lichenysin, but lichenysin producing mutant strain, enhanced aerobic denitrification performance verified the critical role of lichenysin here. Further investigation illustrated that lichenysin promoted the membrane permeability of aerobic denitrifiers, which facilitated the transmembrane transport of nitrate and glucose, and subsequently made more nitrate accessible to NAR and more glucose participate into oxidation for NADH generation respectively. By these means, increased nitrate reduction with less nitrite accumulation and N 2 O emission was realized in the presence of B. licheniformis. Finally, enhancement of B. licheniformis on performance of aerobic denitrifying community from wastewater treatment plant was also observed in laboratory, which suggested the feasibility of its practical application in the future. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocGD276E showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocGD276E exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis. [Display omitted] • Three-dimension structure of B. licheniformis RocG was attained by homology modeling. • Potential mutation sites of RocG were scoped by cMD and RAMD simulations. • The specificity of RocG was altered to NADH-dependent by rational enzyme design. • Cofactor engineering was proven as an effective strategy for increasing γ-PGA production. [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]

Background: Poly-γ-glutamic acid (γ-PGA) is a natural polymer with great potential applications in areas of agriculture, industry, and pharmaceutical. The biodiesel-derived glycerol can be used as an attractive feedstock for γ-PGA production due to its availability and low price; however, insufficient production of γ-PGA from glycerol is limitation. Results: The metabolic pathway of Bacillus licheniformis WX-02 was rewired to improve the efficiency of glycerol assimilation and the supply of NADPH for γ-PGA synthesis. GlpK, GlpX, Zwf, and Tkt1 were found to be the key enzymes for γ-PGA synthesis using glycerol as a feedstock. Through combinational expression of these key enzymes, the γ-PGA titer increased to 19.20 ± 1.57 g/L, which was 1.50-fold of that of the wild-type strain. Then, we studied the flux distributions, gene expression, and intracellular metabolites in WX-02 and the recombinant strain BC4 (over-expression of the above quadruple enzymes). Our results indicated that over-expression of the quadruple enzymes redistributed metabolic flux to γ-PGA synthesis. Furthermore, using crude glycerol as carbon source, the BC4 strain showed a high productivity of 0.38 g/L/h, and produced 18.41 g/L γ-PGA, with a high yield of 0.46 g γ-PGA/g glycerol. Conclusions: The approach to rewiring of metabolic pathways enables B. licheniformis to efficiently synthesize γ-PGA from glycerol. The γ-PGA productivity reported in this work is the highest obtained in glutamate-free medium. The present study demonstrates that the recombinant B. licheniformis strain shows significant potential to produce valuable compounds from crude glycerol. [ABSTRACT FROM AUTHOR]