Development of an Inosine Hyperproducer from Bacillus licheniformis by Systems Metabolic Engineering.

Inosine is widely used in food, chemical, and medicine. This study developed Bacillus licheniformis into an inosine hyperproducer through systems metabolic engineering. First, purine metabolism was activated by deleting inhibitors PurR and YabJ and overexpressing the pur operon. Then, the 5-phosphoribosyl-1-pyrophosphate (PRPP) supply was increased by optimizing the glucose transport system and pentose phosphate pathway, increasing the inosine titer by 97% and decreasing the titers of byproducts by 36%. Next, to prevent the degradation of inosine, genes deoD and pupG coding purine nucleoside phosphorylase were deleted, accumulating 0.91 g/L inosine in the culture medium. Additionally, the downregulation of adenosine 5'-monophosphate (AMP) synthesis pathway increased the inosine titer by 409%. Importantly, enhancing the glycine and aspartate supply increased the inosine titer by 298%. Finally, the guanosine synthesis pathway was blocked, leading to strain IR-8-2 producing 27.41 g/L inosine with a 0.46 g inosine/g glucose yield and a 0.38 g/(L·h) productivity in a shake flask.

CircPTEN suppresses human clear cell renal carcinoma progression and resistance to mTOR inhibitors by targeting epigenetic modification.

Renal cell carcinoma (RCC) is known to be the most commonly diagnosed kidney cancer. Clear cell RCC (ccRCC) represents approximately 85 % of diagnosed RCC cases. Targeted therapeutics, such as multi-targeted tyrosine kinase inhibitors (TKI) and mTOR inhibitors, are widely used in ccRCC therapy. However, patients treated with mTOR and TKI inhibitors easily acquire drug resistance, making the therapy less effective. Here, we demonstrated that circPTEN inhibits the expression of its parental gene PTEN by reducing methylation of the PTEN promotor and inhibits GLUT1 expression by reducing m6A methylation of GLUT1, which suppresses ccRCC progression and resistance to mTOR inhibitors.

Metabolic engineering of Bacillus amyloliquefaciens for efficient production of α-glucosidase inhibitor1-deoxynojirimycin.

Owing to the feature of strong α-glucosidase inhibitory activity, 1-deoxynojirimycin (1-DNJ) has broad application prospects in areas of functional food, biomedicine, etc., and this research wants to construct an efficient strain for 1-DNJ production, basing on Bacillus amyloliquefaciens HZ-12. Firstly, using the temperature-sensitive shuttle plasmid T2 (2)-Ori, gene ptsG in phosphotransferase system (PTS) was weakened by homologous recombination, and non-PTS pathway was strengthened by deleting its repressor gene iolR, and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold, reached 110.72 mg/L. Then, to increase precursor fructose-6-phosphate (F-6-P) supply, phosphofructokinase was weaken, fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement, moreover, regulator gene nanR was deleted, 1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold. Subsequently, promoter of 1-DNJ synthetase cluster was optimized, as well as 5'-UTRs of downstream genes in synthetase cluster, and 1-DNJ produced by the final strain reached 478.62 mg/L. Last but not the least, 1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter, which was the highest yield of 1-DNJ reported to date. Taken together, our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis, this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.

A novel toolbox for precise regulation of gene expression and metabolic engineering in Bacillus licheniformis.

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.

Microbial synthesis of bacitracin: Recent progress, challenges, and prospects.

Microorganisms are important sources of various natural products that have been commercialized for human medicine and animal healthcare. Bacitracin is an important antibacterial natural product predominantly produced by Bacillus licheniformis and Bacillus subtilis, and it is characterized by a broad antimicrobial spectrum, strong activity and low resistance, thus bacitracin is extensively applied in animal feed and veterinary medicine industries. In recent years, various strategies have been proposed to improve bacitracin production. Herein, we systematically describe the regulation of bacitracin biosynthesis in genus Bacillus and its associated mechanism, to provide a theoretical basis for bacitracin overproduction. The metabolic engineering strategies applied for bacitracin production are explored, including improving substrate utilization, using an enlarged precursor amino acid pool, increasing ATP supply and NADPH generation, and engineering transcription regulators. We also present several approaches of fermentation process optimization to facilitate the industrial large-scale production of bacitracin. Finally, the challenges and prospects associated with microbial bacitracin synthesis are discussed to facilitate the establishment of high-yield and low-cost biological factories.

Enhancing the activity of disulfide-bond-containing proteins via promoting disulfide bond formation in Bacillus licheniformis.

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.

Systematic Adaptation of Bacillus licheniformis to 2-Phenylethanol Stress.

The compound 2-phenylethanol (2-PE) is a bulk flavor and fragrance with a rose-like aroma that can be produced by microbial cell factories, but its cellular toxicity inhibits cellular growth and limits strain performance. Specifically, the microbe Bacillus licheniformis has shown a strong tolerance to 2-PE. Understanding these tolerance mechanisms is crucial for achieving the hyperproduction of 2-PE. In this report, the mechanisms of B. licheniformis DW2 resistance to 2-PE were studied by multi-omics technology coupled with physiological and molecular biological approaches. 2-PE induced reactive oxygen species formation and affected nucleic acid, ribosome, and cell wall synthesis. To manage 2-PE stress, the antioxidant and global stress response systems were activated; the repair system of proteins and homeostasis of the ion and osmotic were initiated. Furthermore, the tricarboxylic acid cycle and NADPH synthesis pathways were upregulated; correspondingly, scanning electron microscopy revealed that cell morphology was changed. These results provide deeper insights into the adaptive mechanisms of B. licheniformis to 2-PE and highlight the potential targets for genetic manipulation to enhance 2-PE resistance. IMPORTANCE The ability to tolerate organic solvents is essential for bacteria producing these chemicals with high titer, yield, and productivity. As exemplified by 2-PE, bioproduction of 2-PE represents a promising alternative to chemical synthesis and plant extraction approaches, but its toxicity hinders successful large-scale microbial production. Here, a multi-omics approach is employed to systematically study the mechanisms of B. licheniformis DW2 resistance to 2-PE. As a 2-PE-tolerant strain, B. licheniformis displays multifactorial mechanisms of 2-PE tolerance, including activating global stress response and repair systems, increasing NADPH supply, changing cell morphology and membrane composition, and remodeling metabolic pathways. The current work yields novel insights into the mechanisms of B. licheniformis resistance to 2-PE. This knowledge can also be used as a clue for improving bacterial performances to achieve industrial-scale production of 2-PE and potentially applied to the production of other relevant organic solvents, such as tyrosol and hydroxytyrosol.

Modular Engineering to Enhance Keratinase Production for Biotransformation of Discarded Feathers.

Biotransformation of wasted feathers via feather-degrading enzyme has gained immense popularity, low conversion efficiency hinders its scale application, and the main purpose of this study is to improve feather-degrading enzyme production in Bacillus licheniformis. Firstly, keratinase from Bacillus amyloliquefaciens K11 was attained with the best performance for feather hydrolysis, via screening several extracellular proteases from Bacillus; also, feather powder was proven as the most suitable substrate for determination of feather-degrading enzyme activity. Then, expression elements, including signal peptides and promoters, were optimized, and the combination of signal peptide SPSacC with promoter Pdual3 owned the best performance, keratinase activity aggrandized by 6.21-fold. According to amino acid compositions of keratinase and feeding assays, Ala, Val, and Ser were proven as critical precursors, and strengthening these precursors' supplies via metabolic pathway optimization resulted in a 33.59% increase in the keratinase activity. Furthermore, keratinase activity reached 2210.66 U/mL, up to 56.74-fold from the original activity under the optimized fermentation condition in 3-L fermentor. Finally, the biotransformation process of discarded feathers by the fermented keratinase was optimized, and our results indicated that 90.94% of discarded feathers (16%, w/v) were decomposed in 12 h. Our results suggested that strengthening precursor amino acids' supplies was an efficient strategy for enhanced production of keratinase, and this research provided an efficient strain as well as the biotransformation process for discarded feather re-utilization.

N6-methyladenosine-modified circular RNA QSOX1 promotes colorectal cancer resistance to anti-CTLA-4 therapy through induction of intratumoral regulatory T cells.

BACKGROUND: Colorectal cancer (CRC) is the 3rd most common cancer worldwide. CircRNAs are promising novel biomarkers for CRC. T regulatory (Treg) cells express the immune checkpoint receptor of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and promote tumor immunological tolerance. We therefore investigate the biological functions and mechanisms of circQSOX1 in CRC tumorigenesis; involvement of circQSOX1 in promoting Treg cell-mediated CRC immune escape in anti-CTLA-4 therapy. METHODS: Bioinformatics analyses were performed for circQSOX1expressions, specific binding sites, and N6-methyladenosine (m6A) motifs of circQSOX1, thatwere further validated with a series of experiments. Functions of circQSOX1 in promoting CRC development, Treg cells-based immune escape, and anti-CTLA-4 therapy response were investigated both in vitro and in vivo. RESULTS: High circQSOX1 expression was associated with carcinogenesis and poor clinical outcome of CRC patients. METTL3-mediated RNA m6A modification on circQSOX1 could be read by IGF2BP2 in CRC cells. CircQSOX1 promoted CRC development by regulating miR-326/miR-330-5p/PGAM1 axis. CircQSOX1 regulated glycolysis and promoted immune escape of CRC cells, and inhibits anti-CTLA-4 therapy response in CRC patients. CONCLUSION: m6A-modified circQSOX1 facilitated CRC tumorigenesis by sponging miR-326 and miR-330-5p to promotes PGAM1 expression, which further promoted CRC immune escape by activating glycolysis and inactivating the anti-CTLA-4 therapy response of CRC. Combined treatment with sh-circQSOX1 and anti-CTLA-4 could be a strategy to overcome Treg cell-mediated CRC immune therapy resistance.

Virtual reality training improves accommodative facility and accommodative range.

AIM: To evaluate the effects of virtual reality (VR) training on different parameters of vision. METHODS: Sixty individuals ranged 18-60 years old with asthenopia were randomly divided into short-term (n=40) and long-term (n=20) treatment groups. They were given a specially designed VR training device only once for 15min or 3-4 times a day for 15min each time for 1mo. The visual acuity, spherical equivalent, accommodative range, accommodative facility, pupil size, and visual fatigue were evaluated before (control) and after VR training. RESULTS: The visual acuity, accommodative range, and accommodative facility increased in subjects of the short-term treatment group, whereas their pupil size contracted significantly. No significant changes in spherical equivalent and visual fatigue were observed. The changes in distant vision and corrected visual acuity were positively correlated with those in pupil size, but not with spherical equivalent. The accommodative range and accommodative facility improved significantly in subjects of the long-term treatment group. No significant changes in visual acuity, spherical equivalent, pupil size, and visual fatigue were noted. CONCLUSION: VR training can improve the accommodative range and accommodative facility of human eyes. Although short-term VR training can transiently improve vision, which probably due to bright light adaptation, there is no evidence that it can improve myopia.

PIK3CA mutations-mediated downregulation of circLHFPL2 inhibits colorectal cancer progression via upregulating PTEN.

BACKGROUND: PIK3CA mutation and PTEN suppression lead to tumorigenesis and drug resistance in colorectal cancer (CRC). There is no research on the role of circular RNAs (circRNAs) in regulating PIK3CA mutation and MEK inhibitor resistance in CRC. METHODS: The expression of circLHFPL2 in PIK3CA-mutant and wild-type cells and tissues was quantified by RNA-sequencing and qRT-PCR. CCK-8 assay and colony formation assay were used to evaluate cell viability. Annexin V/PI staining was implemented to assess cell apoptosis. Luciferase assay, biotin-coupled microRNA capture, and RIP assay were used to validate the interaction among potential targets. Western blotting and qRT-PCR assays were used to evaluate the expression of involved targets. Xenograft tumor in a nude mouse model was used to explore the role of circRNAs in vivo. RESULTS: RNA sequencing defined downregulated expression of circLHFPL2 in both PIK3CAH1047R (HCT116) and PIK3CAE545K (DLD1) cells. CircLHFPL2 was also downregulated in PIK3CA-mutant CRC primary cells and tissues, which was correlated with poor prognosis. CircLHFPL2 was mainly localized in the cytoplasm and its downregulation was attributed to the PI3K/AKT signaling pathway activated by phosphorylating Foxo3a. CircLHFPL2 inhibited PI3KCA-Mut CRC progression both in vitro and in vivo. Furthermore, our work indicated that circLHFPL2 acts as a ceRNA to sponge miR-556-5p and miR-1322 in CRC cells and in turn modulate the expression of PTEN. Importantly, circLHFPL2 was able to overcome PIK3CA-mediated MEK inhibitor resistance in CRC cells. CONCLUSIONS: Downregulation of circLHFPL2 sustains the activation of the PI3K/AKT signaling pathway via a positive feedback loop in PIK3CA-mutant CRC. In addition, downregulation of circLHFPL2 leads to MEK inhibitor resistance in CRC. Therefore, targeting circLHFPL2 could be an effective approach for the treatment of CRC patients harboring oncogenic PIK3CA mutations.

Nuclear translocation of p85ß promotes tumorigenesis of PIK3CA helical domain mutant cancer.

PI3Ks consist of p110 catalytic subunits and p85 regulatory subunits. PIK3CA, encoding p110α, is frequently mutated in human cancers. Most PIK3CA mutations are clustered in the helical domain or the kinase domain. Here, we report that p85ß disassociates from p110α helical domain mutant protein and translocates into the nucleus through a nuclear localization sequence (NLS). Nuclear p85ß recruits deubiquitinase USP7 to stabilize EZH1 and EZH2 and enhances H3K27 trimethylation. Knockout of p85ß or p85ß NLS mutant reduces the growth of tumors harboring a PIK3CA helical domain mutation. Our studies illuminate a novel mechanism by which PIK3CA helical domain mutations exert their oncogenic function. Finally, a combination of Alpelisib, a p110α-specific inhibitor, and an EZH inhibitor, Tazemetostat, induces regression of xenograft tumors harboring a PIK3CA helical domain mutation, but not tumors with either a WT PIK3CA or a PIK3CA kinase domain mutation, suggesting that the drug combination could be an effective therapeutic approach for PIK3CA helical domain mutant tumors.

Multilevel metabolic engineering of Bacillus licheniformis for de novo biosynthesis of 2-phenylethanol.

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.

Exosomes as drug delivery system in gastrointestinal cancer.

Gastrointestinal cancer is one of the most common malignancies with relatively high morbidity and mortality. Exosomes are nanosized extracellular vesicles derived from most cells and widely distributed in body fluids. They are natural endogenous nanocarriers with low immunogenicity, high biocompatibility, and natural targeting, and can transport lipids, proteins, DNA, and RNA. Exosomes contain DNA, RNA, proteins, lipids, and other bioactive components, which can play a role in information transmission and regulation of cellular physiological and pathological processes during the progression of gastrointestinal cancer. In this paper, the role of exosomes in gastrointestinal cancers is briefly reviewed, with emphasis on the application of exosomes as drug delivery systems for gastrointestinal cancers. Finally, the challenges faced by exosome-based drug delivery systems are discussed.

Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis.

Terminators serve as the regulatory role in gene transcription termination; however, few researches about terminator optimization have been conducted, which leads to the lack of available and universal terminator for gene expression regulation in Bacillus. To solve this problem and expand synthetic biology toolbox of Bacillus licheniformis, the terminator T1 of endogenous α-amylase gene (amyL) was characterized in this research, with a termination efficiency of 87.81%. Then, we explored and optimized the termination strength of terminator T1 from four aspects: the distance between stop codon and terminator, GC content at the bottom of stem structure, loop size, and U-tract length, and the best terminator T24 was attained by combination optimization strategy, which termination efficiency was increased to 97.97%, better than the commonly used terminator T7 (T7P) from Escherichia coli. Finally, terminator T24 was applied to protein expression, which, respectively, led to 33.00%, 25.93%, and 11.78% increases of green fluorescence intensity, red fluorescence intensity, and keratinase activity, indicating its universality in protein expression. Taken together, this research not only expands a plug-and-play synthetic biology toolbox in B. licheniformis but also provides a reference for the artificial design of versatile intrinsic terminator.

Development and characterization of acidic-pH-tolerant mutants of Zymomonas mobilis through adaptation and next-generation sequencing-based genome resequencing and RNA-Seq.

BACKGROUND: Acid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5 ~ 7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0. RESULTS: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50 ~ 130% enhancement on growth rate, 4 ~ 9 h reduction on fermentation time to consume glucose, and 20 ~ 63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole-genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs), respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role in acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions. CONCLUSION: An acidic-pH-tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

Engineering Expression Cassette of pgdS for Efficient Production of Poly-γ-Glutamic Acids With Specific Molecular Weights in Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is an emerging biopolymer with various applications and γ-PGAs with different molecular weights exhibit distinctive properties. However, studies on the controllable molecular weights of biopolymers are limited. The purpose of this study is to achieve production of γ-PGAs with a wide range of molecular weights through manipulating the expression of γ-PGA depolymerase (PgdS) in Bacillus licheniformis WX-02. Firstly, the expression and secretion of PgdS were regulated through engineering its expression elements (four promoters and eight signal peptides), which generated γ-PGAs with molecular weights ranging from 6.82 × 104 to 1.78 × 106 Da. Subsequently, through combination of promoters with signal peptides, the production of γ-PGAs with a specific molecular weight could be efficiently obtained. Interestingly, the γ-PGA yield increased with the reduced molecular weight in flask cultures (Pearson correlation coefficient of -0.968, P < 0.01). Finally, in batch fermentation, the highest yield of γ-PGA with a weight-average molecular weight of 7.80 × 104 Da reached 39.13 g/L under glutamate-free medium. Collectively, we developed an efficient strategy for one-step production of γ-PGAs with specific molecular weights, which have potential application for industrial production of desirable γ-PGAs.

Efficient synthesis of 2-phenylethanol from L-phenylalanine by engineered Bacillus licheniformis using molasses as carbon source.

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.

Multistep Metabolic Engineering of Bacillus licheniformis To Improve Pulcherriminic Acid Production.

The cyclodipeptide pulcherriminic acid, produced by Bacillus licheniformis, is derived from cyclo(l-Leu-l-Leu) and possesses excellent antibacterial activities. In this study, we achieved the high-level production of pulcherriminic acid via multistep metabolic engineering of B. licheniformis DWc9n*. First, we increased leucine (Leu) supply by overexpressing the ilvBHC-leuABCD operon and ilvD, involved in Leu biosynthesis, to obtain strain W1, and the engineered strain W2 was further attained by the deletion of gene bkdAB, encoding a branched-chain α-keto acid dehydrogenase in W1. As a result, the intracellular Leu content and pulcherriminic acid yield of W2 reached 147.4 mg/g DCW (dry cell weight) and 189.9 mg/liter, which were 227.6% and 48.9% higher than those of DWc9n*, respectively. Second, strain W3 was constructed through overexpressing the leucyl-tRNA synthase gene leuS in W2, and it produced 367.7 mg/liter pulcherriminic acid. Third, the original promoter of the pulcherriminic acid synthetase cluster yvmC-cypX in W3 was replaced with a proven strong promoter, PbacA, to produce the strain W4, and its pulcherriminic acid yield was increased to 507.4 mg/liter. Finally, pulcherriminic acid secretion was strengthened via overexpressing the transporter gene yvmA in W4, resulting in the W4/pHY-yvmA strain, which yielded 556.1 mg/liter pulcherriminic acid, increased by 337.8% compared to DWc9n*, which is currently the highest pulcherriminic acid yield to the best of our knowledge. Taken together, we provided an efficient strategy for enhancing pulcherriminic acid production, which could apply to the high-level production of other cyclodipeptides.IMPORTANCE Pulcherriminic acid is a cyclodipeptide derived from cyclo(l-Leu-l-Leu), which shares the same iron chelation group with hydroxamate sidephores. Generally, pulcherriminic acid-producing strains could be the perfect candidates for antibacterial and anti-plant-pathogenic fungal agents. In this study, we obtained the promising W4/pHY-yvmA pulcherriminic acid-producing strain via a multistep metabolic modification. The engineered W4/pHY-yvmA strain is able to achieve 556.1 mg/liter pulcherriminic acid production, which is the highest yield so far to the best of our knowledge.

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis.

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.