A smart RBS library and its prediction model for robust and accurate fine-tuning of gene expression in Bacillus species.

Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, are important industrial bacteria. However, there is a lack of standardized and predictable genetic tools for convenient and reproducible assembly of genetic modules in Bacillus species to realize their full potential. In this study, we constructed a Ribosome Binding Site (RBS) library in B. licheniformis, which provides incremental regulation of expression levels over a 104-fold range. Additionally, we developed a model to quantify the resulting translation rates. We successfully demonstrated the robust expression of various target genes using the RBS library and showed that the model accurately predicts the translation rates of arbitrary coding genes. Importantly, we also extended the use of the RBS library and prediction model to B. subtilis, B. thuringiensis, and B. amyloliquefacie. The versatility of the RBS library and its prediction model enables quantification of biological behavior, facilitating reliable forward engineering of gene expression.

A new ROS response factor YvmB protects Bacillus licheniformis against oxidative stress under adverse environment.

Bacillus spp., a class of aerobic bacteria, is widely used as a biocontrol microbe in the world. However, the reactive oxygen species (ROS) will accumulate once the aerobic bacteria are exposed to environmental stresses, which can decrease cell activity or lead to cell death. Hydroxyl radical (·OH), the strongest oxide in the ROS, can damage DNA directly, which is generated through Fenton Reaction by H2O2 and free iron. Here, we proved that the synthesis of pulcherriminic acid (PA), an iron chelator produced by Bacillus spp., could reduce DNA damage to protect cells from oxidative stress by sequestrating excess free iron, which enhanced the cell survival rates in stressful conditions (salt, antibiotic, and high temperature). It was worth noting that the synthesis of PA was found to be increased under oxidative stress. Thus, we demonstrated that the YvmB, a direct negative regulator of PA synthesis cluster yvmC-cypX, could be oxidized at cysteine residue (C57) to form a dimer losing the DNA-binding activity, which led to an improvement in PA production. Collectively, our findings highlight that YvmB senses ROS to regulate PA synthesis is one of the evolved proactive defense systems in bacteria against adverse environments.IMPORTANCEUnder environment stress, the electron transfer chain will be perturbed resulting in the accumulation of H2O2 and rapidly transform to ·OH through Fenton Reaction. How do bacteria deal with oxidative stress? At present, several iron chelators have been reported to decrease the ·OH generation by sequestrating iron, while how bacteria control the synthesis of iron chelators to resist oxidative stress is still unclear. Our study found that the synthesis of iron chelator PA is induced by reactive oxygen species (ROS), which means that the synthesis of iron chelator is a proactive defense mechanism against environment stress. Importantly, YvmB is the first response factor found to protect cells by reducing the ROS generation, which present a new perspective in antioxidation studies.

Engineering the Tat-secretion pathway of Bacillus licheniformis for the secretion of cytoplasmic enzyme arginase.

The industrial bacterium Bacillus licheniformis has long been used as a microbial factory for the production of enzymes due to its ability to secrete copious amounts of native extracellular proteins and its generally regarded as safe (GRAS) status. However, most attempts to use B. licheniformis to produce heterologous and cytoplasmic enzymes primarily via the general secretory (Sec) pathway have had limited success. The twin-arginine transport (Tat) pathway offers a promising alternative for the extracellular export of Sec-incompatible proteins because it transports full, correctly folded proteins. However, compared to the Sec pathway, the yields of the Tat pathway have historically been too low for commercial use. To improve the export efficiency of the Tat pathway, we identified the optimal Tat-dependent signal peptides and increased the abundance of the Tat translocases, the signal peptidase (SPase), and the intracellular chaperones. These strategic modifications significantly improved the Tat-dependent secretion of the cytoplasmic enzyme arginase into the culture medium using B. licheniformis. The extracellular enzymatic activity of arginase showed a 5.2-fold increase after these modifications. Moreover, compared to the start strain B. licheniformis 0F3, the production of extracellular GFP was improved by 3.8 times using the strategic modified strain B. licheniformis 0F13, and the extracellular enzymatic activity of SOX had a 1.3-fold increase using the strain B. licheniformis 0F14. This Tat-based production chassis has the potential for enhanced production of Sec-incompatible enzymes, therefore expanding the capability of B. licheniformis as an efficient cellular factory for the production of high-value proteins. KEY POINTS: • Systematic genetic modification of Tat-pathway in B. licheniformis. • Significant enhancement of the secretion capacity of Tat pathway for delivery the cytoplasmic enzyme arginase. • A new platform for efficient extracellular production of Sec-incompatible enzymes.

Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

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.

Engineered multiple translation initiation sites: a novel tool to enhance protein production in Bacillus licheniformis and other industrially relevant bacteria.

Gram-positive bacteria are a nascent platform for synthetic biology and metabolic engineering that can provide new opportunities for the production of biomolecules. However, the lack of standardized methods and genetic parts is a major obstacle towards attaining the acceptance and widespread use of Gram-positive bacterial chassis for industrial bioproduction. In this study, we have engineered a novel mRNA leader sequence containing more than one ribosomal binding site (RBS) which could initiate translation from multiple sites, vastly enhancing the translation efficiency of the Gram-positive industrial strain Bacillus licheniformis. This is the first report elucidating the impact of more than one RBS to initiate translation and enhance protein output in B. licheniformis. We also explored the application of more than one RBS for both intracellular and extracellular protein production in B. licheniformis to demonstrate its efficiency, consistency and potential for biotechnological applications. Moreover, we applied these concepts for use in other industrially relevant Gram-positive bacteria, such as Bacillus subtilis and Corynebacterium glutamicum. In all, a highly efficient and robust broad-host expression element has been designed to strengthen and fine-tune the protein outputs for the use of bioproduction in microbial cell factories.

Praseodymium-Doped Cr2O3 Prepared by In Situ Pyrolysis of MIL-101(Cr) for Highly Efficient Catalytic Oxidation of 1,2-Dichloroethane.

The development of economical catalysts that exhibit both high activity and durability for chlorinated volatile organic compounds (CVOCs) elimination remains a challenge. The oxidizing and acidic sites play a crucial role in the oxidation process of CVOCs; herein, praseodymium (Pr) was introduced into CrOx catalysts via in situ pyrolysis of MIL-101(Cr). With the decomposition of the ligand, a mixed micro-mesoporous structure was formed within the M-Cr catalyst, thereby reducing the contact resistance between catalyst active sites and the 1,2-dichloroethane molecule. Moreover, the synergistic interaction between chromium and praseodymium facilitates Oß species and acidic sites, significantly enhancing the low-temperature catalytic performance and durability of the M-PrCr catalyst for 1,2-dichloroethane (1,2-DCE) oxidation. The M-30PrCr catalyst possess enhanced active oxygen sites and acid sites, thereby exhibiting the highest catalytic activity and stability. This study may provide a novel and promising strategy for practical applications in the elimination of 1,2-DCE.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

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.

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.

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.

Construct a synthetic Entner-Doudoroff pathway in Bacillus licheniformis for enhancing lichenysin production.

Lichenysin, a cyclic lipopeptide biosurfactant produced by Bacillus licheniformis, is composed of aspartate, glutamine, valine, leucine, isoleucine, and branched chain fatty acids. The synthesis of these amino acids and fatty acids requires pyruvate and NADPH as the primary precursor and cofactor. Therefore, a sufficient supply of pyruvate and NADPH is crucial for lichenysin production. This study aimed to increase lichenysin production by constructing a synthetic ED pathway in B. licheniformis WX02 through introducing phosphogluconate dehydratase (encoded by gene edd) and 2-keto-3-deoxygluconate 6-phosphate aldolase (encoded by gene eda) from Escherichia coli. Additionally, the NADP+-dependent glucose-6-phosphate dehydrogenase (encoded by gene zwf) was overexpressed, resulting in an engineered strain WX02/pHY-edda(Ec)-zwf. Analysis of the fermentation process revealed that the concentrations of pyruvate, aspartate, glutamine, valine, leucine, branched-chain fatty acids (iC15:0, aC15:0, iC16:0, iC17:0), and NADPH in WX02/pHY-edda(Ec)-zwf were increased by 77.21%, 80.41%, 85.31%, 141.64%, 44.94%, 35.08%, 38.08%, 19.33%, 21.16%, and 425%, respectively, compared to the control strain WX02/pHY300, which resulted in a 45.43% increase of lichenysin titer. This work took advantage of the ED pathway to increase lichenysin production for the first time, and provides a promising strategy for boosting the productivity of biochemicals that require pyruvate and NADPH as precursor and cofactor.