Metabolic engineering of Bacillus subtilis for high-titer production of menaquinone-7

Menaquinone-7 (MK-7) is a member of vitamin K₂ used for prevention from osteoporosis and cardiovascular calcification. This study constructed Bacillus subtilis strains for high-titer production of MK-7 through metabolic engineering approaches. In B. subtilis, MK-7 biosynthesis was categorized into five modules: glycerol dissociation pathway, shikimate pathway, pyrimidine metabolic pathway, methylerythritol phosphate pathway, and MK-7 pathway. Overexpression of GlpK and GlpD (glycerol dissociation pathway) led to a ~10% increase in the MK-7 titer. Deletion of the genes mgsA and araM increased the MK-7 production by 15%. Furthermore, overexpression of AroG^D146N (shikimate pathway), PyrG^E156K (pyrimidine metabolic pathway), HepS (methylerythritol phosphate pathway), and VHb could also increase the MK-7 titer. Finally, we obtained a recombinant strain BSMK_11 with simultaneous overexpressing the genes glpK, glpD, aroG ^fbr, pyrG ^fbr, hepS, vgb, and knockouting the genes mgsA and araM, and the MK-7 titer reached 281.4 ± 5.0 mg/L (i.e., 12.0 mg/g DCW) in a 5 L fermenter.     

Complete Genome Sequence Analysis of Bacillus subtilis Bbv57, a Promising Biocontrol Agent against Phytopathogens

Plant growth-promoting rhizobacteria (PGPR) are a group of root-associated beneficial bacteria emerging as one of the powerful agents in sustainable plant disease management. Among the PGPR, Bacillus sp. has become a popular biocontrol agent for controlling pests and the diseases of several crops of agricultural and horticultural importance. Understanding the molecular basis of the plant growth-promoting and biocontrol abilities of Bacillus spp. will allow us to develop multifunctional microbial consortia for sustainable agriculture. In our study, we attempted to unravel the genome complexity of the potential biocontrol agent Bacillus subtilis Bbv57 (isolated from the betelvine’s rhizosphere), available at TNAU, Coimbatore. A WGS analysis generated 26 million reads, and a de novo assembly resulted in the generation of 4,302,465 bp genome of Bacillus subtilis Bbv57 containing 4363 coding sequences (CDS), of which 4281 were functionally annotated. An analysis of 16S rRNA revealed its 100% identity to Bacillus subtilis IAM 12118. A detailed data analysis identified the presence of >100 CAZymes and nine gene clusters involved in the production of secondary metabolites that exhibited antimicrobial properties. Further, Bbv57 was found to harbor 282 unique genes in comparison with 19 other Bacillus strains, requiring further exploration.     

纳豆中纳豆枯草杆菌的筛选和纳豆激酶的分离过程研究

采用酪蛋白平板初筛一摇瓶复筛的筛选策略,从日本传统食品纳豆中筛选获得了一株高产纳豆激酶的纳豆枯草菌菌株,与国内公开报道的不同菌株相比,有更好的产酶能力。菌株经过液体发酵后获得含酶发酵液,采用硫酸铵盐析及Sephadex G-75凝胶层析、Sepharose CM Fast Flow离子交换层析对纳豆激酶进行了分离纯化,两步层析的纯化倍数和酶活回收率分别达到4．75、743％和1.32、77％,获得了纯度很高的纳豆激酶。     

核黄素基因工程菌的构建及其发酵的初步研究

构建整合型核黄素质粒pRB63,该质粒含有解调的B.subtilis核黄素操纵子,将其转化入B.subtilisRH13并在染色体上进行适当的扩增后得到RH13::[pRB63]n系列工程菌,其核黄素合成能力随着pRB63扩增程度的增加而增强,最终达到RH13的6～7倍.随后以RH13::[pRB63]n系列工程菌和B.subtilis YB1为亲株进行原生质体融合,筛得重组菌B.subtilis RH33.该菌在含10%葡萄糖或蔗糖的分批发酵中培养64h可产核黄素量4.2 g·L-1.采用以葡萄糖为碳源的流加发酵工艺,24 h可积累核黄素7～8 g·L-1,48 h达11～12 g·L-1,核黄素对葡萄糖的得率为0.056 g·g-1.     

枯草芽孢杆菌XF-1膏状悬浮剂增稠剂的筛选

生物相容性、贮存稳定性和析水性研究表明,先糊化6%可溶性淀粉成初级增稠剂,再在100 mL初级增稠剂中加入1 g枯草芽孢杆菌XF-1原粉和35 g可溶性淀粉所组成的初级悬浮剂,具有很好的热稳定性和耐冷性,XF-1菌体数达9.30×1010 CFU/g。     

枯草芽孢杆菌对黑土中可培养微生物的生态效应

研究了微生物农药枯草芽孢杆菌对东北黑土中可培养微生物的生态影响.其动态变化表明:低质量分数枯草芽孢杆菌对细菌总数没有明显影响,较高质量分数枯草芽孢杆菌町促进细菌总数的显著增加,其中质量分数在3200 mg/kg时,枯草芽孢杆菌对细菌的刺激强度最高,为对照的11倍.枯草芽孢杆菌对土壤中的放线菌也有刺激作用,刺激强度最高时,放线菌数量可增至对照的8.3倍左右.枯草芽孢杆菌对真菌的敏感性较低,只有质量分数高达3200 mg/kg时,才对真菌产生明显刺激作用,最高刺激强度为同时期对照的29倍.     

枯草芽孢杆菌对土壤呼吸作用和脲酶活性的影响

通过模拟实验研究了微生物农药枯草芽孢杆菌对黑土的呼吸作用和脲酶活性的生态毒理效应.结果表明:枯草芽孢杆菌各质量分数处理均表现为对土壤呼吸作用的刺激效应,并且土壤巾枯草芽孢杆菌质量分数越大,对土壤呼吸强度的刺激作用越大,其中最高质量分数(3200 mg/kg干土)处理在第42天时达到最大刺激强度,刺激率为69.1%.与对照相比,除第1天外,所有处理(32～3200 mg/kg干土)对土壤脲酶均表现出刺激效应,其中最高质量分数(3200 mg/kg干土)处理在第28天脲酶活性上升到最高,刺激率达到101.1%.     

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.     

氨基甲酸乙酯水解酶在枯草芽孢杆菌中的表达及发酵优化

将来源于赖氨酸芽孢杆菌SC02的氨基甲酸乙酯水解酶(UH)基因在枯草芽孢杆菌Bacillus subtilis WB600中进行克隆和表达,在枯草芽孢杆菌中实现了UH活性表达,在摇瓶水平通过单因素考察和响应面分析实验对氨基甲酸乙酯水解酶发酵进行优化. 结表明,酶活最高可达到14.20 U/mL,产酶最佳培养基成分为：淀粉10 g/L、磷酸氢二钾9 g/L、麦芽浸膏25 g/L、硫酸镁1 g/L、胰蛋白胨55 g/L,最适发酵温度为37℃,最佳接种量4%. 在3 L发酵罐中采用最优发酵条件,酶活在16 h达到18.03 U/mL.     

Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.     

枯草芽孢杆菌联产纳豆激酶和g-聚谷氨酸

利用实验室保存的纳豆激酶生产菌Bacillus subtilis Natto NLSSe进行了g-聚谷氨酸合成研究,在不添加谷氨酸的培养基中合成了分子量在200~300 万Da的g-聚谷氨酸,表明该菌是谷氨酸非依赖型菌. 合成纳豆激酶的合适碳、氮源分别是蔗糖和大豆蛋白胨,合成g-聚谷氨酸的合适碳、氮源分别是柠檬酸和NH4C1. 通过正交实验研究了碳、氮源对纳豆激酶和g-聚谷氨酸联产的影响,结果表明,增加培养基中大豆蛋白胨及柠檬酸的浓度能分别促进纳豆激酶和g-聚谷氨酸的合成,而不抑制另一产物的合成,有利于纳豆激酶和g-聚谷氨酸的联产. 在大豆蛋白胨10 g/L, NH4C1 9 g/L,柠檬酸15 g/L时,纳豆激酶酶活为121.2 U/mL,g-聚谷氨酸产量为1.1 g/L,均达到了单独合成时的水平.     

肌酐酶在枯草芽孢杆菌中的异源表达及分泌机制

肌酐酶是用于临床检测肾小球滤过功能的关键酶之一,但目前国内肌酐酶的生产量较低无法满足市场需求,多依赖于国外进口。为了解决这一问题,本研究将恶臭假单胞杆菌肌酐酶基因克隆至原核表达载体pMA5实现肌酐酶在枯草芽孢杆菌1A751中的异源表达。随后通过启动子优化,使肌酐酶的蛋白表达量显著提高到1.08mg/mL,并且胞外肌酐酶的比酶活力达到238U/mg。同时发现肌酐酶可不依赖于信号肽即可实现胞外分泌,因此对肌酐酶在枯草芽孢杆菌中的分泌机制进行研究。通过对经典分泌途径和Holin途径的分析排除、利用Calcein-AM/PI双染色法鉴定表达菌株1AGC的细胞膜不完整,最后通过电子显微镜观察结果表明表达菌株表面存在潜在的泄漏位点,因此证实肌酐酶在枯草芽孢杆菌中通过细胞泄漏的方式,释放到胞外培养基中。本文构建了一株基于细胞泄漏的肌酐酶高产菌株,为肌酐酶的表达和潜在工业化应用提供理论基础,同时也为枯草芽孢杆菌泄漏表达系统提供了一定的研究基础。     

Purification and enzymatic properties of the extracellular and constitutive chitosanase produced by Bacillus subtilis RKY3

BACKGROUND: Purification and enzymatic properties of a chitosanase from Bacillus subtilis RKY3 have been investigated to produce a chitooligosaccharide. The enzyme reported was extracellular and constitutive, which was purified by two sequential steps including ammonium sulfate precipitation and ion exchange chromatography. RESULTS: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified chitosanase revealed one single band corresponding to a molecular weight of around 24 kDa. The highest chitosanase activity was found to be at pH 6.0 and at 60 °C. Although the mercaptide forming agents such as Hg²⁺ (10 mmol L^?1) and p‐hydroxymercuribenzoic acid (1 mmol L^?1, 10 mmol L^?1) significantly or totally inhibited the enzyme activity, its activity was enhanced by the presence of 10 mmol L^?1 Mn²⁺. The enzyme showed activity for hydrolysis of soluble chitosan and glycol chitosan, but colloidal chitin, carboxymethyl cellulose, crystalline cellulose, and soluble starch were not hydrolyzed. The analysis of chitosan hydrolysis by thin‐layer chromatography and viscosity variation revealed that the purified enzyme should be endosplitting‐type chitosanase. CONCLUSION: The chitosanase produced by Bacillus subtilis RKY3 was a novel chitosanlytic enzyme with relatively low molecular weight, which is a versatile enzyme for chitosan hydrolysis because it could hydrolyze soluble chitosan into a biofunctional oligosaccharide at a high level. Copyright © 2011 Society of Chemical Industry     

Effect of pH and Importance of Ozone initiated Radical Reactions In Inactivating Bacillus subtilis Spore

The effect of pH on the inactivation of Bacillus subtilis spores with ozone in a batch reactor was examined in association with the role of OH radicals. The exact effect OH radicals have on ozone inactivation of microorganism is not well understood, although a direct reaction of molecular ozone has sometimes been emphasized. This study reports a novel observation that the presence of OH radicals plays a significant role in microbial inactivation. Considering the dependence of the ozone decay rate on pH, the observed C¯ T values for achieving a 2 log inactivation using the modified Chick-Watson model was 40 % lower at pH 8.2 compared to that at pH 5.6. In the presence of OH radical scavengers (tert-butanol), the observed change of C¯ T with pH was within 10%. This difference could be explained by the significant role of OH radicals.     

Specific organic acids enhance the D-ribose productivity of a transketolase-defective Bacillus subtilis strain

When the transketolase-defective and D -ribose-producing Bacillus subtilis strain ATCC 21951 was cultured in a medium provided with certain organic acids, a significantly enhanced D -ribose titre was obtained, while the production of acetoin and 2,3-butanediol drastically decreased. Although the biochemical background of this phenomenon is not completely understood, it makes D -ribose synthesis more efficient and cheaper, the latter due to an easier recovery of D -ribose from the fermentation medium. ©1997 SCI     

Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.     

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.     

枯草芽孢杆菌脂肪酶表达质粒的构建及其表达

以枯草芽孢杆菌168(B.subtilis 168)染色体为模板PCR扩增出P43启动子,与大肠杆菌-枯草杆菌穿梭质粒pUBC19相连得到表达载体pUBC-P43,然后将枯草芽孢杆菌脂肪酶基因lipA克隆到载体pUBC-P43启动子下游,得到重组质粒pUBCPL并转化B.subtilis TZ10.经中性红油脂平板、酶切和PCR方法鉴定得到重组菌TZ10/pUBCPL.宿主菌TZ10是B.subtilis DB104染色体缺失了lipA基因后获得.重组菌经初步发酵,以橄榄油为底物测定发酵上清液最高脂肪酶活力为49.1 U·L-1,而相应菌株DB104发酵最高酶活力仅为11.4 U·L-1.     

Expanding the Functionality of an Autoinduction Device for Repression of Gene Expression in Bacillus subtilis

Autonomous control of gene expression through engineered quorum-sensing processes is broadly applicable to biosynthetic pathways, including simultaneous control of different genes. It is also a powerful tool for balancing growth and production. We had previously engineered a modular autoinduction device for the control of gene expression in B. subtilis. Now, we expand its functionality to repress gene expression autonomously. The engineered R8 promoter responds to AHL accumulation in the culture medium. In a riboflavin-producing strain, the AHL-Lux complex exerts 5-fold repression on the R8-driven expression of the flavokinase/FAD synthetase gene ribC, resulting in a higher titer of the vitamin. We engineered a strain able to autonomously induce and repress different genes simultaneously, demonstrating the potential of the device for use in metabolic engineering.