Biochemical, molecular and in silico characterization of arsenate reductase from Bacillus thuringiensis KPWP1 tolerant to salt, arsenic and a wide range of pH.

The present study characterized aresenate reductase of Bacillus thuringiensis KPWP1, tolerant to salt, arsenate and a wide range of pH during growth. Interestingly, it was found that arsC, arsB and arsR genes involved in arsenate tolerance are distributed in the genome of strain KPWP1. The inducible arsC gene was cloned, expressed and the purified ArsC protein showed profound enzyme activity with the KM and Kcat values as 25 µM and 0.00119 s-1, respectively. In silico studies revealed that in spite of 19-26% differences in gene sequences, the ArsC proteins of Bacillus thuringiensis, Bacillus subtilis and Bacillus cereus are structurally conserved and ArsC structure of strain KPWP1 is close to nature. Docking and analysis of the binding site showed that arsenate ion interacts with three cysteine residues of ArsC and predicts that the ArsC from B. thuringiensis KPWP1 reduces arsenate by using the triple Cys redox relay mechanism.

Enzymatic Synthesis of 3'-5', 3'-5' Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

TipB, a novel cell wall hydrolase, is required for efficient conjugative transfer of pXO16 from Bacillus thuringiensis sv. israelensis.

pXO16, a large plasmid from Bacillus thuringiensis serovar israelensis, exhibits unique features. Not only is pXO16 able to transfer at high frequencies within few minutes, but it is also able to transfer among virtually all members of the Bacillus cereus group. Among the proteins encoded in the tip transfer locus of pXO16, TipB displays an atypical organization compared to known conjugative cell wall hydrolases with the large central soluble lytic transglycosylase (SLT) domain missing from the protein. We constructed a tipB deletion mutant which led to significant reduction in transfer efficiencies in both liquid and filter mating. The initial transfer frequencies could be restored when complementing tipB in trans thus showing the TipB implication in pXO16 conjugative transfer. Additionally, truncated versions of TipB were expressed in Escherichia coli to assess the protein lytic activity. When applied exogenously, TipB-2TM, in which the two N-terminal TM domains were removed, yielded a decrease of ca. 40% in optical density of B. thuringiensis sv. israelensis, a lytic activity that could partially be explained by the C-terminal CHAP-like domain. These results confirm TipB conjugative hydrolase function and provide new insights into pXO16 unique conjugative apparatus.

CdgL is a degenerate nucleotide cyclase domain protein affecting flagellin synthesis and motility in Bacillus thuringiensis.

In Bacillus subtilis, motility genes are expressed in a hierarchical pattern - governed by the σD transcription factor and other proteins such as the EpsE molecular clutch and SlrA/SlrR regulator proteins. In contrast, motile species in the Bacillus cereus group seem to express their motility genes in a non-hierarchical pattern, and less is known about their regulation, also given that no orthologs to σD, EpsE, SlrA or SlrR are found in B. cereus group genomes. Here we show that deletion of cdgL (BTB_RS26690/BTB_c54300) in Bacillus thuringiensis 407 (cry-) resulted in a six-to ten-fold downregulation of the entire motility locus, and loss of flagellar structures and swimming motility. cdgL is unique to the B. cereus group and is found in all phylogenetic clusters in the population except for group I, which comprises isolates of non-motile Bacillus pseudomycoides. Analysis of RNA-Seq data revealed cdgL to be expressed in a three-gene operon with a NupC like nucleoside transporter, and a putative glycosyl transferase for which transposon-based gene inactivation was previously shown to produce a similar phenotype to cdgL deletion. Interestingly, all three proteins were predicted to be membrane-bound and may provide a concerted function in the regulation of B. cereus group motility.

A Thermostable Aluminum-Tolerant Protease Produced by Feather-Degrading Bacillus thuringiensis Isolated from Tea Plantation.

BACKGROUND: Proteases with keratinolytic activity are widely used in biotechnologies. The feather-degrading Bacillus thuringensis isolated from soil sample of a tea plantation produced high level of extracellular keratinase. OBJECTIVE: This study aimed to analyze the properties by biochemical and enzymological methods to gain information for better utilization of the enzyme. METHODS: The enzyme was purified with ion exchange and size exclusion chromatography. The substrate preference, optimal pH and temperature, and the effects of organic solvents and ions were checked. Circular dichroism was performed to compare the secondary structures of the native and apo-enzyme. RESULTS: The enzyme worked best at 50°C, and it was an acidic serine protease with an optimal pH of 6.2. Ions Ca2+ and Mg2+ were essential for its activity. Organic solvents and other metal ions generally deactivated the enzyme in a concentration-dependent manner. However, Mn2+ and DMSO, which were frequently reported as inhibitors of protease, could activate the enzyme at low concentration (0.01 to 2 mmol/L of Mn2+; DMSO <2%, v/v). The enzyme exhibited high resistance to Al3+, which might be explained by the soil properties of its host's residence. Circular dichroism confirmed the contribution of ions to the structure and activity. CONCLUSION: The enzyme was a thermostable aluminum-tolerant serine protease with unique biochemical properties.

[Production of L-2-aminobutyric acid from L-threonine using a trienzyme cascade].

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.

Functional expression of recombinant hybrid enzymes composed of bacterial and insect's chitinase domains in E. coli.

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

Biochemical characterisation and application of keratinase from Bacillus thuringiensis MT1 to enable valorisation of hair wastes through biosynthesis of vitamin B-complex.

This study reports on the exploitation of keratinous hydrolysate by keratinase enzymes to produce vitamin B-complex. Toward this end, keratinase enzyme was produced by Bacillus thuringiensis strain MT1, newly isolated from cattle-yard utilising donkey hairs. Scanning electron microscope (SEM) and Fourier transform infrared spectrophotometer (FTIR) analyses demonstrated hairs disintegration and the disruption of the disulphide bonds of the keratin structure, respectively. The biochemical characterisation of the produced enzyme exhibited optimal activity of 422 U/ml at 50 °C and pH 9 with a molecular mass of 80 kDa. The enzyme activity was entirely deactivated by Ethylenediaminetetraacetic acid (EDTA), implying the existence of a metallokeratinase group. Donkey hairs were thus treated with metallokeratinase, emancipating eight essential and eight more non-essential amino acids, which were identified employing amino acid analyser. These amino acids were subsequently utilised by Saccharomyces cerevisiae strain ATCC 64712, at different concentrations, to produce vitamin B-complex. High-performance liquid chromatography (HPLC) analysis revealed the synthesis of vitamins B1, B2, and B12 at various levels associated with concentrations of supplemented amino acids. This report thus highlights the feasible application of keratinase enzyme as an eco-friendly approach to managing hair waste, and concurrently promotes the implementation of hair-based hydrolysate in vitamin B-complex biosynthesis.

Mixing of Prosopis africana pods and corn cob exerts contrasting effects on the production and quality of Bacillus thuringiensis crude endoglucanase.

Recently, attention has shifted to the use of mixed lignocellulosic substrates for the production of cellulolytic enzymes. However, researchers have focused mainly on achieving increased enzyme yields while neglecting other properties of the enzymes when using such mixtures. In this first-ever report of the application of Prosopis africana pod (PAP) in cellulase production, we investigated the effect of its combination with corn cob (CC), as an inducing carbon source, on the amounts and quality of crude endoglucanase produced by Bacillus thuringiensis SS12. The organism was grown on PAP, CC or their 1:1% w/w mixture (MS) and the crude endoglucanases produced were tested for activity, hydrolytic efficiency, and thermostability. PAP supported the highest enzyme activity (0.138 U/mL) and its endoglucanase was the most effective in hydrolyzing CMC and filter paper while CC-derived endoglucanase was the best for hydrolysis of alkali-pretreated CC. Enzyme activity of MS-derived endoglucanase (0.110 U/mL) was intermediate to that of PAP and CC (0.091 U/mL) and was the most stable at elevated temperatures (70 and 80 °C). It also liberated the least amount of reducing sugars from all tested substrates. Combination of both the substrates, thus, favored enzyme production and thermostability but was detrimental to hydrolytic efficiency.

2-Methylcitrate cycle: a well-regulated controller of Bacillus sporulation.

Bacillus thuringiensis is the most widely used eco-friendly biopesticide, containing two primary determinants of biocontrol, endospore and insecticidal crystal proteins (ICPs). The 2-methylcitrate cycle is a widespread carbon metabolic pathway playing a crucial role in channelling propionyl-CoA, but with poorly understood metabolic regulatory mechanisms. Here, we dissect the transcriptional regulation of the 2-methylcitrate cycle operon prpCDB and report its unprecedented role in controlling the sporulation process of B. thuringiensis. We found that the transcriptional activity of the prp operon encoding the three critical enzymes PrpC, PrpD, and PrpB in the 2-methylcitrate cycle was negatively regulated by the two global transcription factors CcpA and AbrB, while positively regulated by the LysR family regulator CcpC, which jointly account for the fact that the 2-methylcitrate cycle is specifically and highly active in the stationary phase of growth. We also found that the prpD mutant accumulated 2-methylcitrate, the intermediate metabolite of the 2-methylcitrate cycle, which delayed and inhibited sporulation at the early stage. Thus, our results not only revealed sophisticated transcriptional regulatory mechanisms for the metabolic 2-methylcitrate cycle but also identified 2-methylcitrate as a novel regulator of sporulation in B. thuringiensis.

Bisphenol A degradation pathway and associated metabolic networks in Escherichia coli harboring the gene encoding CYP450.

Although bisphenol A (BPA) can be transformed by CYP450, the metabolic networks involved in regulating the transformation processes are not clear. In this study, Escherichia coli harboring the gene encoding CYP450 was used as a model to elucidate the BPA degradation pathway and the associated metabolic network using a proteomic approach. The results showed that CYP450 promotes the transformation of BPA, generating 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol, with hydroquinone and 4-(2-hydroxypropan-2-yl)phenol formed in another pathway. The DNA adducts formed by 1,4-benzoquinone were reduced, and CYP450 played a positive role in cellular homeostasis by promoting the transformation of BPA and mismatch repair. An increase in the synthesis of cell membrane lipids was observed after dislodging BPA. BPA disturbed folate metabolism by decreasing the abundance of dihydrofolate reductase, which inhibited microbial metabolism in the absence of CYP450. The findings of this study revealed the molecular mechanism associated with the metabolic network responsible for pollutant tolerance and degradation.

Development and characterization of chitosan/polyvinyl alcohol polymer material with elastolytic and collagenolytic activities.

The biologically active polymeric material with entrapped peptidase Bacillus thuringiensis var. israelensis with caseinolytic, collagenase and elastase activities was developed as a promising product for medical use. We have evaluated and reported here the following physical-chemical and biochemical characteristics of entrapped enzyme: peptidase/polymer interaction and morphology analyses, film thickness, water content, time of dissolution in water and physiological saline, kinetic of casein hydrolysis and pH- and thermoprofiles of proteolytic activity. Scanning electron microscopy images shows the relative uniformity of the film surface with entrapped peptidase. The released peptidase was characterized by increased proteolytic activity in the acidic (14%-35%) and alkaline (7%-32%) regions. After nine months of storage, peptidase in chitosan/polyvinyl alcohol films retains more than 95% of its initial proteolytic activity. We consider this film as a perspective biotechnological agent in medicine.

A Genome-Centric Approach Reveals a Novel Glycosyltransferase from the GA A07 Strain of Bacillus thuringiensis Responsible for Catalyzing 15-O-Glycosylation of Ganoderic Acid A.

Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15-O-ß-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis. Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes (FQZ25_16345, FQZ25_19840, and FQZ25_19010) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010, were successfully cloned and expressed in a soluble form in Escherichia coli, and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15-O-ß-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (kcat/KM) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates.

Diversity of the Rap-Phr quorum-sensing systems in the Bacillus cereus group.

Bacteria of the Bacillus cereus group colonize several ecological niches and infect different hosts. Bacillus cereus, a ubiquitous species causing food poisoning, Bacillus thuringiensis, an entomopathogen, and Bacillus anthracis, which is highly pathogenic to mammals, are the most important species of this group. These species are closely related genetically, and their specific toxins are encoded by plasmids. The infectious cycle of B. thuringiensis in its insect host is regulated by quorum-sensing systems from the RNPP family. Among them, the Rap-Phr systems, which are well-described in Bacillus subtilis, regulate essential processes, such as sporulation. Given the importance of these systems, we performed a global in silico analysis to investigate their prevalence, distribution, diversity and their role in sporulation in B. cereus group species. The rap-phr genes were identified in all selected strains with 30% located on plasmids, predominantly in B. thuringiensis. Despite a high variability in their sequences, there is a remarkable association between closely related strains and their Rap-Phr profile. Based on the key residues involved in RapH phosphatase activity, we predicted that 32% of the Rap proteins could regulate sporulation by preventing the phosphorylation of Spo0F. These Rap are preferentially located on plasmids and mostly related to B. thuringiensis. The predictions were partially validated by in vivo sporulation experiments suggesting that the residues linked to the phosphatase function are necessary but not sufficient to predict this activity. The wide distribution and diversity of Rap-Phr systems could strictly control the commitment to sporulation and then improve the adaptation capacities of the bacteria to environmental changes.

The crystal structure of the chitinase ChiA74 of Bacillus thuringiensis has a multidomain assembly.

There is no structural information about any chitinase synthesized by Bacillus thuringiensis, the most successful microbial insect larvicide used worldwide. In this study, we solved the 3D structure of the chitinase ChiA74 at 2.26 Å. The crystal structure shows that ChiA74 is composed of a modular arrangement formed by (i) a catalytic region (CD), (ii) a chitinase insertion domain (CID), (iii) a fibronectin type III domain (FnIII), and (iv) a chitin binding domain (CBD). The location of the CBD with respect to the CD has no structural similarity to other chitinases with known structures. The activity of a ChiA74 lacking its secretion signal peptide (ChiA74Δsp) and a truncated version lacking its CBD/FnIII domains (ChiA74Δsp-50) did not have statistical differences in activity against colloidal chitin. However, ChiA74Δsp exhibits 4.5 and 2.0 higher activity than versions lacking the CBD (ChiA74Δsp-60) and CBD/FnIII domains (ChiA74Δsp-50), respectively, when crystalline chitin was used as substrate. Our data suggest that the CBD might plays a significant role in crystalline chitin hydrolysis. We also demonstrated the importance of the catalytic E211 in the CD, as mutants ChiA74ΔspE211N and ChiA74ΔspD207N, E211N were inactive against colloidal and crystalline chitins, chitosan and 4-MU-GlcNAc3. ChiA74 has a processive activity producing oligosaccharides with degree of polymerization (DP) of 1 (GlcNAc) and 2 (GlcNAc2).

A carbohydrate binding module-5 is essential for oxidative cleavage of chitin by a multi-modular lytic polysaccharide monooxygenase from Bacillus thuringiensis serovar kurstaki.

Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards ß-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble ß-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.

CalY is a major virulence factor and a biofilm matrix protein.

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms.

Benzo(a)pyrene degradation by cytochrome P450 hydroxylase and the functional metabolism network of Bacillus thuringiensis.

The relationship between benzo(a)pyrene biodegradation and certain target biomolecules has been investigated. To regulate the degradation process, the associated metabolism network must be clarified. To this end, benzo(a)pyrene degradation, carbon substrate metabolism and exometabolomic mechanism of Bacillus thuringiensis were analyzed. Benzo(a)pyrene was degraded through hydroxylation catalyzed by cytochrome P450 hydroxylase. After the treatment of 0.5 mg L-1 of benzo(a)pyrene by 0.2 g L-1 of cells for 9 d, biosorption and degradation efficiencies were measured at approximately 90% and 80%, respectively. During this process, phospholipid synthesis, glycogen, asparagine, arginine, itaconate and xylose metabolism were significantly downregulated, while glycolysis, pentose phosphate pathway, citrate cycle, amino sugar and nucleotide sugar metabolism were significantly upregulated. These findings offer insight into the biotransformation regulation of polycyclic aromatic hydrocarbons.

A single point mutation in hmgA leads to melanin accumulation in Bacillus thuringiensis BMB181.

Bacillus thuringiensis BMB181 (Bt BMB181), with high melanin production, is an ideal candidate for industrial scale production of light-stable insecticides. However, its melanogenic pathways remain unclear. In the present study, we demonstrated that Bt BMB181 failed to produce melanin after treatment with mesotrione, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the homogentisic acid pathway of melanin synthesis. Heterologous expression experiments suggested that homogentisate-1,2-dioxygenase (HmgA) in Bt BMB171 functions normally, yet HmgA in Bt BMB181 had lost its activity, at least partly. Using the CRISPR-Cas9 system, the hmgA gene in Bt BMB171 was knocked out and the mutant strain gained the ability to produce melanin. Furthermore, the complemented strain reverted to the wild-type phenotype. In addition, overexpression of its own hmgA gene in Bt BMB181 also resulted in failure to produce the pigment. BLAST results indicated that the amino acid alteration (G272E) in HmgA of Bt BMB181 was caused by a single point mutation (815G→ A). The enzyme activity of purified HmgA171 was more than 10-fold higher than that of HmgA181. Finally, we determined that the mutation in hmgA was responsible for melanin accumulation in Bt BMB181. Our results provided new insights into the synthesis and regulation of melanin production in B.thuringiensis and will promote its future industrial application.

Extender Unit Promiscuity and Orthogonal Protein Interactions of an Aminomalonyl-ACP Utilizing Trans-Acyltransferase from Zwittermicin Biosynthesis.

Trans-acting acyltransferases (trans-ATs) are standalone enzymes that select and deliver extender units to polyketide synthase assembly lines. Accordingly, there is interest in leveraging trans-ATs as tools to regioselectively diversify polyketide structures. Yet, little is known regarding the extender unit and acyl carrier protein (ACP) specificity of trans-ATs, particularly those that utilize unusual ACP-linked extender units. For example, the biosynthesis of the antibiotic zwittermicin involves the trans-AT ZmaF, which is responsible for installing a rare ACP-linked aminomalonyl extender unit. Here, we developed a method to access a panel of non-natural and non-native ACP-linked extender units and used it to probe the promiscuity of ZmaF, revealing one of the most promiscuous ATs characterized to date. Furthermore, we demonstrated that ZmaF is highly orthogonal with respect to its ACP specificity, and the ability of ZmaF to trans-complement noncognate PKS modules was also explored. Together, these results set the stage for further engineering ZmaF as a tool for polyketide diversification.