Biobutanol production from sustainable biomass process of anaerobic ABE fermentation for industrial applications.

The growing population increases the need to develop advanced biological methods for utilizing renewable and sustainable resources to produce environmentally friendly biofuels. Currently, energy resources are limited for global demand and are constantly depleting and creating environmental problems. Some higher chain alcohols, like butanol and ethanol, processing similar properties to gasoline, can be alternate sources of biofuel. However, the industrial production of these alcohols remains challenging because they cannot be efficiently produced by microbes naturally. Therefore, butanol is the most interesting biofuel candidate with a higher octane number produced naturally by microbes through Acetone-Butanol-Ethanol fermentation. Feedstock selection as the substrate is the most crucial step in biobutanol production. Lignocellulosic biomass has been widely used to produce cellulosic biobutanol using agricultural wastes and residue. Specific necessary pretreatments, fermentation strategies, bioreactor designing and kinetics, and modeling can also enhance the efficient production of biobutanol. The recent genetic engineering approaches of gene knock in, knock out, and overexpression to manipulate pathways can increase the production of biobutanol in a user friendly host organism. So far various genetic manipulation techniques like antisense RNA, TargeTron Technology and CRISPR have been used to target Clostridium acetobutylicum for biobutanol production. This review summarizes the recent research and development for the efficient production of biobutanol in various aspects.

Providing octane degradation capability to Pseudomonas putida KT2440 through the horizontal acquisition of oct genes located on an integrative and conjugative element.

The extensive use of petrochemicals has produced serious environmental pollution problems; fortunately, bioremediation is considered an efficient way to fight against pollution. In line with Synthetic Biology is that robust microbial chassis with an expanded ability to remove environmental pollutants are desirable. Pseudomonas putida KT2440 is a robust lab microbe that has preserved the ability to survive in the environment and is the natural host for the self-transmissible TOL plasmid, which allows metabolism of toluene and xylenes to central metabolism. We show that the P. putida KT2440 (pWW0) acquired the ability to use octane as the sole C-source after acquisition of an almost 62-kb ICE from a microbial community that harbours an incomplete set of octane metabolism genes. The ICE bears genes for an alkane monooxygenase, a PQQ-dependent alcohol dehydrogenase and aldehyde dehydrogenase but lacks the electron donor enzymes required for the monooxygenase to operate. Host rubredoxin and rubredoxin reductase allow metabolism of octane to octanol. Proteomic assays and mutants unable to grow on octane or octanoic acid revealed that metabolism of octane is mediated by redundant host and ICE enzymes. Octane is oxidized to octanol, octanal and octanoic acid, the latter is subsequently acylated and oxidized to yield acetyl-CoA that is assimilated via the glyoxylate shunt; in fact, a knockout mutant in the aceA gene, encoding isocitrate lyase was unable to grow on octane or octanoic acid.

Biodegradation of binary mixtures of octane with benzene, toluene, ethylbenzene or xylene (BTEX): insights on the potential of Burkholderia, Pseudomonas and Cupriavidus isolates.

The contamination of the environment by crude oil and its by-products, mainly composed of aliphatic and aromatic hydrocarbons, is a widespread problem. Biodegradation by bacteria is one of the processes responsible for the removal of these pollutants. This study was conducted to determine the abilities of Burkholderia sp. B5, Cupriavidus sp. B1, Pseudomonas sp. T1, and another Cupriavidus sp. X5 to degrade binary mixtures of octane (representing aliphatic hydrocarbons) with benzene, toluene, ethylbenzene, or xylene (BTEX as aromatic hydrocarbons) at a final concentration of 100 ppm under aerobic conditions. These strains were isolated from an enriched bacterial consortium (Yabase or Y consortium) that prefer to degrade aromatic hydrocarbon over aliphatic hydrocarbons. We found that B5 degraded all BTEX compounds more rapidly than octane. In contrast, B1, T1 and X5 utilized more of octane over BTX compounds. B5 also preferred to use benzene over octane with varying concentrations of up to 200 mg/l. B5 possesses alkane hydroxylase (alkB) and catechol 2,3-dioxygenase (C23D) genes, which are responsible for the degradation of alkanes and aromatic hydrocarbons, respectively. This study strongly supports our notion that Burkholderia played a key role in the preferential degradation of aromatic hydrocarbons over aliphatic hydrocarbons in the previously characterized Y consortium. The preferential degradation of more toxic aromatic hydrocarbons over aliphatics is crucial in risk-based bioremediation.

Structural Characterization of Diazabicyclooctane ß-Lactam "Enhancers" in Complex with Penicillin-Binding Proteins PBP2 and PBP3 of Pseudomonas aeruginosa.

Multidrug-resistant (MDR) pathogens pose a significant public health threat. A major mechanism of resistance expressed by MDR pathogens is ß-lactamase-mediated degradation of ß-lactam antibiotics. The diazabicyclooctane (DBO) compounds zidebactam and WCK 5153, recognized as ß-lactam "enhancers" due to inhibition of Pseudomonas aeruginosa penicillin-binding protein 2 (PBP2), are also class A and C ß-lactamase inhibitors. To structurally probe their mode of PBP2 inhibition as well as investigate why P. aeruginosa PBP2 is less susceptible to inhibition by ß-lactam antibiotics compared to the Escherichia coli PBP2, we determined the crystal structure of P. aeruginosa PBP2 in complex with WCK 5153. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. The structure suggests a significant role for the diacylhydrazide moiety of WCK 5153 in interacting with the aspartate in the S-X-N/D PBP motif. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with P. aeruginosa PBP3 was explored crystallographically. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. Overall, the studies provide insights into zidebactam and WCK 5153 inhibition of PBP2 compared to their inhibition of PBP3 and the evolutionarily related KPC-2 ß-lactamase. These molecular insights into the dual-target DBOs advance our knowledge regarding further DBO optimization efforts to develop novel potent ß-lactamase-resistant, non-ß-lactam PBP inhibitors.IMPORTANCE Antibiotic resistance is a significant clinical problem. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by ß-lactamases. In this study, we characterized the inhibition by diazabicyclooctanes of penicillin-binding proteins PBP2 and PBP3 from Pseudomonas aeruginosa using protein crystallography and biophysical analyses. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of P. aeruginosa PBP2 to be inhibited by ß-lactam antibiotics, and provide insights that could be used for further antibiotic development.

Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13.

Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.

Discovery of ANT3310, a Novel Broad-Spectrum Serine ß-Lactamase Inhibitor of the Diazabicyclooctane Class, Which Strongly Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacterales and Acinetobacter baumannii.

The diazabicyclooctanes (DBOs) are a class of serine ß-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of ß-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of ß-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.

Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives.

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

Radiosynthesis of the norepinephrine transporter tracer [18 F]NS12137 via copper-mediated 18 F-labelling.

[18 F]NS12137 (exo-3-[(6-[18 F]fluoro-2-pyridyl)oxy]8-azabicyclo[3.2.1]octane) is a highly selective norepinephrine transporter (NET) tracer. NETs are responsible for the reuptake of norepinephrine and dopamine and are linked to several neurodegenerative and neuropsychiatric disorders. The aim of this study was to develop a copper-mediated 18 F-fluorination method for the production of [18 F]NS12137 with straightforward synthesis conditions and high radiochemical yield and molar activity. [18 F]NS12137 was produced in two steps. Radiofluorination of [18 F]NS12137 was performed via a copper-mediated pathway starting with a stannane precursor and using [18 F]F- as the source of the fluorine-18 isotope. Deprotection was performed via acid hydrolysis. The radiofluorination reaction was nearly quantitative as was the deprotection based on HPLC analysis. The radiochemical yield of the synthesis was 15.1 ± 0.5%. Molar activity of [18 F]NS12137 was up to 300 GBq/µmol. The synthesis procedure is straightforward and can easily be automated and adapted for clinical production.

Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli.

BACKGROUND: Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. RESULTS: Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA-D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling. CONCLUSIONS: Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.

Accurate prediction of anti-phagocytic activity of Vibrio vulnificus by measurement of bacterial adherence to hydrocarbonsPrediction of Anti-Phagocytic Activity.

Vibrio vulnificus can cause necrotizing soft tissue infection via exposure through an open wound, and the incubation period in cases of wound infection is only about 16 h. These facts strongly suggest that mechanisms to evade innate immune cell phagocytosis are essential for its pathogenicity. Hydrophobic interaction is one of the binding mechanisms between bacteria and phagocytes. Several factors that maintain cell surface hydrophobicity (CSH) can contribute to anti-phagocytic activity. In this study, we tried to identify V. vulnificus genes involved in maintaining the CSH, in order to elucidate mechanisms of anti-phagocytic activity. We obtained 143 mutants that had lost their ability to proliferate in the host, using signature-tagged transposon basis mutagenesis (STM). The CSH of these mutants was measured by the bacterial adherence to hydrocarbons (BATH) assay. The CSH of only four mutants differed significantly from that of wild type (WT). Of these four mutants, degS mutant (degS::Tn) showed lesser anti-phagocytic activity than WT in the opsonophagocytosis assay, even though degS::Tn showed opaque-type colonies. Furthermore, survival times of mice subcutaneously inoculated with degS::Tn were prolonged. These facts indicated that the BATH assay is a more suitable method of analyzing the anti-phagocytic activity of V. vulnificus than the comparison of colony morphology.

Interspecies interaction of Serratia plymuthica 4Rx13 and Bacillus subtilis B2g alters the emission of sodorifen.

Sodorifen is the major volatile of Serratia plymuthica 4Rx13. It is assumed to be a long-distance communication signal. However, so far the emission patterns of sodorifen had been studied using mono-cultures of S. plymuthica 4Rx13 neglecting that in natura bacteria live in communities. Here, we show that the structured co-cultivation of S. plymuthica 4Rx13 and Bacillus subtilis B2g in a low-diversity model community grown under nutrient-rich conditions led to quantitative changes in sodorifen emission compared to self-paired mono-cultivations. Co-culturing revealed a decreased emission of sodorifen (50%) during exponential growth phase, whereas in the late stationary stage of growth, the amount of headspace sodorifen was increased compared to self-paired mono-cultivation (217% at 500 h of cultivation). Six other compounds that are most probably related to sodorifen or are isomers showed similar emission patterns. These data indicated that S. plymuthica 4Rx13 enhances its communication signal sodorifen as a consequence of interaction with B. subtilis B2g.

Sodorifen Biosynthesis in the Rhizobacterium Serratia plymuthica Involves Methylation and Cyclization of MEP-Derived Farnesyl Pyrophosphate by a SAM-Dependent C-Methyltransferase.

The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (C16H26) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C16 alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with 13C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis. 13C{1H} NMR spectroscopy facilitated the localization of 13C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphate to sodorifen.

Association of Smoking with Metabolic Volatile Organic Compounds in Exhaled Breath.

Lung cancer (LC) screening will be more efficient if it is applied to a well-defined high-risk population. Characteristics including metabolic byproducts may be taken into account to access LC risk more precisely. Breath examination provides a non-invasive method to monitor metabolic byproducts. However, the association between volatile organic compounds (VOCs) in exhaled breath and LC risk or LC risk factors is not studied. Exhaled breath samples from 122 healthy persons, who were given routine annual exam from December 2015 to December 2016, were analyzed using thermal desorption coupled with gas chromatography mass spectrometry (TD-GC-MS). Smoking characteristics, air quality, and other risk factors for lung cancer were collected. Univariate and multivariate analyses were used to evaluate the relationship between VOCs and LC risk factors. 7, 7, 11, and 27 VOCs were correlated with smoking status, smoking intensity, years of smoking, and depth of inhalation, respectively. Exhaled VOCs are related to smoking and might have a potential to evaluate LC risk more precisely. Both an assessment of temporal stability and testing in a prospective study are needed to establish the performance of VOCs such as 2,5-dimethylfuranm and 4-methyloctane as lung cancer risk biomarkers.

Design and synthesis of a novel series of (1'S,2R,4'S)-3H-4'-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2'-bicyclo[2.2.2]octanes] with high affinity for the α7 neuronal nicotinic receptor.

We describe an efficient and convergent synthesis of a series of (1'S,2R,4'S)-3H-4'-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2'-bicyclo[2.2.2]octanes] displaying potency for the α7 nicotinic acetylcholine receptor (nAChR) and good selectivity vs. the related 5-HT3A receptor.

Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts.

BACKGROUND: The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. RESULTS: Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. CONCLUSIONS: This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.

Fungal volatile compounds induce production of the secondary metabolite Sodorifen in Serratia plymuthica PRI-2C.

The ability of bacteria and fungi to communicate with each other is a remarkable aspect of the microbial world. It is recognized that volatile organic compounds (VOCs) act as communication signals, however the molecular responses by bacteria to fungal VOCs remain unknown. Here we perform transcriptomics and proteomics analyses of Serratia plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum. We find that the bacterium responds to fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. Metabolomic analysis of the bacterium exposed to the fungal VOCs, gene cluster comparison, and heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene sodorifen in response to fungal VOCs. These results strongly suggest that VOCs are not only a metabolic waste but important compounds in the long-distance communication between fungi and bacteria.

ω- versus (ω-1)-hydroxylation: Cytochrome P450 4B1 sterics make the call.

Many family 4 cytochrome P450s play key roles in fatty acid hydroxylation at the terminal, or ω, carbon, but the mechanistic basis for this energetically disfavored regiostereochemistry has been less clear. A co-crystal structure of the rabbit family 4 enzyme CYP4B1 with its substrate octane reveals that the propensity for ω-hydroxylation is orchestrated by active-site sterics, partially mediated by an unusual heme-polypeptide ester bond.

Analysis of a new cluster of genes involved in the synthesis of the unique volatile organic compound sodorifen of Serratia plymuthica 4Rx13.

The rhizobacterium Serratia plymuthica 4Rx13 emits the novel and unique volatile sodorifen (C16H26), which has a polymethylated bicyclic structure. Transcriptome analysis revealed that gene SOD_c20750 (annotated as terpene cyclase) is involved in the biosynthesis of sodorifen. Here we show that this gene is located in a small cluster of four genes (SOD_c20750 - SOD_c20780), and the analysis of the knockout mutants demonstrated that SOD_c20760 (annotated as methyltransferase) and SOD_c20780 (annotated as isopentenyl pyrophosphate (IPP) isomerase) are needed for the biosynthesis of sodorifen, while a sodorifen-negative phenotype was not achieved with the SOD_c20770 (annotated as deoxy-xylulose-5-phosphate (DOXP) synthase) mutant. Altogether, the function of this new gene cluster was assigned to the biosynthesis of this structurally unusual volatile compound sodorifen.

The effect of cultivation media and washing whole-cell biocatalysts on monoamine oxidase catalyzed oxidative desymmetrization of 3-azabicyclo[3,3,0]octane.

It is well known that washing whole-cells containing enzyme activities after fermentation, but prior to biocatalysis can improve their activity in the subsequent reaction. In this paper, we quantify the impact of both the fermentation media and cell washing on the performance of whole-cell biocatalysis. The results are illustrated using a recombinant monoamine oxidase (expressed in Escherichia coli, used in resting state) for the oxidative desymmetrization of 3-azabicyclo[3,3,0]octane. It was shown that the need for washing biocatalyst prior to use in a reaction is dependent upon growth medium. Unlike cells grown in LB medium, washing of the cells was essential for cells grown on TB medium. With TB media, washing the cells improved the final conversion by approximately a factor of two. Additionally, over 50-fold improvement was achieved in initial activity. A potential reason for this improvement in activity was identified to be the increase in transfer of substrates across the cell membrane as a result of cell washing.

Evolving P450pyr Monooxygenase for Regio- and Stereoselective Hydroxylations.

P450pyr monooxygenase from Sphingomonas sp. HXN-200 catalysed the regio- and stereoselective hydroxylation at a non-activated carbon atom, a useful but challenging reaction in classic chemistry, with unique substrate specificity for a number of alicyclic compounds. New P450pyr mutants were developed by directed evolution with improved catalytic performance, thus significantly extending the application of the P450pyr monooxygenase family in biohydroxylation to prepare useful and valuable chiral alcohols. Directed evolution of P450pyr created new enzymes with improved S-enantioselectivity or R-enantioselectivity for the hydroxylation of N-benzyl pyrrolidine, enhanced regioselectivity for the hydroxylation of N-benzyl pyrrolidinone, and increased enantioselectivity for the hydroxylation of N-benzyl piperidinone, respectively. Directed evolution of P450pyr generated also mutants with fully altered regioselectivity (from terminal to subterminal) and newly created excellent S-enantioselectivity for the biohydroxylation of n-octane and propylbenzene, respectively, providing new opportunities for the regio- and enantioselective alkane functionalization. New P450pyr mutants were engineered as the first catalyst for highly selective terminal hydroxylation of n-butanol to 1,4-butanediol. Several novel, accurate, sensitive, simple, and HTS assays based on colorimetric or MS detection for measuring the enantio- and/or regioselectivity of hydroxylation were developed and proven to be practical in directed evolution. The P450pyr X-ray structure was obtained and used to guide the evolution. In silico modelling and substrate docking provided some insight into the influence of several important amino acid mutations of the engineered P450pyr mutants on the altered or enhanced regio- and enantioselectivity as well as new substrate acceptance. The obtained information and knowledge is useful for further engineering of P450pyr for other hydroxylations and oxidations.