Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non-yeast signal peptide-mediated cell surface display.

BACKGROUND: Isomaltulose is a 'generally recognized as safe' ingredient and is widely used in the food, pharmaceutical and chemical industries. The exploration and development of efficient technologies is essential for enhancing isomaltulose yield. RESULTS: In the present study, a simple and efficient surface display platform mediated by a non-yeast signal peptide was developed in Yarrowia lipolytica and utilized to efficiently produce isomaltulose from sucrose. We discovered that the signal peptide SP1 of sucrose isomerase from Pantoea dispersa UQ68J (PdSI) could guide SIs anchoring to the cell surface of Y. lipolytica, demonstrating a novel and simple cell surface display strategy. Furthermore, the PdSI expression level was significantly increased through optimizing the promoters and multi-site integrating genes into chromosome. The final strain gained 451.7 g L-1 isomaltulose with a conversion rate of 90.3% and a space-time yield of 50.2 g L-1 h-1. CONCLUSION: The present study provides an efficient way for manufacturing isomaltulose with a high space-time yield. This heterogenous signal peptide-mediated cell surface display strategy featured with small fusion tag (approximately 2.2 kDa of SP1), absence of enzyme leakage in fermentation broth and ample room for optimization, providing a convenient way to construct whole-cell biocatalysts to synthesize other products and broadening the array of molecular toolboxes accessible for engineering Y. lipolytica. © 2024 Society of Chemical Industry.

The wheat growth-promoting traits of Ochrobactrum and Pantoea species, responsible for solubilization of different P sources, are ensured by genes encoding enzymes of multiple P-releasing pathways.

Production and release of organic acids and phosphatase enzymes by microbes are important for inorganic and organic phosphorus cycling in soil. The presence of microorganisms with corresponding traits in the plant rhizosphere lead to improved plant P uptake and ultimately growth promotion. We studied the potential of two rhizosphere-competent strains, Pantoea sp. MR1 and Ochrobactrum sp. SSR, for solubilization of different organic and inorganic P sources in vitro. In a pot experiment we further revealed the impact of the two strains on wheat seedling performance in soil amended with either phytate, rock phosphate or K2HPO4 as solely P source. To directly link P-solubilizing activity to the strain-specific genetic potential, we designed novel primers for glucose dehydrogenase (gcd), phosphatase (pho) and phytase (phy) genes, which are related to the organic and inorganic P solubilization potential. Quantitative tracing of these functional genes in the inoculated soils of the conducted pot experiment further allowed to compare strain abundances in the soil in dependency on the present P source. We observed strain- and P source-dependent patterns of the P solubilization in vitro as well as in the pot experiment, whereby P release, particularly from phytate, was linked to the strain abundance. We further revealed that the activity of microbial phosphatases is determined by the interplay between functional gene abundance, available soil P, and substrate availability. Moreover, positive impacts of microbial seed inoculation on wheat root architecture and aboveground growth parameters were observed. Our results suggest that screening for rhizosphere-competent strains with gcd, pho and phy genes may help to identify new microbial taxa that are able to solubilize and mineralize inorganic as well as organic bound P. Subsequently, the targeted use of corresponding strains may improve P availability in agricultural soils and consequently reduce fertilizer application.

Pantoea stewartii WceF is a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like fold.

Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties of a biofilm and result in a highly viscous environment. Bacterial communities regulate this biofilm state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation of this glycan matrix, however, requires the action of extracellular glycosidases. So far, these were mainly described for bacteriophages that must degrade biofilms for gaining access to host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes the protein WceF within its EPS synthesis cluster. WceF has homologs in various biofilm forming plant pathogens of the Erwinia family. In this work, we show that WceF is a glycosidase active on stewartan, the main P. stewartii EPS biofilm component. WceF has remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed a native trimer of right-handed parallel ß-helices. Despite its similar fold, WceF lacks the high stability found in bacteriophage TSPs. WceF is a stewartan hydrolase and produces oligosaccharides, corresponding to single stewartan repeat units. However, compared with a stewartan-specific glycan hydrolase of bacteriophage origin, WceF showed lectin-like autoagglutination with stewartan, resulting in notably slower EPS cleavage velocities. This emphasizes that the bacterial enzyme WceF has a role in P. stewartii biofilm glycan matrix reorganization clearly different from that of a bacteriophage exopolysaccharide depolymerase.

Construction of recombinant Escherichia coli expressing xylitol-4-dehydrogenase and optimization for enhanced L-xylulose biotransformation from xylitol.

L-Xylulose is a rare ketopentose which inhibits α-glucosidase and is an indicator of hepatitis or liver cirrhosis. This pentose is also a precursor of other rare sugars such as L-xylose, L-ribose or L-lyxose. Recombinant E. coli expressing xylitol-4-dehydrogenase gene of Pantoea ananatis was constructed. A cost-effective culture media were used for L-xylulose production using the recombinant E. coli strain constructed. Response surface methodology was used to optimize these media components for L-xylulose production. A high conversion rate of 96.5% was achieved under an optimized pH and temperature using 20 g/L xylitol, which is the highest among the reports. The recombinant E. coli cells expressing the xdh gene were immobilized in calcium alginate to improve recycling of cells. Effective immobilization was achieved with 2% (w/v) sodium alginate and 3% (w/v) calcium chloride. The immobilized E. coli cells retained good stability and enzyme activity for 9 batches with conversion between 53 and 92% which would be beneficial for economical production of L-xylulose.

Identification and characterization of a novel bacterial carbohydrate esterase from the bacterium Pantoea ananatis Sd-1 with potential for degradation of lignocellulose and pesticides.

OBJECTIVE: Identification and characterization of a novel bacterial carbohydrate esterase (PaCes7) with application potential for lignocellulose and pesticide degradation. RESULTS: PaCes7 was identified from the lignocellulolytic bacterium, Pantoea ananatis Sd-1 as a new carbohydrate esterase. Recombinant PaCes7 heterologously expressed in Escherichia coli showed a clear preference for esters with short-chain fatty acids and exhibited maximum activity towards α-naphthol acetate at 37 °C and pH 7.5. Purified PaCes7 exhibited its catalytic activity under mesophilic conditions and retained more than 40% activity below 30 °C. It displayed a relatively wide pH stability from pH 6-11. Furthermore, the enzyme was strongly resistant to Mg2+, Pb2+, and Co2+ and activated by K+ and Ca2+. Both P. ananatis Sd-1 and PaCes7 could degrade the pesticide carbaryl. Additionally, PaCes7 was shown to work in combination with cellulase and/or xylanase in rice straw degradation. CONCLUSIONS: The data suggest that PaCes7 possesses promising biotechnological potential.

Exploring the Post-translational Enzymology of PaaA by mRNA Display.

PaaA is a RiPP enzyme that catalyzes the transformation of two glutamic acid residues within a substrate peptide into the bicyclic core of Pantocin A. Here, for the first time, we use mRNA display techniques to understand RiPP enzyme-substrate interactions to illuminate PaaA substrate recognition. Additionally, our data revealed insights into the enzymatic timing of glutamic acid modification. The technique developed is quite sensitive and a significant advancement over current RiPP studies and opens the door to enzyme modified mRNA display libraries for natural product-like inhibitor pans.

Investigation into isomerization reaction of phenylalanine aminomutase from Pantoea agglomerans.

Phenylalanine aminomutase (PaPAM) from Pantoea agglomerans is a member of the MIO (4-methylene-imidazol-5-one) family of enzymes, which isomerizes α-phenylalanine to ß-phenylalanine, and could be used to synthesize unnatural ß-arylalanine. However, the mechanism of isomerization reaction is not clear. To investigate the mechanism, the gene (pam), which encodes PaPAM, was first expressed in E.coli, and recombinant PaPAM was prepared using affinity chromatography. Then, 15N-(2S)-α-phenylalanine, (2S)-(3-2H2)-α-phenylalanine and (2S,3S)-[2,3-2H2]-α-phenylalanine were used as substrates to analyze the mechanism of isomerization reaction. The results of MS and NMR showed that the isomerization reaction was performed through the intramolecular exchange of NH2 with pro-3R hydrogen of α-phenylalanine. The PaPAM shuttles the α-NH2 of α-phenylalanine to ß site to replace the pro-3R hydrogen. Simultaneously, the pro-3R hydrogen is shifted to α site to produce ß-phenylalanine. Furthermore, a key residue, Phe at position 455 in the active site, was determined to control the exchange way using molecular docking and sequence alignment of MIO family enzymes. The results indicated that the key 455 Phe residue is involved in changing the binding orientation of the carboxyl group of the intermediate trans-cinnamic acid to control the NH2-H pair exchange.

Surface engineering of a Pantoea agglomerans-derived phenylalanine aminomutase for the improvement of (S)-ß-phenylalanine biosynthesis.

A Pantoea agglomerans-derived phenylalanine aminomutase (PaPAM) was engineered to improve the biocatalytic synthesis of (S)-ß-phenylalanine, which is an important precursor of pharmaceuticals and peptidomimetics. A semi-rational design strategy based on a combination of surface-amino-acid engineering and the amino acid preference of the thermozyme was applied to counteract the enzyme trade-off between improving its activity and stability. The surface glycine, lysine and serine of PaPAM were mutated to alanine, arginine and alanine, respectively. A K340R mutant was screened with a 2.23-fold increased activity and 2.12-fold improved half-life at 50 °C over those of the wild-type PaPAM. These improvements resulted from the more stable enzymatic conformation as well as the more rigid inner loop in K340R. When tested in a whole-cell biocatalytic reaction, the (S)-ß-phenylalanine volumetric productivity of K340R reached 0.47 g/L·h (1.4-fold greater than that of the wild-type PaPAM), and the conversion rate was improved by 17% compared to that of the wild-type PaPAM. The enzymatic properties of K340R and the resulting (S)-ß-phenylalanine production are among the highest reported, and the results indicate that the described strategy is potent for engineering enzymatic stability and activity of PAM.

Pantoea agglomerans YS19 poly(A) polymerase I gene possesses the indole-sensing sequence in the promoter region.

Pantoea agglomerans YS19 is a predominant diazotrophic endophyte with multiple growth-promoting effects on its host plant that was isolated from rice. Indole is confirmed to induce many changes of physiological and biochemical characteristics in bacteria. Although YS19 cannot produce indole, it can sense indole in the environment and be regulated by indole. Here, using gfp as a reporter gene, we constructed a series of recombinant plasmids containing the promoter region of the poly(A) polymerase I gene (pcnB) fused with gfp, and compared the green fluorescence intensity at different concentrations of exogenous indole by a flow cytometer. In this research, we confirmed that exogenous indole significantly inhibited the expression of pcnB by its promoter; the regulation sequence sensitive to indole in the promoter region of the pcnB gene (In-pcnB) was between -129 and -88 bp. In-pcnB is widely distributed and strictly conserved in the same genus. These results suggest novel roles of In-pcnB in P. agglomerans YS19, showing its special relation to the indole regulatory pathway.

Highly regio- and enantioselective synthesis of chiral intermediate for pregabalin using one-pot bienzymatic cascade of nitrilase and amidase.

Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E > 300) towards IBSN at a high substrate concentration of 100 g L-1. Byproduct (S)-3-cyano-5-methyl hexanoic amide ((S)-CMHM) was detected and identified for the first time during the catalytic process. By employing a feasible one-pot bienzymatic cascade of mutant N258D and amidase from Pantoea sp. (Pa-Ami) expressed separately in recombinant Escherichia coli cells, the byproduct (S)-CMHM was eliminated and (S)-CMHA was obtained with a conversion of 45.0% and eep of 99.3%. These results provided the novel plant-derived nitrilase as a promising biocatalyst for (S)-CMHA biosynthesis and demonstrated the feasibility of one-pot bienzymatic cascade reaction for large-scale production of the pregabalin precursor.

Two Novel Acetylesterases from Pantoea dispersa: Recombinant Expression, Purification, and Characterization.

Two novel acetylesterases from Pantoea dispersa, with low amino acid sequence identity between them, were expressed in Escherichia coli with a carboxyl-His6 tail given by the expression plasmid, purified, and characterized. The purified proteins, named Est-1 and Est-2, had a molecular mass of 33 kDa and 37 kDa, respectively. Both proteins presented a modeled structure of homodimers with monomers presenting the α/ß-hydrolase fold, with the catalytic triad Ser-Asp-His present in the active site. The KM for p-nitrophenyl acetate and Vmax values found for Est-1 were of 1.4 ± 0.2 mM and 8.66 ± 0.59 µmol/min and for Est-2 were of 0.36 ± 0.077 mM and 6.13 ± 0.56 µmol/min, respectively. Both enzymes presented an optimum pH of 7.0. The optimum temperature for Est-1 was 40 °C and for Est-2 was 50 °C. The temperatures in which the enzymes Est-1 and Est-2 lost half of their activity (T50) were 44.1 and 58.9 °C, respectively. SDS, EDTA, and PMSF significantly inhibited the enzymes. The two purified enzymes also presented activity against triacetin and were able to deacetylate the carbohydrates pectin and xylan, with higher activity against pectin. Thus, they could be considered as carbohydrate esterases.

Comparative analysis of two non-specific nucleases of the phospholipase D family from the plant pathogen competitor bacterium Pantoea agglomerans.

Bacterial non-specific nucleases of the phospholipase D family are widely distributed among the members of the Enterobacteriaceae. Each genome mainly contains a single copy of a gene encoding a phospholipase D family protein. However, two distantly related isozymes (< 40% identity at the protein level) were identified by BLAST-analyses in the plant pathogenic competitor enterobacterium Pantoea agglomerans. The two nucleases PaNuc-1 and PaNuc-2 were produced in Escherichia coli. Identical gene constructs and expression conditions resulted in the production of PaNuc-1 in soluble form, while PaNuc-2 remained insoluble in inclusion bodies. PaNuc-2 was refolded and both proteins were purified by a combination of affinity and ion exchange chromatography. Proteolytic removal of the HIS-tag allowed the characterization of pure and mature tag-less proteins. Enzymatic properties of both isozymes revealed that they are non-specific nucleases, displaying activities against RNA, single- and double-stranded genomic DNA as well as circular plasmids. However, their biochemical activity profiles were clearly different, with PaNuc-1 being optimally active at 70 °C and pH 7.0, while PaNuc-2 was most active at 45 °C and pH 7.0. The enzymes retained > 90% nuclease activity at EDTA concentrations of 4 mM (PaNuc-2) and 20 mM (PaNuc-1), respectively. Different enzymatic properties suggest that the roles of PaNuc-1 and PaNuc-2 differ in the cell and might be the result of functional diversification after an ancient gene duplication event took place. The fact that both enzymes could be easily produced in recombinant form and their tolerance against metal ion chelators in combination with a broad substrate promiscuity might pave the way to versatile biotechnological applications.

Overexpression of secreted sucrose isomerase in Yarrowia lipolytica and its application in isomaltulose production after immobilization.

Isomaltulose production by bacterial fermentation was limited, due to generation of undesirable products and reduced yields. Isomaltulose production using sucrose isomerase (SIase) catalyzed methods was expected to be more applicable, but was hampered by low SIase activity and lack of a secreted SIase producer. Here, we aimed to obtain high levels of secreted SIase by overexpressing the SIase gene from Pantoea dispersa UQ68J in Yarrowia lipolytica, a successful host for efficient secretory expression, with a newly characterized strong constitutive promoter. After optimization of the culture medium, the engineered strain JD secreted SIase with an activity of 49.3 U/mL. The recombinant SIase was effectively immobilized onto polyvinyl alcohol-alginate, and the enzymatic activity recovery rate was up to 82.4%. The stability of the SIase was significantly improved by immobilization. Batch production of isomaltulose catalyzed by the immobilized SIase was performed under optimal conditions, generating 620.7 g/L isomaltulose with a yield of 0.96 g/g. The conversion rate of sucrose after 13 batches remained above 90%. These results demonstrated that the proposed SIase expression and immobilization method was promising in the industrial production of isomaltulose.

Purification and identification of an aflatoxin B1 degradation enzyme from Pantoea sp. T6.

Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus and other fungi. Soil, crops, food, feed, etc. were susceptible to aflatoxin B1 contamination, which caused adverse economic and health consequences. It is necessary to search for microorganisms or microbial enzymes that can degrade AFB1. The degradation activity of AFB1 by cell-free supernatant (68.30%) of isolate Pantoea sp. T6 was significantly higher (P < 0.05) than viable bacterial cells (4.87%) and intracellular cell extracts (3.68%). The supernatant's AFB1 degradation activity was reduced from 68.30% to 5.33% in treatment with protease K and sodium dodecyl sulphate (SDS). An extracellular enzyme from the supernatant was secreted by Pantoea sp. T6 and was named as Pantoea aflatoxin degradation enzyme (PADE), which was obtained using Diethylaminoethanol (DEAE)-Sepharose chromatography. PADE was further purified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The PADE, outer membrane protein A, was identified by mass spectrometry and molecular mass was 38180.1Da. The optimum temperature and pH for the reaction of PADE with AFB1 were 40 °C and 7.0, respectively. These finding showing that the PADE, which was isolated from the supernatant of Pantoea sp. T6, has the ability to degrade AFB1, and may have potential application for aflatoxin B1 reduction in the food and feed industry.

Structure-Based Engineering of Amidase from Pantoea sp. for Efficient 2-Chloronicotinic Acid Biosynthesis.

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with kcat/Km values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 gproduct liter-1 day-1 These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships.IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.

Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity.

Astaxanthin is a reddish keto-carotenoid classified as a xanthophyll found in various microbes and marine organisms. As a powerful antioxidant having up to 100 times more potency than other carotenoids such as ß-carotene, lutein, and lycopene, astaxanthin is a versatile compound utilized in animal feed, food pigment, health promotion and cosmetic industry. Here, we report development of metabolically engineered Escherichia coli capable of producing astaxanthin to a high concentration with high productivity. First, the heterologous crt genes (crtE, crtY, crtI, crtB, and crtZ) from Pantoea ananatis and the truncated BKT gene (trCrBKT) from Chlamydomonas reinhardtii were introduced to construct the astaxanthin biosynthetic pathway. Then, eight different fusion tags were examined by attaching them to the N- or C-terminus of the trCrBKT membrane protein to allow stable expression and to efficiently guide trCrBKT to the E. coli membrane. When the signal peptide of OmpF and TrxA were tagged to the N-terminus and C-terminus of trCrBKT, respectively, astaxanthin production reached 12.90 mg/L (equivalent to 3.84 mg/gDCW), which was 2.08-fold higher than that obtained without tagging. Upon optimization of culture conditions, this engineered strain WLGB-RPP harboring pAX15 produced 332.23 mg/L (5.38 mg/gDCW) of astaxanthin with the productivity of 3.79 mg/L/h by fed-batch fermentation. In order to further increase astaxanthin production, in silico flux variability scanning based on enforced objective flux (FVSEOF) was performed to identify gene overexpression targets. The engineered strain WLGB-RPP (pAX15, pTrc-ispDF) which simultaneously overexpressing the ispD and ispF genes identified by FVSEOF produced astaxanthin to a higher concentration of 377.10 mg/L (6.26 mg/gDCW) with a productivity of 9.20 mg/L/h upon induction with 1 mM IPTG. When cells were induced with 0.5 mM IPTG to reduce the metabolic burden, astaxanthin concentration further increased to 432.82 mg/L (7.12 mg/gDCW) with a productivity of 9.62 mg/L/h. To more stably maintain plasmid during the fed-batch fermentation of WLGB-RPP (pAX15, pTrc-ispDF), the post-segregational killing hok/sok system was introduced. This strain produced 385.04 mg/L (6.98 mg/gDCW) of astaxanthin with a productivity of 7.86 mg/L/h upon induction with 0.5 mM IPTG. The strategies reported here will be useful for the enhanced production of astaxanthin and related carotenoid products by engineered E. coli strains.

Insights into ß-ketoacyl-chain recognition for ß-ketoacyl-ACP utilizing AHL synthases.

Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and acyl-homoserine lactone biosynthetic pathways are important targets for developing antimicrobial, anticancer and antiparasitic compounds. Published reports on successful isolation of beta-ketoacyl-ACPs in a laboratory remain scarce to date and thus most beta-ketoacyl-ACP utilizing enzymes are routinely characterized using small molecule substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the systematic investigation into the electronic, geometric and spatial aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl-ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum signal synthases.

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3-Phosphate Synthase.

Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site-directed mutagenesis (SDM). The mutant significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile and Pro 102 to Ser. These mutation sites in E.coli have been studied as significant active sites of glyphosate resistance. However, in our research they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate-resistant plants.

Identification and characterization of a novel bacterial pyranose 2-oxidase from the lignocellulolytic bacterium Pantoea ananatis Sd-1.

OBJECTIVE: To identify and characterize a novel bacterial pyranose 2-oxidase (P2Ox) and investigate its potential use in lignin degradation applications. RESULTS: A new bacterial P2Ox (PaP2Ox) enzyme was identified in the lignocellulolytic bacterium Pantoea ananatis Sd-1. The PaP2Ox open reading frame was cloned, and the encoded protein was heterologously expressed in an Escherichia coli expression system. Unlike another reported bacterial P2Ox enzyme, the purified PaP2Ox exhibits a homotetrameric spatial conformation that is similar to fungal P2Oxs, with each subunit having a molecular mass of 65 kDa. The recombinant PaP2Ox exhibits maximum activity at 50 °C and pH 6.5 with D-glucose as its preferred substrate. In addition, this enzyme was shown to work in combination with bacterial laccase in lignin degradation. CONCLUSIONS: The bacterial enzyme PaP2Ox has potential use in ligninolytic systems and shows promising value in industrial biotechnological applications.

Metabolic engineering of Pseudomonas taiwanensis VLB120 with minimal genomic modifications for high-yield phenol production.

Aromatic chemicals are important building blocks for the production of a multitude of everyday commodities. Currently, aromatics production relies almost exclusively on petrochemical processes. To achieve sustainability, alternative synthesis methods need to be developed. Here, we strived for an efficient production of phenol, a model aromatic compound of industrial relevance, from renewable carbon sources using the solvent-tolerant biocatalyst Pseudomonas taiwanensis VLB120. First, multiple catabolic routes for the degradation of aromatics and related compounds were inactivated, thereby obtaining the chassis strain P. taiwanensis VLB120Δ5 incapable of growing on 4-hydroxybenzoate (ΔpobA), tyrosine (Δhpd), and quinate (ΔquiC, ΔquiC1, ΔquiC2). In this context, a novel gene contributing to the quinate catabolism was identified (quiC2). Second, we employed a combination of reverse- and forward engineering to increase metabolic flux towards the product, using leads obtained from the analysis of aromatics producing Pseudomonas putida strains previously generated by mutagenesis. Phenol production was enabled by the heterologous expression of a codon-optimized and chromosomally integrated tyrosine phenol-lyase encoding gene from Pantoea agglomerans AJ2985 (PaTPL2). The genomic modification of endogenous genes encoding TrpEP290S, AroF-1P148L, and PheAT310I, and the deletion of pykA improved phenol production 17-fold, while also minimizing the burden caused by plasmids and auxotrophies. The additional overexpression of known bottleneck enzymes (AroGfbr, TyrAfbr) derived from Escherichia coli further enhanced phenol titers. The best producing strain P. taiwanensis VLB120Δ5-TPL36 reached yields of 15.8% and 18.5% (Cmol/Cmol) phenol from glucose and glycerol, respectively, in a mineral medium without addition of complex nutrients. This is the highest yield ever reported for microbially produced phenol.