Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay.

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol.

Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters.

As concerns increase regarding sustainable industries and environmental pollutions caused by the accumulation of non-degradable plastic wastes, bio-based polymers, particularly biodegradable plastics, have attracted considerable attention as potential candidates for solving these problems by substituting petroleum-based plastics. Among these candidates, polyhydroxyalkanoates (PHAs), natural polyesters that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and material properties similar to those of commodity plastics. Accordingly, substantial efforts have been made to gain a better understanding of mechanisms related to the biosynthesis and properties of PHAs and to develop natural and recombinant microorganisms that can efficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent advances in biotechnology, including those related to evolutionary engineering, synthetic biology, and systems biology, can provide efficient and effective tools and strategies that reduce time, labor, and costs to develop microbial platform strains that produce desired chemicals and materials. Adopting these technologies in a systematic manner has enabled microbial fermentative production of non-natural polyesters such as poly(lactate) [PLA], poly(lactate-co-glycolate) [PLGA], and even polyesters consisting of aromatic monomers from renewable biomass-derived carbohydrates, which can be widely used in current chemical industries. In this review, we present an overview of strain development for the production of various important natural PHAs, which will give the reader an insight into the recent advances and provide indicators for the future direction of engineering microorganisms as plastic cell factories. On the basis of our current understanding of PHA biosynthesis systems, we discuss recent advances in the approaches adopted for strain development in the production of non-natural polyesters, notably 2-hydroxycarboxylic acid-containing polymers, with particular reference to systems metabolic engineering strategies.

Metabolic engineering of Corynebacterium glutamicum for the production of glutaric acid, a C5 dicarboxylic acid platform chemical.

Corynebacterium glutamicum was metabolically engineered for the production of glutaric acid, a C5 dicarboxylic acid that can be used as platform building block chemical for nylons and plasticizers. C. glutamicum gabT and gabD genes and Pseudomonas putida davT and davD genes encoding 5-aminovalerate transaminase and glutarate semialdehyde dehydrogenase, respectively, were examined in C. glutamicum for the construction of a glutaric acid biosynthesis pathway along with P. putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. The glutaric acid biosynthesis pathway constructed in recombinant C. glutamicum was engineered by examining strong synthetic promoters PH30 and PH36, C. glutamicum codon-optimized davTDBA genes, and modification of davB gene with an N-terminal His6-tag to improve the production of glutaric acid. It was found that use of N-terminal His6-tagged DavB was most suitable for the production of glutaric acid from glucose. Fed-batch fermentation using the final engineered C. glutamicum H30_GAHis strain, expressing davTDA genes along with davB fused with His6-tag at N-terminus could produce 24.5 g/L of glutaric acid with low accumulation of l-lysine (1.7 g/L), wherein 5-AVA accumulation was not observed during fermentation.

Enhanced production of glutaric acid by NADH oxidase and GabD-reinforced bioconversion from l-lysine.

Glutaric acid is a promising alternative chemical to phthalate plasticizer since it can be produced by the bioconversion of lysine. Though, recent studies have enabled the high-yield production of its precursor, 5-aminovaleric acid (AMV), glutaric acid production via the AMV pathway has been limited by the need for cofactors. Introduction of NAD(P)H oxidase (Nox) with GabTD enzyme remarkably diminished the demand for oxidized nicotinamide adenine dinucleotide (NAD+ ). Supply of oxygen through vigorous shaking had a significant effect on the conversion of AMV with a reduced requirement of NAD + . A high conversion rate was achieved in Nox coupled GabTD reaction under optimized expression vector, terrific broth (TB), and pH 8.5 at high cell density. Supplementary expression of GabD resulted in the production of 353 ± 35 mM glutaric acid with 88.3 ± 8.7% conversion from 400 mM AMV. Moreover, the reaction with a higher concentration of AMV could produce 528 ± 21 mM glutaric acid with 66.0 ± 2.7% conversion. In addition, the co-biotransformation strategy of GabTD and DavBA whole cells could produce 282 mM glutaric acid with 70.8% conversion from lysine, compared to the 111 mM glutaric acid yield from the combined GabTD-DavBA system.

Engineering a short, aldolase-based pathway for (R)-1,3-butanediol production in Escherichia coli.

Microbial processes can produce a wide range of compounds; however, producing complex and long chain hydrocarbons remains a challenge. Aldol condensation offers a direct route to synthesize these challenging chemistries and can be catalyzed by microbes using aldolases. Deoxyribose-5-phosphate aldolase (DERA) condenses aldehydes and/or ketones to ß-hydroxyaldehydes, which can be further converted to value-added chemicals such as a precursor to cholesterol-lowering drugs. Here, we implement a short, aldolase-based pathway in Escherichia coli to produce (R)-1,3-BDO from glucose, an essential component of pharmaceutical products and cosmetics. First, we expressed a three step heterologous pathway from pyruvate to produce 0.3 g/L of (R)-1,3-BDO with a yield of 11.2 mg/g of glucose in wild-type E. coli K12 MG1655. We used a systems metabolic engineering approach to improve (R)-1,3-BDO titer and yield by: 1) identifying and reducing major by-products: ethanol, acetoin, and 2,3-butanediol; 2) increasing pathway flux through DERA to reduce accumulation of toxic acetaldehyde. We then implemented a two-stage fermentation process to improve (R)-1,3-BDO titer by 8-fold to 2.4 g/L and yield by 5-fold to 56 mg/g of glucose (11% of maximum theoretical yield) in strain BD24, by controlling pH to 7 and higher dissolved oxygen level. Furthermore, this study highlights the potential of the aldolase chemistry to synthesize diverse products directly from renewable resources in microbes.

Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum strains from empty fruit bunch biosugar solution.

BACKGROUND: Recent interest has been focused on the production of platform chemicals from renewable biomass due to increasing concerns on global warming and depletion of fossil fuel reserves. Microbial production of platform chemicals in biorefineries has been suggested to be a promising solution for these problems. Gamma-aminobutyrate (GABA), a versatile bulk chemical used in food and pharmaceutical industry, is also used as a key monomer for nylon 4. GABA can be biologically produced by decarboxylation of glutamate. RESULTS: In this study, we examined high glutamate-producing Corynebacterium glutamicum strains as hosts for enhanced production of GABA from glucose and xylose as carbon sources. An Escherichia coli gadB mutant with a broad pH range of activity and E. coli xylAB genes were expressed under the control of a synthetic H36 promoter. When empty fruit bunch (EFB) solution was used as carbon source (45 g/L glucose and 5 g/L xylose), 12.54 ± 0.07 g/L GABA was produced by recombinant C. glutamicum H36GD1852 expressing E. coli gadB mutant gene and xylAB genes. Batch fermentation of the same strain resulted in the production of 35.47 g/L of GABA when EFB solution was added to support 90 g/L glucose and 10 g/L xylose. CONCLUSIONS: This is the first report of GABA production by recombinant C. glutamicum strains from co-utilization of glucose and xylose from EFB solution. Recombinant C. glutamicum strains developed in this study should be useful for an efficient and sustainable production of GABA from lignocellulosic biomasses.

Metabolic engineering of Corynebacterium glutamicum for fermentative production of chemicals in biorefinery.

Bio-based production of industrially important chemicals provides an eco-friendly alternative to current petrochemical-based processes. Because of the limited supply of fossil fuel reserves, various technologies utilizing microbial host strains for the sustainable production of platform chemicals from renewable biomass have been developed. Corynebacterium glutamicum is a non-pathogenic industrial microbial species traditionally used for L-glutamate and L-lysine production. It is a promising species for industrial production of bio-based chemicals because of its flexible metabolism that allows the utilization of a broad spectrum of carbon sources and the production of various amino acids. Classical breeding, systems, synthetic biology, and metabolic engineering approaches have been used to improve its applications, ranging from traditional amino-acid production to modern biorefinery systems for production of value-added platform chemicals. This review describes recent advances in the development of genetic engineering tools and techniques for the establishment and optimization of metabolic pathways for bio-based production of major C2-C6 platform chemicals using recombinant C. glutamicum.

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies.

The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in kcat/Km Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates.IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.

Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.

BACKGROUND: Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock. RESULTS: Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is similar to the initial concentration of xylose during the fed-batch fermentation suggesting that xylose consumption is not inhibited during fermentation. Finally, recombinant R. eutorpha NCIMB11599 expressing the E. coli xylAB gene was examined for the production of P(3HB) from the hydrolysate solution of sunflower stalk. The hydrolysate solution of sunflower stalk was prepared as a model lignocellulosic biomass, which contains 78.8 g/L of glucose, 26.9 g/L of xylose, and small amount of 4.8 g/L of galactose and mannose. When recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes was cultured in a nitrogen limited chemically defined medium containing 23.1 g/L of hydrolysate solution of sunflower stalk, which corresponds to 16.8 g/L of glucose and 5.9 g/L of xylose, it completely consumed glucose and xylose in the sunflower stalk based medium resulting in the production of 7.86 g/L of P(3HB) with a P(3HB) content of 72.53 wt%. CONCLUSIONS: Ralstonia eutropha was successfully engineered to utilize xylose as a sole carbon source as well as to co-utilize it in the presence of glucose for the synthesis of P(3HB). In addition, R. eutropha engineered to utilized xylose could synthesize P(3HB) from the sunflower stalk hydrolysate solution containing glucose and xylose as major sugars, which suggests that xylose utilizing R. eutropha developed in this study should be useful for development of lignocellulose based microbial processes.

Metabolic engineering of Corynebacterium glutamicum for enhanced production of 5-aminovaleric acid.

BACKGROUND: 5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical that can be used for the synthesis of polymers and other chemicals of industrial interest. Enzymatic conversion of L-lysine to 5AVA has been achieved by employing lysine 2-monooxygenase encoded by the davB gene and 5-aminovaleramidase encoded by the davA gene. Additionally, a recombinant Escherichia coli strain expressing the davB and davA genes has been developed for bioconversion of L-lysine to 5AVA. To use glucose and xylose derived from lignocellulosic biomass as substrates, rather than L-lysine as a substrate, we previously examined direct fermentative production of 5AVA from glucose by metabolically engineered E. coli strains. However, the yield and productivity of 5AVA achieved by recombinant E. coli strains remain very low. Thus, Corynebacterium glutamicum, a highly efficient L-lysine producing microorganism, should be useful in the development of direct fermentative production of 5AVA using L-lysine as a precursor for 5AVA. Here, we report the development of metabolically engineered C. glutamicum strains for enhanced fermentative production of 5AVA from glucose. RESULTS: Various expression vectors containing different promoters and origins of replication were examined for optimal expression of Pseudomonas putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. Among them, expression of the C. glutamicum codon-optimized davA gene fused with His6-Tag at its N-Terminal and the davB gene as an operon under a strong synthetic H36 promoter (plasmid p36davAB3) in C. glutamicum enabled the most efficient production of 5AVA. Flask culture and fed-batch culture of this strain produced 6.9 and 19.7 g/L (together with 11.9 g/L glutaric acid as major byproduct) of 5AVA, respectively. Homology modeling suggested that endogenous gamma-aminobutyrate aminotransferase encoded by the gabT gene might be responsible for the conversion of 5AVA to glutaric acid in recombinant C. glutamicum. Fed-batch culture of a C. glutamicum gabT mutant-harboring p36davAB3 produced 33.1 g/L 5AVA with much reduced (2.0 g/L) production of glutaric acid. CONCLUSIONS: Corynebacterium glutamicum was successfully engineered to produce 5AVA from glucose by optimizing the expression of two key enzymes, lysine 2-monooxygenase and delta-aminovaleramidase. In addition, production of glutaric acid, a major byproduct, was significantly reduced by employing C. glutamicum gabT mutant as a host strain. The metabolically engineered C. glutamicum strains developed in this study should be useful for enhanced fermentative production of the novel C5 platform chemical 5AVA from renewable resources.

Construction of heterologous gene expression cassettes for the development of recombinant Clostridium beijerinckii.

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

Structural insights into the efficient CO2-reducing activity of an NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA.

CO2 fixation is thought to be one of the key factors in mitigating global warming. Of the various methods for removing CO2, the NAD-dependent formate dehydrogenase from Candida boidinii (CbFDH) has been widely used in various biological CO2-reduction systems; however, practical applications of CbFDH have often been impeded owing to its low CO2-reducing activity. It has recently been demonstrated that the NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA (TsFDH) has a higher CO2-reducing activity compared with CbFDH. The crystal structure of TsFDH revealed that the biological unit in the asymmetric unit has two conformations, i.e. open (NAD(+)-unbound) and closed (NAD(+)-bound) forms. Three major differences are observed in the crystal structures of TsFDH and CbFDH. Firstly, hole 2 in TsFDH is blocked by helix α20, whereas it is not blocked in CbFDH. Secondly, the sizes of holes 1 and 2 are larger in TsFDH than in CbFDH. Thirdly, Lys287 in TsFDH, which is crucial for the capture of formate and its subsequent delivery to the active site, is an alanine in CbFDH. A computational simulation suggested that the higher CO2-reducing activity of TsFDH is owing to its lower free-energy barrier to CO2 reduction than in CbFDH.

Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack.

Development of engineered Escherichia coli whole-cell biocatalysts for high-level conversion of L-lysine into cadaverine.

A whole-cell biocatalytic system for the production of cadaverine from L-lysine has been developed. Among the investigated lysine decarboxylases from different microorganisms, Escherichia coli LdcC showed the best performance on cadaverine synthesis when E. coli XL1-Blue was used as the host strain. Six different strains of E. coli expressing E. coli LdcC were investigated and recombinant E. coli XL1-Blue, BL21(DE3) and W were chosen for further investigation since they showed higher conversion yield of lysine into cadaverine. The effects of substrate pH, substrate concentrations, buffering conditions, and biocatalyst concentrations have been investigated. Finally, recombinant E. coli XL1-Blue concentrated to an OD(600) of 50, converted 192.6 g/L (1317 mM) of crude lysine solution, obtained from an actual lysine manufacturing process, to 133.7 g/L (1308 mM) of cadaverine with a molar yield of 99.90 %. The whole-cell biocatalytic system described herein is expected to be applicable to the development of industrial bionylon production process.

Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from Staphylococcus aureus.

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.

Enhancing the activity of Bacillus circulans xylanase by modulating the flexibility of the hinge region.

Enzymes undergo multiple conformational changes in solution, and these dynamics are considered to play a critical role in enzyme activity. Hinge-bending motions, resulting from reciprocal movements of dynamical quasi-rigid bodies, are thought to be related to turnover rate and are affected by the physical properties of the hinge regions. In this study, hinge identification and flexibility modification of the regions by mutagenesis were conducted to explore the relationship between hinge flexibility and catalytic activity. Bacillus circulans xylanase was selected for the identification and mutation of the hinge regions. As a result, turnover rate (V(max)) was improved approximately twofold in mutants that have more rigid hinge structure, despite the decrease in K(m) and V(max)/K(m). This result indicates that the rigidly mutated hinge has positive effects on B. circulans xylanase activity.

Tryptophan-Based Hyperproduction of Bioindigo by Combinatorial Overexpression of Two Different Tryptophan Transporters.

Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an E. coli whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl ß-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing aroP and tnaB plasmid with tnaA and FMO produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.

Statistical Analysis of the Role of Cavity Flexibility in Thermostability of Proteins.

Conventional statistical investigations have primarily focused on the comparison of the simple one-dimensional characteristics of protein cavities, such as number, surface area, and volume. These studies have failed to discern the crucial distinctions in cavity properties between thermophilic and mesophilic proteins that contribute to protein thermostability. In this study, the significance of cavity properties, i.e., flexibility and location, in protein thermostability was investigated by comparing structural differences between homologous thermophilic and mesophilic proteins. Three dimensions of protein structure were categorized into three regions (core, boundary, and surface) and a comparative analysis of cavity properties using this structural index was conducted. The statistical analysis revealed that cavity flexibility is closely related to protein thermostability. The core cavities of thermophilic proteins were less flexible than those of mesophilic proteins (averaged B' factor values, -0.6484 and -0.5111), which might be less deleterious to protein thermostability. Thermophilic proteins exhibited fewer cavities in the boundary and surface regions. Notably, cavities in mesophilic proteins, across all regions, exhibited greater flexibility than those in thermophilic proteins (>95% probability). The increased flexibility of cavities in the boundary and surface regions of mesophilic proteins, as opposed to thermophilic proteins, may compromise stability. Recent protein engineering investigations involving mesophilic xylanase and protease showed results consistent with the findings of this study, suggesting that the manipulation of flexible cavities in the surface region can enhance thermostability. Consequently, our findings suggest that a rational or computational approach to the design of flexible cavities in surface or boundary regions could serve as an effective strategy to enhance the thermostability of mesophilic proteins.