Rui-Zhi Han

Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation

In this study, an effective corn stover (CS) pretreatment method was developed for biobutanol fermentation. Deep eutectic solvents (DESs), consisted of quaternary ammonium salts and hydrogen donors, display similar properties to room temperature ionic liquid. Seven DESs with different hydrogen donors were facilely synthesized. Choline chloride:formic acid (ChCl:formic acid), an acidic DES, displayed excellent performance in the pretreatment of corn stover by removal of hemicellulose and lignin as confirmed by SEM, FTIR and XRD analysis. After optimization, glucose released from pretreated CS reached 17.0 g L(-1) and yield of 99%. The CS hydrolysate was successfully utilized in butanol fermentation by Clostridium saccharobutylicum DSM 13864, achieving butanol titer of 5.63 g L(-1) with a yield of 0.17 g g(-1) total sugar and productivity of 0.12 g L(-1)h(-1). This study demonstrates DES could be used as a promising and biocompatible pretreatment method for the conversion of lignocellulosic biomass into biofuel.

Biobutanol production from corn stover hydrolysate pretreated with recycled ionic liquid by Clostridium saccharobutylicum DSM 13864.

In this study, corn stover (CS) hydrolysates, pretreated by fresh and recycled ionic liquid (IL) [Bmim][Cl], were utilized in butanol fermentation by Clostridium saccharobutylicum DSM 13864. An efficient CS pretreatment procedure using [Bmim][Cl] was developed, giving a glucose concentration of 18.7 g L(-1) using ten times recycled [Bmim][Cl], representing about 77% of that produced with fresh IL (24.2 g L(-1)). Fermentation of hydrolysate I (pretreated by fresh IL) resulted in 7.4 g L(-1) butanol with a yield of 0.21 g g total-sugar(-1) and a productivity of 0.11 g L(-1)h(-1), while 7.9 g L(-1) butanol was achieved in fermentation using hydrolysate II (pretreated by ten times reused IL) with similar levels of acetone and ethanol, as well as yield and productivity. This study provides evidence for the efficient utilization of IL in CS pretreatment for biobutanol fermentation.

Significantly improved solvent tolerance of Escherichia coli by global transcription machinery engineering

BackgroundEscherichia coli has emerged as a promising platform microorganism to produce biofuels and fine chemicals of industrial interests. Certain obstacles however remain to be overcome, among which organic-solvent tolerance is a crucial one.ResultsWe used global transcription machinery engineering (gTME) to improve the organic-solvent tolerance (OST) of E. coli JM109. A mutant library of σ70 encoded by rpoD was screened under cyclohexane pressure. E. coli JM109 strain harboring σ70 mutant C9 was identified with capability of tolerating 69xa0% cyclohexane. The rpoD mutant contains three amino-acid substitutes and a stop-codon mutation, resulting a truncated sequence containing regions σ1.1 and σ1.2. Total protein difference produced by E. coli JM109 strain harboring C9 was examined with 2D-PAGE, and 204 high-abundant proteins showed over twofold variation under different solvent stress.ConclusionsOur results show that several genes (gapA, sdhB, pepB and dppA) play critical roles in enhanced solvent tolerance of E. coli, mainly involving in maintaining higher intracellular energy level under solvent stress. Global transcription machinery engineering is therefore a feasible and efficient approach for engineering strain with enhanced OST-phenotype.

PEGylation and pharmacological characterization of a potential anti-tumor drug, an engineered arginine deiminase originated from Pseudomonas plecoglossicida.

Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for arginine-auxotrophic tumors. PEGylation is one of the best methods to formulate a bioconjugated protein with extended physical stability and reduced immunogenicity. Here, PEGylation and pharmacological properties of an engineered ADI originated from Pseudomonas plecoglossicida were studied. Among polyethylene glycol (PEG) reagents with succinimidyl ester groups varying in size and linkers, three PEGylated products with high yield and catalytic activity were further characterized, named ADI-SS(20 kDa), ADI-SC(20 kDa), and ADI-SPA(20 kDa). In the pharmacodynamic/pharmacokinetic (PD/PK) studies with ADI-SPA(20 kDa), a remarkable improvement in circulating half-life compared with native ADI was observed. ADI-SPA(20 kDa) injections via intravenous, intramuscular and subcutaneous routes all exhibited superior efficacy than native ADI on depleting serum arginine. Additionally, our results demonstrated that single ADI-SPA(20 kDa) administration of 5u2009U/mouse via intravenous injection could maintain serum arginine at an undetectable level for 5 days with a half-life of 53.2u2009h, representing 11-fold improvement in half-life than that of the native ADI. In a mice H22 hepatocarcinoma model, ADI-SPA(20 kDa) dosage of 5u2009U per 5 days showed an inhibition rate of 95.02% on tumor growth during 15-day treatments.

Soluble Expression of (+)-γ-Lactamase in Bacillus subtilis for the Enantioselective Preparation of Abacavir Precursor

Chiral Vince lactam (γ-lactam) is an important precursor of many carbocyclic nucleoside analogues and pharmaceuticals. Here, a (+)-γ-lactamase encoding gene delm from Delftia sp. CGMCC 5755 was identified through genome hunting. To achieve its soluble and functional expression, Escherichia coli and Bacillus subtilis expression systems were introduced. Compared with E. coli system, recombinant (+)-γ-lactamase showed improved protein solubility and catalytic activity in B. subtilis 168. Reaction conditions for enantioselective resolution of γ-lactam were optimized to be at 30xa0°C, pH 9.0, and 300xa0rpm when employing the recombinant B. subtilis 168/pMA5-delm whole cells. Kinetic analysis showed that the apparent Vmax and Km were 0.595xa0mmol/(minu2009·u2009gDCW) and 378xa0mmol/L, respectively. No obvious substrate inhibition was observed. In a 500-mL reaction system, enantioselective resolution of 100xa0g/L γ-lactam was achieved with 10xa0g/L dry cells, resulting in 55.2xa0% conversion and 99xa0% ee of (−)-γ-lactam. All above suggested that recombinant B. subtilis 168/pMA5-delm could potentially be applied in the preparation of optically pure (−)-γ-lactam.

Simultaneous saccharification and fermentation of dilute alkaline-pretreated corn stover for enhanced butanol production by Clostridium saccharobutylicum DSM 13864

Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF.

Characterization and Soluble Expression of d-Hydantoinase from Pseudomonas fluorescens for the Synthesis of d-Amino Acids

An active d-hydantoinase from Pseudomonas fluorescens was heterogeneously overexpressed in Escherichia coli BL21(DE3) and designated as d-PfHYD. Sequence and consensus analysis suggests that d-PfHYD belongs to the dihydropyrimidinase/hydantoinase family and possesses catalytic residues for metal ion and hydantoin binding. d-PfHYD was purified to homogeneity by nickel affinity chromatography for characterization. d-PfHYD is a homotetramer with molecular weight of 215xa0kDa and specific activity of 20.9xa0Uxa0mg−1. d-PfHYD showed the highest activity at pH 9.0 and 60xa0°C. Metal ions such as Mn2+, Fe2+, and Fe3+ could activate d-PfHYD with 20xa0% improvement. Substrate specificity analysis revealed that purified d-PfHYD preferred aliphatic to aromatic 5′-monosubstituted hydantoins. Among various strategies tested, chaperone GroES-GroEL was efficient in improving the soluble expression of d-PfHYD. Employing 1.0xa0gxa0L−1 recombinant E. coli BL21(DE3)-pET28-hyd/pGRO7 dry cells, 100xa0mM isobutyl hydantoin was converted into d-isoleucine with 98.7xa0% enantiomeric excess (ee), isolation yield of 78.3xa0%, and substrate to biocatalyst ratio of 15.6. Our results suggest that recombinant d-PfHYD could be potentially applied in the synthesis of d-amino acids.

Carbonyl group-dependent high-throughput screening and enzymatic characterization of diaromatic ketone reductase

We have developed a carbonyl group-dependent colorimetric method for assay of carbonyl reductases using inexpensive 2,4-dinitrophenylhydrazine (DNPH). This DNPH method has high sensitivity and low background disturbance, and can be used for high-throughput screening of carbonyl reductases toward various ketones including diaromatic ketones, aliphatic ketones/diketones, and aromatic ketones. For 1-(4′-chlorophenyl)-1-(pyridine-2′-yl)-methyl ketone (CPMK), a characteristic absorbance peak at around 500 nm was observed for the product of CPMK and DNPH with a molar absorbance coefficient of 13u2006750 L mol−1 cm−1. Significantly, this method is also amendable to whole-cell systems for facile high-throughput screening and substrate specificity profiling. In random mutagenesis of a diaromatic ketone reductase KpADH, three variants (M131F, S196Y and S237A) with improved activity toward CPMK were identified using the DNPH method. The substrate specificity of KpADH and its variants toward sixteen prochiral ketones was well characterized. Furthermore, kinetics and molecular docking analyses of KpADH and its variants were performed to elucidate the potential catalytic and enantioselective mechanisms. Consequently, our study provides a rapid screening and characterization method for carbonyl reductases with potential applications in the preparation of optically active secondary alcohols.

Efficient access to L-phenylglycine using a newly identified amino acid dehydrogenase from Bacillus clausii

An amino acid dehydrogenase from Bacillus clausii (BcAADH) was identified and overexpressed in Escherichia coli BL21(DE3) for the preparation of L-phenylglycine from benzoylformic acid. Recombinant BcAADH was purified to homogeneity and characterized. BcAADH could catalyse reductive amination and oxidative deamination at optimum pHs of 9.5 and 10.5. Furthermore, BcAADH has a broad substrate spectrum, displaying activities toward various aromatic and aliphatic keto acids. When coexpressed with glucose dehydrogenase from Bacillus megaterium, the potential application of BcAADH in the preparation of L-phenylglycine was investigated at a high substrate loading and low biocatalyst addition. As much as 400 mM benzoylformic acid could be fully reduced into L-phenylglycine within 6 h at >99.9% ee. With merely 0.5 g DCW L−1, 200 mM benzoylformic acid was completely reduced, resulting in a substrate to biocatalyst ratio of 60 g g−1, environmental factor of 4.7 and 91.7% isolation yield at gram scale. This study provides guidance for the application of BcAADH in the synthesis of chiral non-natural amino acids.

Enhanced curdlan production with nitrogen feeding during polysaccharide synthesis by Rhizobium radiobacter

Curdlan is a secondary metabolite synthesized by Agrobacterium sp. and some other bacteria. A newly isolated exopolysaccharide-producing strain was identified to be Rhizobium radiobacter CGMCC 12099. The polysaccharide product was confirmed to be curdlan with a molecule weight of 1.4×10(5)Da, and its molecular structure was determined by HPLC and infrared spectrum. Although nitrogen source is necessary for cell reproduction, curdlan production is largely dependent on nitrogen limitation, as well as cell vitality. Here, a nitrogen feeding strategy was investigated to elevate the curdlan production by R. radiobacter. The optimal concentration and addition time of (NH4)2HPO4 were investigated. The results showed that the enhanced cell density was correlated to the amount of (NH4)2HPO4 added. Also, nitrogen addition in earlier fermentation stage was beneficial to the cell growth and curdlan production. Furthermore, continuously feeding strategy was employed by feeding (NH4)2HPO4 at a constant rate of 1.24g/h at 35(th)h of fermentation for 9h, achieving a final curdlan production of 65.27g/L, productivity of 0.544g/L/h and glucose conversion rate of 38.89%. The curdlan production was improved by 2.1 times compared with that without nitrogen addition. This study provides a feasible and cheap nitrogen feeding strategy to enhance curdlan production.