Jin-Jun Dong

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l(-1). In batch fermentation, 45.5 g l(-1) succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l(-1) of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l(-1) at a rate of 1.21 g l(-1) h(-1) after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.

Succinic acid production from corn stover by simultaneous saccharification and fermentation using Actinobacillus succinogenes

Simultaneous saccharification and fermentation (SSF) technique was applied for succinic acid production by Actinobacillus succinogenes in a 5-l stirred bioreactor with corn stover as the raw material. The process parameters of SSF, including corn stover pretreatment condition, substrate concentration, enzyme loading and fermentation temperature were investigated. Results indicated that pretreating corn stover with diluted alkaline was beneficial for the succinic acid production, and succinic acid yield could be significantly increased when adding the cellulase supplemented with cellobiase. The maximal succinic acid concentration and yield could reach 47.4 g/l and 0.72 g/g-substrate, respectively. The corresponding operation conditions were summarized as follows: SSF operation at 38 °C for 48 h, diluted alkaline pretreated corn stover as substrate with concentration of 70 g/l, enzyme loading of 20FPU cellulase and 10 U cellobiase per gram substrate. This result suggested an industrial potential of succinic acid production by using SSF and corn stover.

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.

Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug

Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, Km and kcat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

Detoxification of furfural residues hydrolysate for butanol fermentation by Clostridium saccharobutylicum DSM 13864

The toxicity of furfural residues (FRs) hydrolysate is a major obstacle in its application. This work focused on the detoxification of FRs hydrolysate and its application in butanol fermentation. Combination of activated carbon and resin 717 was appropriate for the detoxification of hydrolysate. Mixed sterilization of FRs hydrolysate and corn steep liquor (CSL) was better than the separate ones, since proteins in CSL could adsorb and remove toxic components during sterilization. The results further confirmed that simultaneous sterilization of activated carbon + resin and fermentation medium was more efficient for detoxification and butanol production, in which 76.4% of phenolic compounds and 99.3% of Maillard reaction products were removed, 8.48 g/L butanol and 12.61 g/L total solvent were obtained. This study provides feasible and economic approaches for the detoxification of FRs hydrolysate and its application in butanol production.

Identification of d-carbamoylase for biocatalytic cascade synthesis of d-tryptophan featuring high enantioselectivity

In this study, an enantioselective d-carbamoylase (AcHyuC) was identified from Arthrobacter crystallopoietes with optimum pH of 8.5, much more compatible with hydantoinase process than other reported d-N-carbamoylases. AcHyuC has a substrate preference for aromatic carbamoyl-compounds. The dynamic kinetic resolution (DKR) cascade was developed by combining this AcHyuC with hydantoin racemase from Arthrobacter aurescens (AaHyuA) and d-hydantoinase from Agrobacterium tumefaciens (AtHyuH) for enantioselective resolution of l-indolylmethylhydantoin into d-Trp. The optimum pH of DKR cascade reaction was determined to be 8.0, and PEG 400 could facilitate the reaction. As much as 80mM l-indolylmethylhydantoin could be fully converted to d-Trp within 12h at 0.5L scale, with 99.4% yield, >99.9% e.e. and productivity of 36.6gL-1d-1. This study provides a new d-carbamoylase compatible with the DKR cascade for efficient production of optically pure d-Trp from l-indolylmethylhydantoin.

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.

High production of genistein diglucoside derivative using cyclodextrin glycosyltransferase from Paenibacillus macerans

Genistein has been regarded as one important soy isoflavone with multiple health benefits, whereas its applications are limited by the low hydrophilicity. To improve the water solubility, codon optimized cyclodextrin glycosyltransferase from Paenibacillus macerans was employed for genistein transglycosylation in this study. At least four transglycosylation products were produced and identified by HPLC and LC–MS: genistein monoglucoside, diglucoside, triglucoside, and tetraglucoside derivatives. Obviously, the yields of genistein monoglucoside and genistein diglucoside exhibited great superiority compared with other two products. To maximize the yield of genistein diglucoside, various reaction conditions such as genistein dissolvents, glycosyl donors, substrates concentrations and ratios, enzyme concentrations, reaction pH, temperature, and time were optimized. Finally, the yield of genistein diglucoside was enhanced by 1.5-fold under the optimum reaction system. Our study demonstrates that the production of genistein diglucoside could be specifically enhanced, which is one important genistein derivative with better water solubility and stability.

Engineering coenzyme A-dependent pathway from Clostridium saccharobutylicum in Escherichia coli for butanol production

Clostridium saccharobutylicum has been proved to be efficient in butanol fermentation from various feedstocks. Whereas, lack of genetic manipulation system has severely hindered the engineering of C. saccharobutylicum for more extensive applications. In this study, recombinant Escherichia coli harboring heterologous coenzyme A-dependent pathway from C. saccharobutylicum DSM 13864 was constructed, which consisted of solventogenic pathway genes: acetoacetyl-CoA thiolase (thlA), aldehyde/alcohol dehydrogenase (adhE2) and bcs-operon (crt-bcd1-etfB2-fixB2-hbd). Then, a butanol titer of 67mg/L was attained. After replacing thlA with acetyl-CoA acetyltransferase (atoB) from E. coli and deleting the competitive branch genes lactate dehydrogenase (ldhA), aldehyde/alcohol dehydrogenase (adhE1) and fumarate reductase (frdBC), the butanol titer was successfully improved for 3.8-fold (254mg/L). Under the optimum fermentation conditions, the final butanol titer reached 584mg/L after 120h. This result demonstrates the feasibility of adapting CoA-dependent solventogenic pathway from C. saccharobutylicum in E. coli for butanol synthesis.