Effects of cell division protein-encoding genes knockout on solvent formation and cell morphology in Clostridium acetobutylicum / 生物工程学报

Clostridium acetobutylicum is an important strain for bio-butanol formation. In recent years, gene-editing technology is widely used for developing the hyper-butanol-production strains. In this study, three genes (cac1251, cac2118 and cac2125) encoding cell division proteins (RodA, DivIVA and DivIB) in C. acetobutylicum were knocked out. The cac2118-knockout strain had changed its cell morphology to spherical-shape during the solventogenesis, and obtained a higher butanol yield of 0.19 g/g, increasing by 5.5%, compared with the wild type strain. The glucose utilization and butanol production of cac1251-knockout strain decreased by 33.9% and 56.3%, compared the with wild type strain, reaching to 47.3 g/L and 5.6 g/L. The cac1251-knockout strain and cac2125-knockout strain exhibited poor cell growth with cell optical density decreased by 40.4% and 38.3%, respectively, compared with that of the wild type strain. The results indicate that cell division protein DivIVA made the differences in the regulation of cell morphology and size. Cell division proteins RodA and DivIB played significant roles in the regulation of cell division, and affected cell growth, as well as solventogenesis metabolism.

Advances in the synthesis of biobutanol by consolidated bioprocessing from lignocellulose / 生物工程学报

Consolidated bioprocessing (CBP) is a multi-step process in a bioreactor, which completes hydrolase production, enzymatic hydrolysis, and microbial fermentation. It is considered to be the most promising process for the production of second-generation biofuels because of its simple steps and low cost. Due to the complexity of lignocellulose degradation and the butanol synthesis pathway, few wild microorganisms can directly utilize lignocellulose to synthesize butanol. With the development of synthetic biology, single-bacterium directly synthesizes butanol using lignocellulose by introducing a butanol synthesis pathway in the cellulolytic Clostridium. However, there are still some problems such as heavy metabolic load of single bacterium and low butanol yield. Co-culture can relieve the metabolic burden of single bacterium through the division of labor in different strains and can further improve the efficiency of butanol synthesis. This review analyzes the recent research progress in the synthesis of biobutanol using lignocellulose by consolidated bioprocessing from both the single-bacterium strategy and co-culture strategy, to provide a reference for the research of butanol and other biofuels.

Acetone­butanol­ethanol fermentation from sugarcane bagasse hydrolysates: utilization of C5 and C6 sugars

Background: Fuels and chemicals from renewable feedstocks have a growing demand, and acetone, butanol and ethanol (ABE) are some relevant examples. These molecules can be produced by the bacterial fermentation process using hydrolysates generated from lignocellulosic biomass as sugarcane bagasse, one of the most abundant sources of lignocellulosic biomass in Brazil. It originates as a residue in mills and distilleries in the production of sugar and ethanol. Results: In the present work, two strategies to generate hydrolysates of sugarcane bagasse were adopted. The fermentation of the first hydrolysate by Clostridium acetobutylicum DSM 6228 resulted in final concentrations of butanol, acetone and ethanol of 6.4, 4.5 and 0.6 g/L, respectively. On the other hand, the second hydrolysate presented better results (averages of 9.1, 5.5 and 0.8 g/L, respectively), even without the need for nutrient supplementation, since key elements were already present in the medium. The productivity (QP) and yield (YP/S) of the solvents with second hydrolysate were 0.5 g/L•h-1 and 0.4 g/g, respectively. Conclusions: The results described herein open new perspectives for the production of important molecules from residual lignocellulosic biomass for the fuel and chemical industries within the context of second-generation biorefinery.

Fermentation optimization based on cell self-adaptation to environmental stress - a review / 生物工程学报

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Selection of Schizochytrium limacinum mutants based on butanol tolerance

Background: Mutation breeding is one of the most important routes to achieving high docosahexaenoic acid (DHA) productivity using Schizochytrium. However, few selection strategies have been reported that aim to generate a high DHA content in Schizochytrium lipids. Results: First, culture temperature altered the butanol tolerance of Schizochytrium limacinum B4D1. Second, S. limacinum E8 was obtained by selecting mutants with high butanol tolerance. This mutant exhibited a 17.97% lower proportion of DHA than the parent strain S. limacinum B4D1. Third, a negative selection strategy was designed in which S. limacinum F6, a mutant with poor butanol tolerance, was obtained. The proportion of DHA in S. limacinum F6 was 11.22% higher than that of parent strain S. limacinum B4D1. Finally, the performances of S. limacinum B4D1, E8 and F6 were compared. These three strains had different fatty acid profiles, but there was no statistical difference in their biomasses and lipid yields. Conclusion: It was feasible to identified the relative DHA content of S. limacinum mutants based on their butanol tolerance.

Optimization of 1,2,4-butanetriol synthetic pathway in Escherichia coli / 生物工程学报

1,2,4-Butanetriol (BT) is an important non-natural chemical with a variety of industrial applications. A recombinant Escherichia coli biosynthesizing BT from D-xylose was constructed by heterologously expressing xdh and mdlC, and knocking out competing pathway genes including xylA, xylB, yjhE, yagH and ycdW. To optimize BT synthesis pathway, the third catalytic step that catalyzes the decarboxylation reaction of 3-deoxy-D-glycero-pentulosonic acid was identified as a potential bottleneck. Consequently, 2-keto acid decarboxylases from three different microorganisms were screened, and the kivD gene from Lactococcus lactis was found to increase BT titer by 191%. The improved strain BW-025 reached a final BT titer of 2.38 g/L under optimized transformation conditions. Attempts on synthetic pathway optimization were also made by fine-tuning the expression levels of each enzyme involved in the whole pathway based on BW-025. As a result, an xdh overexpressed recombinant strain, BW-074 was finally generated, with 48.62% higher BT production than that of BW-025.

Anti-proliferative effects of ginsenosides extracted from mountain ginseng on lung cancer / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer.</p><p><b>METHODS</b>The inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis.</p><p><b>RESULTS</b>The abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed.</p><p><b>CONCLUSION</b>Major ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.</p>

Improvement of butanol production by Escherichia coli via Tn5 transposon mediated mutagenesis / 生物工程学报

For engineering an efficient butanol-producing Escherichia coli strain, many efforts have been paid on the known genes or pathways based on current knowledge. However, many genes in the genome could also contribute to butanol production in an unexpected way. In this work, we used Tn5 transposon to construct a mutant library including 1 196 strains in a previously engineered butanol-producing E. coli strain. To screen the strains with improved titer of butanol production, we developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader, because pyruvate is the precursor of butanol and its concentration is inversely correlated with butanol in the fermentation broth. Using this method, we successfully screened three mutants with increased butanol titer. The insertion sites of Tn5 transposon was in the ORFs of pykA, tdk, and cadC by inverse PCR and sequencing. These found genes would be efficient targets for further strain improvement. And the genome scanning strategy described here will be helpful for other microbial cell factory construction.

Anti-inflammatory Activity of Butanolic Extract of Albizia lebbeck.

Albizia lebbeck is a tree widely distributed in India and is also found in South Africa, South America and Australia. As in Indian traditional system as folk medicine, this plant is used to treat several inflammatory pathologies such as asthma, arthritis and burns. Study of other species this same genus has demonstrated an anti-inflammatory activity of crude extract, which, in some work has been attributed to the presence of saponins. In order to confirm these findings a study of phytochemical profile was realized and a rich extract in saponins, butanolic extract, was obtained and its anti-inflammatory activity was evaluated through measured by inhibition of carrageenan-induced mouse paw oedema, using dexamethasone as reference compound. The extract exhibited a moderate control of the both phase of inflammation, provoking an inhibition of edema formation. However, the butanolic extract exhibited lesser activity than reference compound dexamethasone. The results obtained suggest a significant antiinflamatory property of the butanolic extract of Albizia lebbeck, justifying the use of this plant in the traditional medicine for the treatment of inflammatory conditions and confirm their saponins as bioactive product.

Effect of non-ionic surfactants on butanol production with Clostridium acetobutylicum ATCC 824 / 生物工程学报

The low butanol concentration of acetone-butanol-ethanol fermentation causes uneconomical product recovery. In this work, the effect of small molecule non-ionic surfactants on butanol fermentation was evaluated, using laboratory stocks of Clostridium acetobutylicum ATCC 824. Non-ionic surfactants substantially increased butanol production when additive amount was higher than 1% (W/W). Butanol concentration reached 16.9 g/L with 5% (W/W) Tween 80 and 100 g/L glucose in a 5 L fermenter. It was found that surfactants micelle solubilization capacity to butanol was very limited, indicating that butanol could hardly enter the surfactants micelle. Butanol production improvement was probably caused by cell surface hydrophobicity change due to surfactants adsorption.

Effects of chemically modified sugarcane bagasse on butanol production by immobilized Clostridium acetobutylicum XY16 / 生物工程学报

Sugarcane bagasse modified by polyethylenimine (PEI) and glutaraldehyde (GA) was used as a carrier to immobilize Clostridium acetobutylicum XY16 in the process of butanol production. The effects of chemically modified sugarcane bagasse on batch and repeat-batch fermentations were investigated. Batch fermentation was conducted with an addition of 10 g/L modified sugarcane bagasse and 60 g/L glucose, resulting in a high solvent concentration of 21.67 g/L and productivity of 0.60 g/(L x h) with the treatment of 4 g/L PEI and 1 g/L GA. Compared to the fermentations by free cells and immobilized cells on unmodified sugarcane bagasse, the productivity increased 130.8% and 66.7%, respectively. The fibrous-bed bioreactor also maintained a stable butanol production during repeat-batch fermentations, achieving a maximum productivity of 0.83 g/(L x h) with a high yield of 0.42 g/g.

Study of reaction parameters and kinetics of esterification of lauric acid with butanol by immobilized Candida antarctica lipase.

Esterification of lauric acid with n-butanol, catalyzed by immobilized Candida antarctica lipase (CAL) in aqueous-organic biphasic solvent system was studied. Effects of various reaction parameters on esterification were investigated, such as type and amount of solvent, amount of buffer, pH, temperature, speed of agitation, amount of enzyme, butanol and lauric acid. The most suitable reaction conditions for esterification were observed at 50ºC and pH 7.0 using 5000 μmoles of lauric acid, 7000 μmoles of butanol, 0.25 ml phosphate buffer, 1 ml of isooctane as the solvent and 50 mg of immobilized enzyme in the reaction medium at agitation speed of 150 rpm. Maximum esterification of 96.36% was acheived in 600 min of reaction time at n-butanol to lauric acid molar ratio of 1: 0.7. Kinetic study for the esterification of lauric acid with n-butanol using immobilized CAL was carried out and the kinetic constants were estimated by using non-linear regression method. The estimated value of Michaelis kinetic constants for butanol (KmBt) and acid (KmAc) were 451.56 (M) and 4.7 × 10-7(M), respectively and the value of dissociation constant (KBt) of the butanol-lipase complex was 9.41 × 107(M). The estimated constants agreed fairly well with literature data.

Butanol production from corn stover hydrolysate with in-situ liquid-liquid extraction / 生物工程学报

Butanol production from corn stover hydrolysates (CSH) with in-situ liquid-liquid extraction was studied to enhance the production and reduce the fermentation cost. Oleyl alcohol was selected as the suitable solvent and added at the initial fermentation time with the ratio of 1:1 (oleyl alcohol: fermentation broth, V/V). Under this condition, butanol and ABE from CSH with 32.1 g/L total sugars were 3.28 and 4.72 g/L, which were 958.1% and 742.9% higher than those of the controls, respectively. Butanol and ABE production from CSH of 49.7 g/L total sugars after detoxification by ion exchange resin D301 coupled with extraction fermentation were 10.34 g/L and 14.72 g/L with an ABE yield of 0.31 g/g (g ABE/g utilized sugar), which were equal to those of glucose and xylose mixture fermentation. The detoxification and extraction fermentation technology of cellulosic butanol production would provide a crucial technical support to the industrialized production of cellulosic butanol.

Development in molecular genetic manipulation of solventogenic clostridia / 生物工程学报

Solventogenic clostridia are important industrial microorganisms. Optimization of the fermentation performance of solventogenic clostridia, through genetic modification, has always been considered as the main topic involved in solvents production. However, due to the incomplete genetic tools, no research breakthroughs have been achieved. In recent years, with the development of new technologies and methods (e.g. TargeTron gene knockout, large DNA fragment integration method), great progresses have been made towards genetic engineering solventogenic clostridia. In this review, we summarize the development of the genetic tools for solventogenic clostridial species, and simultaneously point out the shortages of the existing technologies in efficiency and comprehensiveness. Therefore, optimization of the existing technologies in gene inactivation in clostridia, such as establishing homologous exchange-based gene deletion and exchange, is still imperative; and in parallel, new genetic tools (e.g. multiplex genome editing, targeted or random multi-copy gene integration) should also be timely developed.

Key enzymes in butanol fermentation by a facultative anaerobe Bacillus sp. TSH1 / 生物工程学报

Bacillus sp. TSH1 is a butanol-producing microorganism newly isolated in our laboratory; it can grow and ferment under facultative anaerobic conditions, while sharing similar fermentation pathways and products with Clostridium acetobutylicum. To illustrate the relationships between the products and the enzyme activities in Bacillus sp. TSH1, key butanol- and ethanol-forming enzymes were studied, including butyraldehyde dehydrogenase, butanol dehydrogenase and alcohol dehydrogenase. The activities of the three enzymes increased rapidly after the initiation of fermentation. Activities of three enzymes peaked before 21 h, and simultaneously, product concentrations also began to increase gradually. The maximum activity of alcohol dehydrogenase was 0.054 U/mg at 12 h, butyraldehyde dehydrogenase 0.035 U/mg at 21 h and butanol dehydrogenase 0.055 U/mg at 15 h. The enzyme activities then decreased, but remained constant at a low level after 24 h, while the concentrations of butanol, acetone, and ethanol continued increasing until the end of the fermentation. The results will attribute to the understanding of the butanol metabolic mechanism, and provide a reference for further study of a facultative Bacillus metabolic pathway.

Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.

Chemical constituents from n-butanol extract of aerial part of Polygala sibirica / 中国中药杂志

Nine compounds were isolated from the n-butanol extract of the aerial parts of Polygala sibirica by various column chromatographic methods. Their structures were identified by MS and NMR spectroscopic data as sibiricaxanthone F (1), amentoflavone (2), linarin (3), zigu-glucoside I (4), 3, 6'-disinapoyl sucrose (5), tenuifoliside A (6), 2, 4, 4-trimethyl-3-formyl-6-hy-droxy-2, 5-cyclohexadien-1-one (7), lanierone (8), and aralia cerebroside (9) , respectively. Compounds 2, 3, 4, 7, 8 were isolated from the genus Ploygala for the first time, and compound 9 was firstly isolated from the title plant.

Butanol pathway construction and promoter optimization in Escherichia coli / 生物工程学报

Promoter optimization is a useful tool in synthetic biology. Fusing promoters of various strengths to genes is a good method to get the best gene overexpression level. Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications. At present, multiple metabolic engineering strategies have been attempted for butanol production in non-native host Escherichia coli. But there were little work on promoter optimization. We fused thlA (thiolase) with strong promoter Alper PLTetO1 or weak promoter Alper BB, operon with strong promoter Braatsch 20 or weak promoter Braatsch 10 by fast assemble method DNA assembler in Escherichia coli. The experimental results showed thlA with strong promoter Alper PLTetO1, operon with weak promoter Braatsch 10 got best butanol concentration 28 mg/L, which increased at least 3-5 fold compared with other combination.

Screening of Clostridium strains through ribosome engineering for improved butanol production / 生物工程学报

We used ribosome engineering technology, with which antibiotic-resistant strains are resulted from mutations on microbial ribosome, to screen a high butanol-producing Clostridium strain. A novel mutant strain S3 with high butanol production and tolerance was obtained from the original Clostridium acetobutylicum L7 with the presence of mutagen of streptomycin. Butanol of 12.48 g/L and ethanol of 1.70 g/L were achieved in S3, 11.2% and 50%, respectively higher than the parent strain. The conversion rate of glucose to butanol increased from 0.19 to 0.22, and fermentation time was 9 h shorter. This caused an increase in butanol productivity by 30.5%, reaching 0.24 g/(Lh). The mutant butanol tolerance was increased from 12 g/L to 14 g/L, the viscosity of fermentation broth was dramatically decreased to 4 mPa/s, 60% lower than the parent strain. In addition, the genetic stability of mutant strain S3 was also favorable. These results demonstrate that ribosome engineering technology may be a promising process for developing high butanol-producing strains.