Optimization of hydrogen production in Enterobacter aerogenes by Complex I peripheral fragments destruction and maeA overexpression.

As a concentrated energy source with high added value, hydrogen has great development prospects, with special emphasis on sustainable microbial production as a replacement for traditional fossil fuels. In this study, λ-Red recombination was used to alter the activity of Complex I by single and combined knockout of nuoE, nuoF and nuoG. In addition, the conversion of malic to pyruvic acid was promoted by overexpressing the maeA gene, which could increase the content of NADH and formic acid in the bacterial cells. Compared to the original strain, hydrogen production was 65% higher in the optimized strain IAM1183-EFG/M, in which the flux of the formic acid pathway was increased by 257%, the flux of the NADH pathway was increased by 13%, and the content of metabolites also changed significantly. In further bioreactor, the total hydrogen production of the scale-up IAM1183-EFG/M after 44 h of fermentation was 4.76 L, which increased by 18% compared with the starting strain. This study provides a new direction for future exploration of microbial hydrogen production by combinatorial modification of multiple genes.

Metabolic regulation and optimization of oxygen supply enhance the 2,3-butanediol yield of the novel Klebsiella sp. isolate FSoil 024.

BACKGROUND: Biogenic 2,3-butanediol (2,3-BDO) is a high-value-added compound that can be used as a liquid fuel and a platform chemical. Bioproduction of 2,3-BDO is an environmentally friendly choice. METHOD AND RESULTS: Three recombinant derivatives of the novel Klebsiella sp. isolate FSoil 024 (WT) were constructed via different strategies including deletion of lactate dehydrogenase by λ-Red homologous recombination technology, overexpression of the small-noncoding RNA RyhB and a combination of both. The 2,3-BDO productivity of the mutants increased by 61.3%-79%, and WT-Δldh/ryhB displayed the highest 2,3-BDO yield of 42.36 mM after 24 h of shake-flask fermentation. Glucose was shown as the best carbon source for 2,3-BDO production by WT-Δldh/ryhB. In addition, higher oxygenation was favorable for ideal product synthesis. The maximal 2,3-BDO yield of WT and WT-Δldh/ryhB were increased by 23.3% and 52.5% respectively compared to the control group in the presence of 70% oxygen (V:V' = O2 :(O2 + N2 )). CONCLUSION AND IMPLICATIONS: According to the present study, deletion of lactate dehydrogenase, RyhB overexpression, and manipulation of oxygen supply showed great impacts on cell growth, 2,3-BDO productivity and cellular metabolism of the novel isolated strain Klebsiella sp. FSoil 024. This work would also provide insights for promoting 2,3-BDO biosynthesis for industrial applications.

Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains.

The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some "scissors plasmids" via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96-100%), rather than the non-replication initiation region (88-92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles.

Comparison of metabolism substances in Cordyceps sinensis and Cordyceps militaris cultivated with tussah pupa based on LC-MS.

The objective of our study was to compare the chemical composition of Cordyceps sinensis (C. sinensis) and Cordyceps militaris (C. militaris) cultivated with tussah pupa by using metabonomics technology in order to clarify the similarity and difference of the two medicinal materials from the whole metabolite level. The results showed that there were 25 different metabolites among the 69 metabolites that were highly expressed in C. militaris cultivated with tussah pupa compared with C. sinensis in both positive and negative ion modes. Analysis results of partial differential metabolites pathways indicated that 16 differential metabolites were involved in multiple pathways, such as histidine metabolism, arginine biosynthesis, tyrosine metabolism, glyoxylate and dicarboxylate metabolism, phenylpropanoid biosynthesis, pyruvate metabolism, etc. Therefore, the composition of C. militaris cultivated with tussah pupa had significant advantage comparing with C. sinensis, which demonstrated that high-priced C. sinensis could be substituted with C. militaris cultivated with tussah pupa to some extent. PRACTICAL APPLICATIONS: This study comprehensively compared the chemical composition of Cordyceps sinensis (C. sinensis) and Cordyceps militaris (C. militaris) cultivated with tussah pupa by using metabonomics technology in order to clarify the similarity and difference of the two medicinal materials from the whole metabolite level. The experimental results provide a theoretical basis and scientific support for whether C. sinensis can be substituted with C. militaris cultivated with tussah pupa in clinical practice.

Effect of mechanical vibration on postharvest quality and volatile compounds of blueberry fruit.

The objective of the study was to investigate the effect of mechanical vibration on postharvest quality and volatile compounds in blueberries (Vaccinium spp. Berkeley). Ethylene production, respiratory rate, firmness, decay incidence, soluble solids content (SSC), titratable acid content (TAC), flavonoid content, total phenols content (TPC), enzyme activity and volatile compounds of blueberry fruit were determined. Results showed that mechanical vibration resulted in the increase of ethylene production, respiratory rate, decay incidence and the decrease of firmness, enzyme activity, SSC, TAC, flavonoid content, TPC in blueberries comparing with the control. Moreover, mechanical vibration improved the relative content of alcohols and decreased the relative content of esters in comparison with the control group. In conclusion, the effect of transport vibration on blueberry quality was conspicuous. The quality of blueberry fruit gradually decreased with the extension of vibration time during the transportation, which seriously reduced storage life and commodity value of blueberries.

Carotenoid Production by Caterpillar Medicinal Mushrooms, Cordyceps militaris (Ascomycetes), under Different Culture Conditions.

Cordyceps militaris, a well-known edible and medicinal species, is a potential source of natural carotenoids. The optimized conditions for carotenoid production are important for high-quality cultivation since carotenoid contents are the key factors for the appearance of fruiting bodies. In this study, carotenoid production by C. militaris under different culture conditions was compared. It was found that two-stage culture and fruiting body cultivation were suitable for the carotenoid production. Light is one of the important factors for carotenoid accumulation in this fungus and carotenoid can be produced by continuous shaking under light irradiation. Blue light with intensity of 17.3-64.3 lux can promote carotenoid production under the three tested culture methods. The carotenoid content was 4410.42 ± 30.83 and 3747.92 ± 178.13 µg g-1 under blue light intensity of 64.3 lux for the velum under two-stage culture and fruiting body cultivation, respectively. Oat medium may be suitable for the cultivation of fruiting bodies due to its high carotenoid production compared with other grains such as rice, wheat, and millet. Oxygen vector and oxidative stress induced by hydrogen peroxide can promote carotenoid production significantly under the two-stage culture. The carotenoid extracted from C. militaris showed strong antioxidant activity with IC50 value of 0.219 ± 0.01 mg ml-1, even higher than that of BHT, a positive control. This study will benefit the high-quality cultivation of this fungus and confirm that the carotenoid from C. militaris has the potential to be developed as a health food.

Evaluation of 1-methylcyclopropene (1-MCP) treatment combined with nano-packaging on quality of pleurotus eryngii.

The aim of the present study was to investigate the effect of 1-methylcyclopropene (1-MCP) combined with nano-packaging on quality of pleurotus eryngii stored at 4 ± 1 °C for 12 days. Texture, respiration rate, soluble protein, soluble sugar, weight loss, soluble solid, malondialdehyde (MDA) content, polyphenol oxidase (PPO), superoxide dismutase (SOD) and catalase (CAT) activity of treated pleurotus eryngii were determined. The results suggested that 1-MCP treatment combined with nano-packaging reduce respiration rate, weight loss and MDA content, delayed the decrease of soluble protein content, and maintained soluble sugar and soluble solid content of treated pleurotus eryngii during the storage at 4 ± 1 °C compared with the untreated samples. The efficiency of the combined treatment (1-MCP + nano-packaging) was better than that of 1-MCP or nano-packaging alone. Moreover, 1-MCP plus nano-packaging treatment effectively improved SOD and CAT activities, and suppressed the increase of PPO activity in pleurotus eryngii. Therefore, present results indicated that 1-MCP plus nano-packaging treatment may be an effective technology on maintaining commercial quality and lengthening shelf life of pleurotus eryngii.