Biosynthesis of α-lipoic acid in Gluconobacter oxydans increases the production of vitamin C by one-step fermentation / 生物工程学报

In a one-step fermentation system of vitamin C production with Gluconobacter oxydans and Ketogulonicigenium vulgare, a functional module of α-lipoic acid biosynthesis was constructed in G. oxydans. The engineered G. oxydans was co-cultured with K. vulgare to enhance the growth and 2-keto-L-gulonic acid (2-KGA) production of K. vulgare. This one-step fermentation system alleviated the growth inhibition during the mono-culture of K. vulgare and strengthened the interaction between the two bacteria. Moreover, the yield of vitamin C precursor (2-KGA) increased to 73.34 g/L (the control group was 59.09 g/L), and the conversion of D-sorbitol to 2-KGA increased to 86.0%. This study provides a new idea for further optimizing the one-step fermentation system of vitamin C production.

Isolation and partial characterization of a new moderate thermophilic Albidovulum sp. SLM16 with transaminase activity from Deception Island, Antarctica

BACKGROUND: A moderately thermophilic, slightly halophilic, aerobic, Gram-stain negative, bacterial strain, SLM16, was isolated from a mixed of seawater-sand-sediment sample collected from a coastal fumarole located in Whalers Bay, Deception Island, Antarctica. The aim was to screen for thermophilic microorganisms able to degrade primary amines and search for amine transaminase activity for potential industrial application. RESULTS: Identification and partial characterization of the microorganism SLM16 were carried out by means of morphological, physiological and biochemical tests along with molecular methods. Cells of strain SLM16 were non-motile irregular rods of 1.5-2.5 µm long and 0.3-0.45 µm wide. Growth occurred in the presence of 0.5-5.5% NaCl within temperature range of 35-55 °C and pH range of 5.5-9.5, respectively. The DNA G+C composition, estimated from ftsY gene, was 66% mol. Phylogenetic analysis using de 16S rRNA gene sequence showed that strain SLM16 belongs to the marine bacterial genus Albidovulum. CONCLUSION: Strain SLM16 is a moderate thermophilic Gram negative microorganisms which belongs to the marine bacterial genus Albidovulum and is closely related to Albidovulum inexpectatum species based on phylogenetic analysis. Additionally, amine-transaminase activity towards the arylaliphatic amine α-methylbenzylamine was detected.

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production

Abstract A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.

Fitness analysis between the L-sorbosone dehydrogenase modules and Ketogulonigenium vulgare chassis / 生物工程学报

Ketogulonigenium vulgare is an acid-producing strain in the process of two-step vitamin C fermentation. L-sorbosone dehydrogenase (SNDH) is one of the key enzymes during the biosynthesis of 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. However, the catalytic mechanism of SNDH is unclear. According to the whole genome sequencing of K. vulgare, two genes encoding sorbosone dehydrogenases, one derived from the chromosome (named as sndhg) and one from plasmid (named as sndhp), were introduced into an industrial strain K. vulgare. The overexpression of gene sndhg had hardly effect on 2-KGA production, and the overexpression of gene sndhp produced an obvious byproduct in the fermentation broth. Combinational expression of sndhg/sndhp with pqqA (obtaining sndhg-pqqA and sndhp-pqqA modules) in K. vulgare resulted in the similar fermentation phenotype to two previous strains. After serial sub-cultivation of co-cultured Bacillus endophyticus with each engineered K. vulgare for 50 d, the conversion rate of 2-KGA increased by 15.4%, 179%, 0.65% and 125% compared with that of the parental K. vulgare with B. endophyticus. This study shows that adaptive evolution of microbial consortium is an effective strategy to increase the fitness between functional modules and chassis, thus quickly getting better strains for production of 2-KGA.

Purification and structural elucidation of exoploysaccharide from a new marine bacterium Lentibacter algarum ZXM100T / 生物工程学报

Exopolysaccharide La0.1-1 was extracted from the broth of a marine bacterium Lentibacter algarum ZXM100T isolated from the seawater in the coastal region of Qingdao and purified by Q Sepharose Fast Flow ion-exchange chromatography and Superdex 75 gel-permeation chromatography. Its physiochemical properties and primary structural characters were investigated by chemical analysis together with high performance liquid chromatography (HPLC), high performance gel permeation chromatography (HPGPC) and gas chromatography and mass spectrometry (GC-MS). The results show that the total sugar content of the exoploysaccharide La0.1-1 was about 66% with an average molecular weight at 12.0 kDa. La0.1-1 is mainly composed of Gal, Man, GlcN at the ratio of 1.35:1.1:1.0. Results of GC-MS and NMR demonstrate that the exopolysaccharide La0.1-1 mainly exists with the beta configuration. The primary linkage styles are --> 2)-Manp(1 --> and --> 3)-Galp(1 --> with a small amount of --> 4)-Galp(--> 1 and --> 4)-Manp(1 --> linkages. The linkage mode of GlcN is --> 4)GlcN(1 --> and terminal linkage. The exopolysaccharide has mainly a linear sructure with a few branches linked to 0-6 of --> 2)-Manp(1 --> and 0-4 or 0-6 of --> 3)-Galp(1 -->. 1D-NMR data also revealed that La0.1-1 is substituted by certain acetyl; the acetyl is mainly linked to N-2 of GlcN. The exopolysaccharides of the bacterium of Lentibacter genus is reported for the first time, and an exopolysaccharide with novel structure was obtained, which enriched marine polysaccharide resources.

Enhancement of 2-keto-L-gulonic acid production using three-stage pH control strategy / 生物工程学报

The aim of this study was to improve the 2-keto-L-gulonic acid (2-KLG) production efficiency by Ketogulonicigenium vulgare and Bacillus megaterium by using multi-stage pH control strategy. The effect of pH on the cell growths and 2-KLG production showed that the optimum pH for K. vulgare and B. megaterium cell growth were 6.0 and 8.0, respectively, while the optimum pH for 2-KLG production was 7.0. Based on the above results, we developed a three-stage pH control strategy: the pH was kept at 8.0 during the first 8 h, then decreased to 6.0 for the following 12 h, and maintained at 7.0 to the end of fermentation. With this strategy, the titer, productivity of 2-KLG and L-sorbose consumption rate were achieved at 77.3 g/L, 1.38 g/(L x h) and 1.42 g/(L x h), respectively, which were 9.7%, 33.2% and 25.7% higher than the corresponding values of the single pH (pH 7.0) control model.

Enhancing 2-keto-L-gulonic acid production under hyperosmotic stress by adding sucrose / 生物工程学报

This study aimed to further enhance 2-keto-L-gulonic acid (2-KLG) production efficiency. A strategy for enhancing Ketogulonigenium vulgare growth and 2-KLG production by improving B. megaterium growth with sucrose was developed based on the time course of osmolality during 2-KLG industrial scale fermentation and effects of osmolality on cells growth and 2-KLG production. Results showed that the accumulation of 2-KLG and the feeding of alkaline matter led to an osmolality rise of 832 mOsmol/kg in the culture broth. High osmotic stress (1 250 mOsmol/kg) made the growth ofB. megaterium and K. vulgare decreased 15.4% and 31.7%, respectively, and consequently the titer and productivity of 2-KLG reduced 67.5% and 69.3%, respectively. When supplement sucrose under high osmotic condition (1 250 mOsmol/kg), B. megaterium growth was significantly improved, with the result that 2-KLG production was increased 87%. Furthermore, by applying this sucrose addition strategy further to batch fermentation in 3 L fermentor, the productivity of 2-KLG increased 10.4%, and the duration of fermentation declined 10.8%. The results presented here provide a potential strategy for enhancing the target metabolites produced by mixed strains at environmental stress.