D-(+)-pantothenic acid (DPA) has been widely used in feed and food additives and pharmaceutical industries. D(-)-pantolactone (DPL) is a key intermediate in the production of DPA. The bio-asymmetric synthesis of DPL from ketopantoyl lactone (KPL) by conjugated polyketone reductases is an attractive route. In this study, high expression of a conjugated polyketone reductase from Candida glabrata (Cg/CPR) in Escherichia coli was achieved after optimization of vectors. The optimum temperature and pH of purified Cg/CPR were 40 degrees C and 4.5, respectively. The Michaelis-Menten constant K-m and V-max of purified Cg/CPR for KPL are 73.09 mu M and 2.29 mu mol/mg(-1) s(-1), respectively. To circumvent the spontaneous degradation of KPL in biocatalytic reaction of synthesizing DPL, the reaction was performed by a strategy combing fed-batch addition of whole cell catalyst with substrate semi-continuous feeding. At the final KPL loading of 1000 mM, DPL with an isolated yield of 94.24 % was obtained after 13-h reaction and enantiomeric excess (e.e.) achieved > 99 %. The space-time yield of DPL reached to 228 g L-1 d(-1).

Background Ophiocordyceps sinensis is an important traditional Chinese medicine for its comprehensive active ingredients, such as cordycepin, cordycepic acid, and Cordyceps polysaccharide. O. sinensis zjut , a special strain isolated from O. sinensis, has similar pharmacological functions to wild O. sinensis . Currently, O. sinensis with artificial cultivation has been widely studied, but systematic fundamental research at protein levels has not been determined. Results Proteomes of O. sinensis zjut at different culture periods (growth period, 3rd day; pre-stable period, 6th day; and stable period, 9th day) were relatively quantified by relative isotope markers and absolute quantitative technology. In total, 4005 proteins were obtained and further annotated with Gene Ontology, Kyoto Encyclopedia of Genes and Genomes database. Based on the result of the annotations, metabolic pathways of active ingredients, amino acids and fatty acid were constructed, and the related enzymes were exhibited. Subsequently, comparative proteomics of O. sinensis zjut identified the differentially expressed proteins (DEPs) by growth in different culture periods, to find the important proteins involved in metabolic pathways of active ingredients. 605 DEPs between 6d-VS-3d, 1188 DEPs between 9d-VS-3d, and 428 DEPs between 9d-VS-6d were obtained, respectively. Conclusion This work provided scientific basis to study protein profile and comparison of protein expression levels of O. sinensis zjut, and it will be helpful for metabolic engineering works to active ingredients for exploration, application and improvement of this fungus.

Ophiocordyceps sinensis has been used as a traditional medicine or healthy food in China for thousands of years. Hirsutella sinensis was reported as the only correct anamorph of O. sinensis . It is reported that the laboratory-grown H. sinensis mycelium has similar clinical efficacy and less associated toxicity compared to the wild O. sinensis . The research of the H. sinensis is becoming more and more important and urgent. To gain deeper insight into the biological and pharmacological mechanisms, we sequenced the genome of H. sinensis . The genome of H. sinensis (102.72 Mb) was obtained for the first time, with > 99% coverage. 10,200 protein-encoding genes were predicted based on the genome sequence. A detailed secondary metabolism analysis and structure verification of the main ingredients were performed, and the biosynthesis pathways of seven ingredients (mannitol, cordycepin, purine nucleotides, pyrimidine nucleotides, unsaturated fatty acid, cordyceps polysaccharide and sphingolipid) were predicted and drawn. Furthermore, infection process and mechanism of H. sinensis were studied and elaborated in this article. The enzymes involved in the infection mechanism were also predicted, cloned and expressed to verify the mechanism. The genes and proteins were predicted and annotated based on the genome sequence. The pathways of several active components in H. sinensis were predicted and key enzymes were confirmed. The work presented here would improve the understanding of the genetic basis of this organism, and contribute to further research, production and application of H. sinensis .

To enhance nucleoside production in Hirsutella sinensis , the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase , inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase , uridine nucleosidase , uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis.

Two novel family 18 chitinases, chiA and chiH, were identified and cloned from the transcriptome of H. sinensis based on the transcriptome sequence data. The recombinant chitinases were overexpressed in Escherichia coli BL21, subsequently purified and functionally characterized. The optimal temperature and pH for chiA were 55 °C and 5.0, respectively, and those for chiH were 50 °C and 5.0, respectively. The highest enzyme activities of 11.5 U/mg and 8.1 U/mg were obtained for chiA and chiH, respectively, when colloidal chitin was used as the substrate with Ba2+. chiA exhibited higher Vmax of 1.94 μmol/μg/h and kcat of 1.443 S-1 than those of chiH with Vmax of 1.63 μmol/μg/h and kcat of 1.175 S-1, and both were efficient towards colloidal chitin compared with other typical family 18 chitinases. Substrate specificity and gene expression analyses indicated that chiA and chiH preferred substrates containing N-acetyl groups, such as colloidal chitin and glycol chitin, while no activity was detected toward laminarin, cellobiose, carboxymethyl cellulose and starch. The work presented here would aid in the understanding and performance of future studies on the infection mechanism of H. sinensis.

Hirsutella sinensis is considered as the only correct anamorph of Ophiocordyceps sinensis. To improve cordycepin and cordycepic acid production in H. sinensis, the biosynthetic pathways of cordycepin and cordycepic acid were predicted, and verified by cloning and expressing genes involved in these pathways, respectively. Then, 5′-nucleotidase participating in biosynthetic pathway of cordycepin, hexokinase, and glucose phosphate isomerase involved in biosynthetic pathway of cordycepic acid, were demonstrated playing important roles in the corresponding biosynthetic pathway by real-time PCR, accompanying with significantly up-regulated 15.03-, 5.27-, and 3.94-fold, respectively. Moreover, the metabolic regulation of H. sinensis was performed. As expected, cordycepin production reached 1.09 mg/g when additional substrate of 5′-nucleotidase was 4 mg/mL, resulting in an increase of 201.1 % compared with the control. In the same way, cordycepic acid production reached 26.6 and 23.4 % by adding substrate of hexokinase or glucose phosphate isomerase, leading to a rise of 77.3 and 55.1 %, respectively. To date, this is the first time to improve cordycepin and cordycepic acid production through metabolic regulation based on biosynthetic pathway analysis, and metabolic regulation is proved as a simple and effective way to enhance the output of cordycepin and cordycepic acid in submerged cultivation of H. sinensis.

The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2 mM of manganese sulfate on 0 day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6 days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis.

Background: Ophiocordyceps sinensis, a worm and fungus combined mixture which Hirsutella sinensis is parasitic on the caterpillar body, has been used as a traditional medicine or healthy food in China for thousands of years. H. sinensis is reported as the only correct anamorph of O. sinensis and its main active ingredients are similar to the natural O. sinensis.Results: H. sinensis L0106, asexual strain of O. sinensis, was isolated and identified in this study. Three transcriptomes of H. sinensis at different cultivation periods (growth period 3d, pre-stable period 6d and stable period 9d) were sequenced for the first time by RNA-Seq method, and 25,511 unigenes (3d), 25,214 unigenes (6d) and 16,245 unigenes (9d) were assembled and obtained, respectively. These unigenes of the three samples were further assembled into 20,822 unigenes (All), and 62.3 percent of unigenes (All) could be annotated based on protein databases. Subsequently, the genes and enzymes involved in the biosynthesis of the active ingredients according to the sequencing and annotation results were predicted. Based on the predictions, we further investigated the interaction of different pathway networks and the corresponding enzymes. Furthermore, the differentially expressed genes (DEGs) of H. sinensis grown during different developmental stages (3d-VS-6d, 3d-VS-9d and 6d-VS-9d) were globally detected and analyzed based on the data from RNA-Seq, and 764 DEGs between 3d and 6d, 1,869 DEGs between 3d and 9d, and 770 DEGs between 6d and 9d were found, respectively.Conclusions: This work presented here would aid in understanding and carrying out future studies on the genetic basis of H. sinensis and contribute to the further artificial production and application of this organism. This study provided a substantial contribution and basis to further characterize the gene expression profiles of H. sinensis in the metabolic pathways of active ingredients.