Fluorescence and antioxidant activity of heterologous expression of phycocyanin and allophycocyanin from Arthrospira platensis.

Phycocyanin and allophycocyanin are important active substances in Arthrospira platensis, because of their fluorescent characteristic and antioxidant capacity. In order to solve the problem of insufficient production and inconvenient modification of natural protein, recombinant expression was performed and the fluorescence activity and antioxidant activity was analyzed to meet the demand for phycocyanin and allophycocyanin. A total of seven recombinant strains were constructed in this study, including individual phycocyanin or allophycocyanin, co-expression of phycocyanin-allophycocyanin, and their co-expression with chromophore, and the expression strain for individual chromophore. Different molecular weights of phycocyanin and allophycocyanin were detected in the recombinant strains, which indicated the different polymers expressed. Through mass spectrometry identification, phycocyanin and allophycocyanin may form a dimer of 66 kDa and a polymer of 300 kDa. The results of fluorescence detection showed that phycocyanin and allophycocyanin combined with phycocyanobilin to show fluorescence activity. The fluorescence peak of recombinant phycocyanin was mainly concentrated at 640 nm, which was similar to natural phycocyanin, the fluorescence peak of purified recombinant allophycocyanin was at about 642 nm. The fluorescence peak of the co-expressed recombinant phycocyanin-allophycocyanin is located at 640 nm, and the fluorescence intensity is between the recombinant phycocyanin and the recombinant allophycocyanin. After purification, the fluorescence peak of the recombinant phycocyanin is more concentrated and the fluorescence intensity is higher, which is about 1.3 times of recombinant phycocyanin-allophycocyanin, 2.8 times of recombinant allophycocyanin, indicating that phycocyanin may be more suitable to be used as fluorescence probe in medicine. The antioxidant capacity was measured by using total antioxidant capacity (T-AOC) and DPPH (2,2'-diphenyl-1-triphenylhydrazino) free radical scavenging method, and the recombinant phycobiliprotein showed antioxidant activity. Phycocyanobilin also has certain antioxidant activity and could enhance the antioxidant activity of phycobiliprotein to a certain extent. Recombinant phycocyanin-allophycocyanin polymer has stronger T-AOC, which is about 1.17-2.25 times that of the other five recombinant proteins. And recombinant phycocyanin has stronger DPPH antioxidant activity, which is about 1.2-2.5 times that of the other five recombinant proteins. This study laid the foundation for the application of recombinant phycocyanin and allophycocyanin in medical detection and drug development.

Expression of fatty acid related gene promotes astaxanthin heterologous production in Chlamydomonas reinhardtii.

Natural astaxanthin is a high-value ketone carotenoid mainly derived from Haematococcus pluvialis, which is an excellent antioxidant for human consumption. To study the role of lipids in accumulation of astaxanthin, the H. pluvialis-derived astaxanthin synthesis pathway genes (ß-carotene ketolase gene, BKT and ß-carotene hydroxylase gene, BCH) and fatty acid elongation gene (mitochondrial trans-2-enoyl-coa reductase gene, MECR) were heterologously co-expressed in C. reinhardtii. Zeaxanthin, the precursor of astaxanthin synthesis, was significantly increased after BKT and BCH were expressed. In contrast, the α-carotene that competes with astaxanthin synthesis for lycopene decreased significantly. This redistribution of carbon flow was conducive to the synthesis of astaxanthin. In addition, the transformant only expressed astaxanthin metabolism related genes (BKT, BCH) would lead to an increase in total lipid, a decrease in monounsaturated fatty acids and an increase in polyunsaturated fatty acids. On this basis, the expression of MECR gene further increased the total lipid, and the relative content of different fatty acids also changed. The astaxanthin content of algal strains transformed with BKT and BCH genes was nearly 50% higher than that of the wild type. On this basis, the astaxanthin content of transformants expressing MECR gene related to long-chain fatty acid synthesis was increased by 227.5%. In this study, an astaxanthin production model similar to H. pluvialis by combining carotenoid metabolism and fatty acid metabolism was constructed in C. reinhardtii. The results suggest that the increase in astaxanthin is indeed linked to the regulation of fatty acid metabolism, and this link may involve the type of fatty acids and the dynamics of astaxanthin ester in cells. The strategy of promoting the synthesis of fatty acids has potential to achieve efficient production of astaxanthin in C. reinhardtii.

Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis.

Molecular cloning and expression analysis of a new bilin lyase: the cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the ß-subunit of phycocyanin in Arthrospira platensis FACHB314.

To study the assembly of phycocyanin ß subunit, the gene cpcT was first cloned from Arthrospira platensis FACHB314. To explore the function of cpcT, the DNA of phycocyanin ß subunit and cpcT were transformed into Escherichia coli BL21 with the plasmid pET-hox1-pcyA, which contained the genes hemeoxygenase 1 (Hox1) and ferredoxin oxidoreductase (PcyA) needed to produce phycocyanobilin. The transformed strains showed specific phycocyanin fluorescence, and the fluorescence intensity was stronger than the strains with only phycocyanin ß subunit, indicating that CpcT can promote the assembly of phycocyanin to generate fluorescence. To study the possible binding sites of apo-phycocyanin and phycocyanobilin, the Cys-82 and Cys-153 of the ß subunit were individually mutated, giving two kinds of mutants. The results show that Cys-153 maybe the active site for ß subunit binding to phycocyanobilins, which is catalyzed by CpcT in A. platensis FACHB314.

Application of the yeast-surface-display system for orally administered salmon calcitonin and safety assessment.

High manufacturing costs and oral delivery are the constraints in clinical application of calcitonin. We selected surface-displayed Saccharomyces cerevisiae as a low-cost and safe carrier for oral delivery of salmon calcitonin (sCT). The sCT DNA fragment, optimized according to the codon preference of S. cerevisiae, was synthesized and cloned into the plasmid M-pYD1 to yield recombinant yAGA2-sCT, which was induced to express sCT by galactose for 0, 12, and 24 h. sCT expression was detected on the cell surface by indirect immunofluorescence and peaked at 12 h. About 65% recombinants expressed sCT on flow cytometry. The in vivo and in vitro activity of recombinant sCT was determined by detecting bioactivity of antiosteoclastic absorption on bone wafers and orally administering yAGA2-sCT to Wistar rats, respectively. For safety assessment of yAGA2-sCT, we observed abnormalities, morbidity, and mortality and determined body weight, serum chemistry parameters, hematological parameters, and organ weight. In vitro bioactivity of the recombinant sCT was similar to that of commercial sCT, Miacalcic; oral administration of 5 g/kg yAGA2-sCT induced a long-term hypocalcemic effect in Wistar rats and no adverse effects. This study demonstrates that yAGA2-sCT anchoring sCT protein on a S. cerevisiae surface has potential for low-cost and safe oral delivery of sCT.