Photoclick reaction for rapid and simple fluorescence detection of itaconic acid and its derivatives in fungal cultures.

Itaconic acid (IA) and its derivatives produced by fungi have significant potential as industrial feedstocks. We recently developed a method for the detection of these compounds based on their terminal C-C double bonds. However, the presence of reducing agents, such as glucose and other fungal metabolites, leads to undesirable side reactions, and consequently, deteriorates the detection specificity. Therefore, we developed a fluorescence detection method for IA and its derivatives underpinned by a photoclick reaction. The photoclick reaction between conjugated IA and 5-(4-methoxyphenyl)-2-phenyl-2H-tetrazole under UV irradiation affords a fluorescent product. No fluorescence was detected when succinic acid was subjected to the reaction, indicating that a terminal C-C double bond is required to induce fluorescence. Optimal reaction conditions were determined to be a combination of 80% final dimethyl sulfoxide concentration, 30-s UV irradiation, and a pH of 2. Two weeks after the reaction at 4 °C, 89.0% of the initial intensity was retained, indicating that the reaction product was relatively stable. Glucose and kojic acid did not induce fluorescence after the reaction, indicating that these reducing agents did not affect fluorescence. IA was detected in a culture of Aspergillus terreus, and its quantification using the photoclick reaction was in agreement with the results obtained using high-performance liquid chromatography analysis. Interestingly, the IA derivative avenaciolide present in submillimolar quantities was also detectable in a culture of Aspergillus avenaceus using this method. The established method will enable the development of high-throughput screening methods to identify fungi that produce IA and its derivatives.

Production of glycerate from glucose using engineered Escherichiacoli.

In this study, glycerate was produced from glucose using engineered Escherichia coli BW25113. Plasmid pSR3 carrying gpd1 and gpp2 encoding two isoforms of glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae and plasmid pLB2 carrying aldO encoding alditol oxidase from Streptomyces violaceoruber were introduced into E. coli to enable the production of glycerate from glucose via glycerol. Disruptions of garK and glxK genes in the E. coli genome were performed to minimize the consumption of glycerate produced. As a result, E. coli carrying these plasmids could produce nearly three times higher concentration of glycerate (0.50 ± 0.01 g/L) from 10 g/L glucose compared to E. coli EG_2 (0.14 ± 0.02 g/L). In M9 medium, disruption of garK and glxK resulted in an impaired growth rate with low production of glycerate, while supplementation of 0.5 g/L casamino acids and 0.5 g/L manganese sulfate to the medium replenished the growth rate and elevated the glycerate titer. Further disruption of glpF, encoding a glycerol transporter, increased the glycerate production to 0.80 ± 0.00 g/L. MR2 medium improved the glycerate production titers and specific productivities of E. coli EG_4, EG_5, and EG_6. Upscale production of glycerate was carried out in a jar fermentor with MR2 medium using E. coli EG_6, resulting in an improvement in glycerate production up to 2.37 ± 0.46 g/L with specific productivity at 0.34 ± 0.11 g-glycerate/g-cells. These results indicate that E. coli is an appropriate host for glycerate production from glucose.