Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser–His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.

Widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems; thus, the enzymatic degradation of PET can be a promising solution. Although PETase from Ideonalla sakaiensis (IsPETase) has been reported to have the highest PET degradation activity under mild conditions of all PET-degrading enzymes reported to date, its low thermal stability limits its ability for efficient and practical enzymatic degradation of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy using several IsPETase variants that were screened for high thermal stability to improve PET degradation activity. In particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized β6-β7 connecting loop and extended subsite IIc, had a T m value that was increased by 8.81 °C and PET degradation activity was enhanced by 14-fold at 40 °C in comparison with IsPETaseWT. The designed structural modifications were further verified through structure determination of the variants, and high thermal stability was further confirmed by a heat-inactivation experiment. The proposed strategy and developed variants represent an important advancement for achieving the complete biodegradation of PET under mild conditions.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms.

Highly efficient organic/inorganic hybrid perovskite light-emitting diodes (PeLEDs) based on graphene anode are developed for the first time. Chemically inert graphene avoids quenching of excitons by diffused metal atom species from indium tin oxide. The flexible PeLEDs with graphene anode on plastic substrate show good bending stability; they provide an alternative and reliable flexible electrode for highly efficient flexible PeLEDs.

Tomato yellow leaf curl virus (TYLCV) is one of the most well-known tomato-infecting begomoviruses and transmitted by Bemisia tabaci. Seed transmission has previously been reported for some RNA viruses, but TYLCV has not previously been described as a seed-borne virus. In 2013 and 2014, without whitefly-mediated transmission, TYLCV was detected in young tomato plants germinated from fallen fruits produced from TYLCV-infected tomato plants in the previous cultivation season. In addition, TYLCV-Israel (TYLCV-IL) was also detected in seeds and their seedlings of TYLCV-infected tomato plants that were infected by both viruliferous whitefly-mediated transmission and agro-inoculation. The seed infectivity was 20–100%, respectively, and the average transmission rate to seedlings was also 84.62% and 80.77%, respectively. TYLCV-tolerant tomatoes also produced TYLCV-infected seeds, but the amount of viral genome was less than seen in TYLCV-susceptible tomato plants. When tomato plants germinated from TYLCV-infected seeds, non-viruliferous whiteflies and healthy tomato plants were placed in an insect cage together, TYLCV was detected from whiteflies as well as receiver tomato plants six weeks later. Taken together, TYLCV-IL can be transmitted via seeds, and tomato plants germinated from TYLCV-infected seeds can be an inoculum source of TYLCV. This is the first report about TYLCV seed transmission in tomato.