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Wild-type cytochrome P450 CYP102A1 from Bacillus megaterium is a highly efficient monooxygenase for the oxidation of long-chain fatty acids. The unique features of CYP102A1, such as high catalytic activity, expression yield, regio- and stereoselectivity, and self-sufficiency in electron transfer as a fusion protein, afford the requirements for an ideal biocatalyst. In the past three decades, remarkable progress has been made in engineering CYP102A1 for applications in drug discovery, biosynthesis, and biotechnology. The repertoire of engineered CYP102A1 variants has grown tremendously, whereas the substrate repertoire is avalanched to encompass alkanes, alkenes, aromatics, organic solvents, pharmaceuticals, drugs, and many more. In this article, we highlight the major advances in the past five years in our understanding of the structure and function of CYP102A1 and the methodologies used to engineer CYP102A1 for novel applications. The objective is to provide a succinct review of the latest developments with reference to the body of CYP102A1-related literature.

CRISPR–Cas9 is the state-of-the-art programmable genome-editing tool widely used in many areas. For safe therapeutic applications in clinical medicine, its off-target effect must be dramatically minimized. In recent years, extensive studies have been conducted to improve the gene-editing specificity of the most popular CRISPR–Cas9 nucleases using different strategies. In this review, we summarize and discuss these strategies and achievements, with a major focus on improving the gene-editing specificity through Cas9 protein engineering.

Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347’s role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth.

Melampsora larici-populina (Mlp), the causal agent of Populus leaf rust, secretes an array of effectors into the host through the haustorium to gain nutrients and suppress immunity. The precise mechanisms by which these effectors promote virulence remain unclear. To address this question, we developed a transgenic Arabidopsis line expressing a candidate effector, Mlp124357. Constitutive expression of the effector increased plant susceptibility to pathogens. A GxxxG motif present in Mlp124357 is required for its subcellular localization at the vacuolar membrane of the plant cell, as replacement of the glycine residues with alanines led to the delocalization of Mlp124357 to the nucleus and cytoplasm. We used immunoprecipitation and mass spectrometry (MS) to identify Mlp124357 interaction partners. Only one of the putative interaction partners knock-out line caused delocalization of the effector, indicating that Arabidopsis protein disulfide isomerase-11 (AtPDI-11) is required for the effector localization. This interaction was further confirmed by a complementation test, a yeast-two hybrid assay and a molecular modeling experiment. Moreover, localization results and infection assays suggest that AtPDI-11 act as a helper for Mlp124357. In summary, our findings established that one of Mlp effectors resides at the vacuole surface and modulates plant susceptibility.

Alismatis Rhizoma (AMR) is a well-known natural medicine with a long history in Chinese medicine and has been commonly used for treating a wide range of ailments related to dysuria, edema, nephropathy, hyperlipidaemia, diabetes, inflammation as well as tumors in clinical applications. Most beneficial effects of AMR are attributed to the presence of protostane terpenoids, the major active ingredients of Alismatis Rhizoma (AMR). In this study, a systematic high performance liquid chromatography/diode-array detector/quadrupole-time-of-flight mass spectrometry (HPLC-DAD-Q-TOF MS) and ultra-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC-QqQ MS) method was developed for qualitative and quantitative analyses of the major AMR triterpenoids. First, a total of 25 triterpenoid components, including 24 known compounds and one new compound were identified by comparison with UV spectra, molecular ions and fragmentation behaviors of reference standards or the literature. Second, an efficient method was established for the rapid simultaneous determination of 14 representative triterpenoids by UPLC-QqQ MS. Forty-three batches of AMR were analyzed with linearity (r, 0.9980–0.9999), intra-day precision (RSD, 1.18%–3.79%), inter-day precision (RSD, 1.53%–3.96%), stability (RSD, 1.32%–3.97%), repeatability (RSD, 2.21%–4.25%), and recovery (98.11%–103.8%). These results indicated that new approaches combining HPLC-DAD-Q-TOF MS and UPLC-QqQ MS are applicable in the qualitative and quantitative analysis of AMR.

This paper advances the design of Rod Pre-strained Dielectric Elastomer Actuators (RP-DEAs) in their capability to generate comparatively large static actuation forces with increased lifetime via optimized electrode arrangements. RP-DEAs utilize thin stiff rods to constrain the expansion of the elastomer and maintain the in-plane pre-strain in the rod longitudinal direction. The aim is to study both the force output and the durability of the RP-DEA. Initial design of the RP-DEA had poor durability, however, it generated significantly larger force compared with the conventional DEA due to the effects of pre-strain and rod constraints. The durability study identifies the in-electro-active-region (in-AR) lead contact and the non-uniform deformation of the structure as causes of pre-mature failure of the RP-DEA. An optimized AR configuration is proposed to avoid actuating undesired areas in the structure. The results show that with the optimized AR, the RP-DEA can be effectively stabilized and survive operation at least four times longer than with a conventional electrode arrangement. Finally, a Finite Element simulation was also performed to demonstrate that such AR design and optimization can be guided by analyzing the DEA structure in the state of pre-activation.