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Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source. Here, we established-for the first time-production of the phenylalanine-derived benzylglucosinolate (BGLS) in Saccharomyces cerevisiae using two different engineering strategies: stable genome integration versus plasmid-based introduction of the biosynthetic genes. Although the plasmid-engineered strain showed a tendency to generate higher expression level of each gene (except CYP83B1) in the biosynthetic pathway, the genome-engineered strain produced 8.4-fold higher BGLS yield compared to the plasmid-engineered strain. Additionally, we optimized the genome-engineered strain by overexpressing the entry point genes CYP79A2 and CYP83B1, resulting in a 2-fold increase in BGLS production but also a 4.8-fold increase in the level of the last intermediate desulfo-benzylglucosinolate (dsBGLS). We applied several approaches to alleviate the metabolic bottleneck in the step where dsBGLS is converted to BGLS by sulfotransferase, SOT16 dependent on 39-phosphoadenosine-59-phosphosulfate (PAPS). BGLS production increased 1.7-fold by overexpressing SOT16 and 1.7-fold by introducing APS kinase, APK1, from Arabidopsis thaliana involved in the PAPS regeneration cycle. Modulating the endogenous sulfur assimilatory pathway through overexpression of MET3 and MET14 resulted in 2.4-fold to 12.81 mmol/L (=5.2 mg/L) for BGLS production. IMPORTANCE Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated wit

Halophytes tolerate high salinity levels that would kill conventional crops. Understanding salt tolerance mechanisms will provide clues for breeding salt-tolerant plants. Many halophytes such as quinoa (Chenopodium quinoa) are covered by a layer of epidermal bladder cells (EBCs) that are thought to mediate salt tolerance by serving as salt dumps. We isolated an epidermal bladder cell-free quinoa mutant (ebcf) that completely lacked EBCs and was mutated in REBC and REBC-like1. This mutant showed no loss of salt stress tolerance. When wild-type quinoa plants were exposed to saline soil, EBCs accumulated K + as the major cation, in quantities far exceeding those for Na + . Emerging leaves densely packed with EBCs had the lowest Na + content, whereas old leaves with deflated EBCs served as Na + sinks. When the leaves expanded, K + was recycled from EBCs, resulting in turgor loss that led to a progressive deflation of EBCs. Our findings suggest that EBCs in young leaves serve as a K + -powered hydrodynamic system that functions as a water sink for solute storage. Na + accumulates within old leaves that subsequently wilt and are shed. This mechanism improves the survival of quinoa under high salinity.

2-Phenylethylglucosinolate (2PE) derived from homophenylalanine is present in plants of the Brassicales order as a defense compound. It is associated with multiple biological properties, including deterrent effects on pests and antimicrobial and health-promoting functions, due to its hydrolysis product 2-phenylethyl isothiocyanate, which confers 2PE as a potential application in agriculture and industry. In this study, we characterized the putative key genes for 2PE biosynthesis from Barbarea vulgaris W.T. Aiton and demonstrated the feasibility of engineering 2PE production in Nicotiana benthamiana Domin. We used different combinations of genes from B. vulgaris and Arabidopsis thaliana (L.) Heynh. to demonstrate that: (i) BvBCAT4 performed more efficiently than AtBCAT4 in biosynthesis of both homophenylalanine and dihomomethionine; (ii) MAM1 enzymes were critical for the chain-elongated profile, while CYP79F enzymes accepted both chain-elongated methionine and homophenylalanine; (iii) aliphatic but not aromatic core structure pathway catalyzed the 2PE biosynthesis; (iv) a chimeric pathway containing BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in a two-fold increase in 2PE production compared with the B. vulgaris-specific chain elongation pathway; and (v) profiles of chain-elongated products and glucosinolates partially mirrored the profiles in the gene donor plant, but were wider in N. benthamiana than in the native plants. Our study provides a strategy to produce the important homophenylalanine and 2PE in a heterologous host. Furthermore, chimeric engineering of the complex 2PE biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes – a prerequisite for understanding biochemical evolution. The new-to-nature gene combinations have the potential for application in biotechnological and plant breeding.

Glucosinolates (GLS) are amino acid-derived defense compounds characteristic of the Brassicales order. They have received increased attention since they have been shown to and several types of cancer. These health beneficial effects have made GLSs attractive as dietary supplements. However, the concentration of GLSs is often low and highly regulated in plants natively producing GLSs. This has primed a desire to produce GLSs in microorganisms to provide a rich source and enable intake of a well-defined dose. In this thesis, I focus on the chain-elongated phenylalanine-derived 2-phenylethyl GLS, phenylalanine-derived benzyl GLS and chain-elongated methionine-derived 4- methylsulfinylbutyl GLS, which have been reported to have health beneficial effects. I identified the enzymes in the 2-phenylethyl GLS pathway and successfully produced 2- phenylethyl GLS in Nicotiana benthamiana. I deciphered that the enzymes BCAT4 and MAM1 in the chain elongation from Barbarea vulgaris are critical in the profile of chainelongated products. For the first time, I characterized the role of members of CYP79C family, CYP79C1 and CYP79C2 in GLS biosynthesis. Even though the genes are not expressed in Arabidopsis heterologous expression shows that they are involved in the production of 2- methylpropyl GLS, 1-methylpropyl GLS, benzyl GLS and indolyl-3-methyl GLS. Additionally, I engineered the pathway of benzyl GLS in yeast and optimized the production by modulating sulfur metabolism, increasing the activity of sulfotransferase SOT16 and enhancing the flux at the entry point to the pathway. Finally, I attempted to produce the precursor of 4-methylsulfinylbutyl GLS, dihomo-methionine, in yeast by expressing the chain elongation pathway of Arabidopsis. The one-cycle product homo-methionine but notdihomo-methionine was produced, which may be attributed to the absence of the large subunit of isopropylmalate isomerase as identified by targeted proteomics.

This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible with uracil-containing non-clonal DNA fragments obtained from any synthesizing company. The protocol drastically reduces time and handling between receiving the synthesized DNA fragments and transforming with vector and DNA fragment(s).

Yongxia Chen,1,3,* Ziying Yang,1,* Chao Liu,1 Cuiwei Wang,1 Shunxin Zhao,2 Jing Yang,1 Hongfan Sun,1 Zhengpu Zhang,2 Deling Kong,1 Cunxian Song1 1Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, 2Institute of Polymer Chemistry, Nankai University, 3Center for Medical Device Evaluation of Tianjin, Tianjin, People's Republic of China  *These authors contributed equally to this work Background: Star-shaped polymers provide more terminal groups, and are promising for application in drug-delivery systems. Methods: A new series of six-arm star-shaped poly(lactic-co-glycolic acid) (6-s-PLGA) was synthesized by ring-opening polymerization. The structure and properties of the 6-s-PLGA were characterized by carbon-13 nuclear magnetic resonance spectroscopy, infrared spectroscopy, gel permeation chromatography, and differential scanning calorimetry. Then, paclitaxel-loaded six-arm star-shaped poly(lactic-co-glycolic acid) nanoparticles (6-s-PLGA-PTX-NPs) were prepared under the conditions optimized by the orthogonal testing. High-performance liquid chromatography was used to analyze the nanoparticles' encapsulation efficiency and drug-loading capacity, dynamic light scattering was used to determine their size and size distribution, and transmission electron microscopy was used to evaluate their morphology. The release performance of the 6-s-PLGA-PTX-NPs in vitro and the cytostatic effect of 6-s-PLGA-PTX-NPs were investigated in comparison with paclitaxel-loaded linear poly(lactic-co-glycolic acid) nanoparticles (L-PLGA-PTX-NPs). Results: The results of carbon-13 nuclear magnetic resonance spectroscopy and infrared spectroscopy suggest that the polymerization was successfully initiated by inositol and confirm the structure of 6-s-PLGA. The molecular weights of a series of 6-s-PLGAs had a ratio corresponding to the molar ratio of raw materials to initi