High production of valencene in Saccharomyces cerevisiae through metabolic engineering.

BACKGROUND: The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive. RESULTS: Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield. CONCLUSIONS: This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.

Folate Receptor-Targeted and GSH-Responsive Carboxymethyl Chitosan Nanoparticles Containing Covalently Entrapped 6-Mercaptopurine for Enhanced Intracellular Drug Delivery in Leukemia.

For enhanced intracellular accumulation of 6-mercaptopurine (6-MP) in leukemia, a folate receptor-targeted and glutathione (GSH)-responsive polymeric prodrug nanoparticle was made. The nanoparticles were prepared by conjugating 6-MP to carboxymethyl chitosan via a GSH-sensitive carbonyl vinyl sulfide linkage, ultrasonic self-assembly and surface decoration with folate. The TEM graphs shows that the as-synthesized nanoparticles are spherical with a particle size of 170~220 nm. In vitro drug release of nanoparticles demonstrated acceptable stability in PBS containing 20 µM GSH at pH 7.4. However, the cumulative drug release rate of the samples containing 20 mM and 10 mM GSH medium reached 78.9% and 64.8%, respectively, in pH 5.0 at 20 h. This indicated that this nano-sized system is highly sensitive to GSH. The inhibition ratio of folate-modified nanoparticles compared to unmodified nanoparticles was higher in cancer cells (human promyelocytic leukemia cells, HL-60) while their cytotoxicity was lower in normal cells (mouse fibroblast cell lines, L929). Furthermore, in vitro cancer cell incubation studies confirmed that folate-modified nanoparticles therapeutics were significantly more effective than unmodified nanoparticles therapeutics. Our results suggest that folate receptor-targeting and GSH-stimulation can significantly elevate tumour intracellular drug release. Therefore, folate-modified nanoparticles containing chemoradiotherapy is a potential treatment for leukemia therapy.

Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production.

ε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

High-throughput three-dimensional chemotactic assays reveal steepness-dependent complexity in neuronal sensation to molecular gradients.

Many cellular programs of neural development are under combinatorial regulation by different chemoattractive or chemorepulsive factors. Here, we describe a microfluidic platform that utilizes well-controlled three-dimensional (3D) diffusion to generate molecular gradients of varied steepness in a large array of hydrogel cylinders, allowing high-throughput 3D chemotactic assays for mechanistic dissection of steepness-dependent neuronal chemotaxis. Using this platform, we examine neuronal sensitivity to the steepness of gradient composed of netrin-1, nerve growth factor, or semaphorin3A (Sema3A) proteins, and reveal dramatic diversity and complexity in the associated chemotactic regulation of neuronal development. Particularly for Sema3A, we find that serine/threonine kinase-11 and glycogen synthase kinase-3 signaling pathways are differentially involved in steepness-dependent chemotactic regulation of coordinated neurite repellence and neuronal migration. These results provide insights to the critical role of gradient steepness in neuronal chemotaxis, and also prove the technique as an expandable platform for studying other chemoresponsive cellular systems.