With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.

Benign prostatic hyperplasia (BPH) is one of the most common urinary diseases affecting men, generally after the age of 50. The prevalence of this multifactorial disease increases with age. With aging, the plasma level of testosterone decreases, as well as the testosterone/estrogen ratio, resulting in increased estrogen activity, which may facilitate the hyperplasia of the prostate cells. Another theory focuses on dihydrotestosterone (DHT) and the activity of the enzyme 5α-reductase, which converts testosterone to DHT. In older men, the activity of this enzyme increases, leading to a decreased testosterone/DHT ratio. DHT may promote prostate cell growth, resulting in hyperplasia. Some medicinal plants and their compounds act by modulating this enzyme, and have the above-mentioned targets. This review focuses on herbal drugs that are most widely used in the treatment of BPH, including pumpkin seed, willow herb, tomato, maritime pine bark, Pygeum africanum bark, rye pollen, saw palmetto fruit, and nettle root, highlighting the latest results of preclinical and clinical studies, as well as safety issues. In addition, the pharmaceutical care and other therapeutic options of BPH, including pharmacotherapy and surgical options, are discussed, summarizing and comparing the advantages and disadvantages of each therapy.

Terpenoids represent a large class of natural products with significant commercial applications. These chemicals are currently mainly obtained through extraction from plants and microbes or through chemical synthesis. However, these sources often face challenges of unsustainability and low productivity. In order to address these issues, Escherichia coli and yeast have been metabolic engineered to produce non-native terpenoids. With recent reports of engineering yeast metabolism to produce several terpenoids at high yields, it has become possible to establish commercial yeast production of terpenoids that find applications as perfume ingredients, pharmaceuticals and advanced biofuels. In this review, we describe the strategies to rewire the yeast pathway for terpenoid biosynthesis. Recent advances will be discussed together with challenges and perspectives of yeast as a cell factory to produce different terpenoids.

PCR‐mediated homologous recombination is a powerful approach to introduce epitope tags into the chromosomal loci at the N‐terminus or the C‐terminus of targeted genes. Although strategies of C‐terminal epitope tagging of target genes at their loci are simple and widely used in yeast, C‐terminal epitope tagging is not practical for all proteins. For example, a C‐terminal tag may affect protein function or a protein may get cleaved or processed, resulting in the loss of the epitope tag. Therefore, N‐terminal epitope tagging may be necessary to resolve these problems. In some cases, an epitope tagging strategy is used to introduce a heterologous promoter with the epitope tag at the N‐terminus of a gene of interest. The potential issue with this strategy is that the tagged gene is not expressed at the endogenous level. Another strategy after integration is to excise the selection marker, using the Cre‐LoxP system, leaving the epitope tagged gene expressed from the endogenous promoter. However, N‐terminal epitope tagging of essential genes using this strategy requires a diploid strain followed by tetrad dissection. Here we present 14 new plasmids for N‐terminal tagging, which combines two previous strategies for epitope tagging in a haploid strain. These ‘N‐ICE’ plasmids were constructed so that non‐essential and essential genes can be N‐terminally 3 × FLAG tagged and expressed from an inducible promoter (GAL1), constitutive promoters (CYC1 or PYK1) or the endogenous promoter. We have validated the N‐ICE plasmid system by N‐terminal tagging two non‐essential genes (SET1 and SET2) and two essential genes (ERG11 and PKC1). Copyright © 2016 John Wiley & Sons, Ltd.