The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.DOI: http://dx.doi.org/10.7554/eLife.03703.001

Protein misfolding is monitored by a variety of cellular "quality control" systems. Endoplasmic reticulum (ER) quality control handles misfolded secretory and membrane proteins and is well characterized. However, less is known about the quality control of misfolded cytosolic proteins (CytoQC). To study CytoQC, we have employed a genetic system in Saccharomyces cerevisiae using a transplantable degron, CL1 (1). Attachment of CL1 to the cytosolic protein Ura3p destabilizes Ura3p, targeting it for rapid proteasomal degradation. We have performed a comprehensive analysis of Ura3p-CL1 degradation requirements. As shown previously, we observe that the ER-localized ubiquitin E2 (Ubc6p, Ubc7p, and Cue1p) and E3 (Doa10p) machinery involved in ER-associated degradation (ERAD) are also responsible for the degradation of the cytosolic substrate Ura3p-CL1. Importantly, we find that the cytosol/ER membrane-localized chaperones Ydj1p and Ssa1p, known to be necessary for the ERAD of membrane proteins with misfolded cytosolic domains, are also required for the ubiquitination and degradation of Ura3p-CL1. In addition, we show a role for the Cdc48p-Npl4p-Ufd1p complex in the degradation of Ura3p-CL1. When ubiquitination is blocked, a portion of Ura3p-CL1 is ER membrane-localized. Furthermore, access to the cytosolic face of the ER is required for the degradation of CL1 degroncontaining proteins. The ER is distributed throughout the cytosol, and our data, together with previous studies, suggest that the cytosolic face of the ER membrane serves as a "platform" for the degradation of Ura3p-CL1, which may also be the case for other CytoQC substrates.Mutation, errors in transcription or translation, and cellular stress can cause alterations in amino acids that may prevent proteins from attaining their properly folded, native conformations. Protein "quality control" is an essential process monitoring protein folding, ultimately targeting misfolded proteins for degradation via the ubiquitin-proteasome system. The importance of protein quality control is best exemplified by the numerous human diseases that can result from protein misfolding due to mutational or physiological causes and include cystic fibrosis, Parkinson disease, and ␣ 1 -antitrypsin deficiency (2, 3).Distinct protein quality control systems appear to exist in various cellular compartments, including the nucleus, mitochondria, and endoplasmic reticulum (ER), 2 with the bestcharacterized system being ER quality control (4 -7). Studies of ER quality control and, in particular, ER-associated degradation (ERAD) have revealed discrete chaperone and ubiquitination machinery required for the recognition and ubiquitination of different classes of misfolded secretory or membrane proteins. Much of this work has been greatly aided by the use of "model" ER quality control substrates, such as CPY* or Ste6p*, in the yeast Saccharomyces cerevisiae (8 -12). For example, it has become clear that model membrane proteins with misfolded cytosolic domains (called ERAD-C substrates), such as St...

Oleaginous yeast are promising organisms for the production of lipid-based chemicals and fuels from simple sugars. In this work, we explored Rhodosporidium toruloides for the production of lipid-based products. This oleaginous yeast natively produces lipids at high titers and can grow on glucose and xylose. As a first step, we sequenced the genomes of two strains, IFO0880, and IFO0559, and generated draft assemblies and annotations. We then used this information to engineer two R. toruloides strains for increased lipid production by over-expressing the native acetyl-CoA carboxylase and diacylglycerol acyltransferase genes using Agrobacterium tumefaciens mediated transformation. Our best strain, derived from IFO0880, was able to produce 16.4 ± 1.1 g/L lipid from 70 g/L glucose and 9.5 ± 1.3 g/L lipid from 70 g/L xylose in shake-flask experiments. This work represents one of the first examples of metabolic engineering in R. toruloides and establishes this yeast as a new platform for production of fatty-acid derived products.