CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.

Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for Fast Engineering of Heterologous Productions in Saccharomyces cerevisiae.

Nowadays, the yeast Saccharomyces cerevisiae is the platform of choice for demonstrating the proof of concept of the production of metabolites with a complex structure. However, introducing heterologous genes and rewiring the endogenous metabolism is still not standardized enough, affecting negatively the readiness-to-market of such metabolites. We developed the Easy Modular Integrative fuSion-ready Expression (Easy-MISE) toolkit, which is a novel combination of synthetic biology tools based on a single Golden Gate multiplasmid assembly meant to further ameliorate the rational predictability and flexibility of the process of yeast engineering. Thanks to an improved cloning screening strategy, double and independent transcription units are easily assembled and subsequently integrated into previously characterized loci. Moreover, the devices can be tagged for localization. This design allows for a higher degree of modularity and increases the flexibility of the engineering strategy. We show with a case study how the developed toolkit accelerates the construction and the analysis of the intermediate and the final engineered yeast strains, leaving space to better characterize the heterologous biosynthetic pathway in the final host and, overall, to improve the fermentation performances. Different S. cerevisiae strains were built harboring different versions of the biochemical pathway toward glucobrassicin (GLB) production, an indolyl-methyl glucosinolate. In the end, we could demonstrate that in the tested conditions the best-producing strain leads to a final concentration of GLB of 9.80 ± 0.267 mg/L, a titer 10-fold higher than the best result previously reported in the literature.

pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted double-strand breaks are controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming, and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome editing in S. cerevisiae, pCEC-red. This tool allows to (i) transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and (ii) insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and (iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (∼90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.

Conversion of sugar beet residues into lipids by Lipomyces starkeyi for biodiesel production.

BACKGROUND: Lipids from oleaginous yeasts emerged as a sustainable alternative to vegetable oils and animal fat to produce biodiesel, the biodegradable and environmentally friendly counterpart of petro-diesel fuel. To develop economically viable microbial processes, the use of residual feedstocks as growth and production substrates is required. RESULTS: In this work we investigated sugar beet pulp (SBP) and molasses, the main residues of sugar beet processing, as sustainable substrates for the growth and lipid accumulation by the oleaginous yeast Lipomyces starkeyi. We observed that in hydrolysed SBP the yeast cultures reached a limited biomass, cellular lipid content, lipid production and yield (2.5 g/L, 19.2%, 0.5 g/L and 0.08 g/g, respectively). To increase the initial sugar availability, cells were grown in SBP blended with molasses. Under batch cultivation, the cellular lipid content was more than doubled (47.2%) in the presence of 6% molasses. Under pulsed-feeding cultivation, final biomass, cellular lipid content, lipid production and lipid yield were further improved, reaching respectively 20.5 g/L, 49.2%, 9.7 g/L and 0.178 g/g. Finally, we observed that SBP can be used instead of ammonium sulphate to fulfil yeasts nitrogen requirement in molasses-based media for microbial oil production. CONCLUSIONS: This study demonstrates for the first time that SBP and molasses can be blended to create a feedstock for the sustainable production of lipids by L. starkeyi. The data obtained pave the way to further improve lipid production by designing a fed-batch process in bioreactor.