An improved CRISPRi system in Pichia pastoris.

CRISPR interference (CRISPRi) has been developed and widely used for gene repression in various hosts. Here we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional repressor domains. The CRISPRi system shows strong repression of eGFP, with the highest efficiency of 85%. Repression of native genes is demonstrated by targeting AOX1 promoter. AOX1 is efficiently repressed and the mutant strains show much slower growth in methanol medium. Effects of gRNA expression and processing on CRISPRi efficiency is also investigated. It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression, and Csy4 cleavage shows higher repression efficiency. However, gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of eGFP. By expression of two gRNAs targeting promoters of eGFP and AOX1 in an array together with Cys4 recognition sites, both genes can be repressed simultaneously. Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes (FAA1 and FAA2). Both genes are efficiently repressed, demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in P. pastoris.

Enhancing wine ester biosynthesis in mixed Hanseniaspora uvarum/Saccharomyces cerevisiae fermentation by nitrogen nutrient addition.

The dynamic changes of wine ester production during mixed fermentation with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae F5 was investigated at different levels and timings of nitrogen nutrient addition. Nitrogen additions were performed by supplementing yeast assimilable nitrogen (YAN) into a synthetic grape must with defined composition. Ester precursors and extracellular metabolites involved in ester synthesis were analyzed throughout the fermentation. Results showed that nitrogen additions covering 50-200 mg/L YAN at the point of yeast inoculation slightly affected yeast competition and ester profiles. Interestingly, when YAN was supplemented in the mid-stage, the survival of H. uvarum Yun268 was enhanced, resulting in more than a 2-fold increase in the levels of higher alcohol acetates compared to that at the initial stage. Furthermore, carbon fluxes may be redistributed in the central pathway, which contributed to the production of medium-chain fatty acids and eventually triggered a 1.2-fold elevation in corresponding ethyl ester levels.