4.7 Article

Development of CRISPR/Cas9-Based Genome Editing Tools for Polyploid Yeast Cyberlindnera jadinii and Its Application in Engineering Heterologous Steroid-Producing Strains

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ACS SYNTHETIC BIOLOGY
卷 -, 期 -, 页码 -

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AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.3c00278

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Cyberlindnera jadinii; CRISPR/Cas9; heterologoussteroids; campesterol; cholesterol

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This study developed an efficient CRISPR/Cas9 toolkit to address challenges in genetic manipulation of the polyploid genome of industrial yeast Cyberlindnera jadinii. The toolkit achieved high efficiency in single-gene knockdown and integration of exogenous genes. The research demonstrated the potential of C. jadinii in producing heterologous sterols and successfully increased the production of campesterol and cholesterol through shake-flask fermentation and high-cell-density fed-batch fermentation.
In this study, a suite of efficient CRISPR/Cas9 tools was developed to overcome the genetic manipulation challenges posed by the polyploid genome of industrial yeast Cyberlindnera jadinii. The developed CRISPR/Cas9 system can achieve a 100% single-gene knockdown efficiency in strain NBRC0988. Moreover, the integration of a single exogenous gene into the target locus using a 50 bp homology arm achieved near-100% efficiency. The efficiency of simultaneous integration of three genes into the chromosome is strongly influenced by the length of the homology arm, with the highest integration efficiency of 62.5% obtained when selecting a homology arm of about 500 bp. By utilizing the CRISPR/Cas system, this study demonstrated the potential of C. jadinii in producing heterologous sterols. Through shake-flask fermentation, the engineered strains produced 92.1 and 81.8 mg/L of campesterol and cholesterol, respectively. Furthermore, the production levels of these two sterols were further enhanced through high-cell-density fed-batch fermentation in a 5 L bioreactor. The highest titer of campesterol reached 807 mg/L [biomass OD600 = 294, productivity of 6.73 mg/(Lh)]. The titer of cholesterol reached 1.52 g/L [biomass OD600 = 380, productivity of 9.06 mg/(Lh)], marking the first gram-scale production of steroidal compounds in C. jadinii.

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