Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms

Abstract

There is a growing interest in establishing the methylotrophic yeast Pichia pastoris as microbial cell factories for producing fuels, chemicals, and natural products, particularly with methanol as the feedstock. Although CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technology has been established for the integration of multigene biosynthetic pathways, long (500−1000 bp) homology arms are generally required, probably due to low homologous recombination (HR) efficiency in P. pastoris. To achieve efficient genome integration of heterologous genes with short homology arms, we aimed to enhance HR efficiency by introducing the recombination machinery from Saccharomyces cerevisiae. First, we overexpressed HR related genes, including RAD52, RAD59, MRE11, and SAE2, and evaluated their effects on genome integration efficiency. Then, we constructed HR efficiency enhanced P. pastoris, which enabled single-, two-, and three-loci integration of heterologous gene expression cassettes with ∼40 bp homology arms with efficiencies as high as 100%, ∼98%, and ∼81%, respectively. Finally, we demonstrated the construction of β-carotene producing strain and the optimization of betaxanthin producing strain in a single step. The HR efficiency enhanced P. pastoris strains can be used for the construction of robust cell factories, and our machinery engineering strategy can be employed for the modification of other nonconventional yeasts.

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

“…17 With the flourishing development of synthetic biology and the advantages in expressing rate-limiting enzymes (e.g., polyketide synthase, terpene synthase, and cytochrome P450 enzyme) 18 to high levels, there is a growing interest in establishing K. phaffii as a chassis for large-scale production of natural products. 19 Currently, a panel of natural product biosynthetic pathways have been reconstituted in K. phaffii, including but not limited to (+)-valencene, (+)-nootkatone, 20−22 lycopene, 23 β-carotene, 24 astaxanthin, 25,26 betaxanthin, 27 menaquinone-4, 28 lovastatin, 29 and dammarenediol-II. Nevertheless, there has been no report on the biosynthesis of αsantalene in K. phaffii.…”

Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris

“…We and others previously showed that overexpression of HR machinery genes such as RAD52 remarkably enhance the HR efficiency in yeast and mammalian cells [ 15 , 17 , 26 , 27 ], which however resulted in low colony numbers and brought the difficulties in picking out the positive clones. One possible reason is that HR is considerably slower than NHEJ and the NHEJ can save more cells from DSB event.…”

“…1). Recently, we and others tried to engineer the recombination machinery to improve the HR repair process by overexpressing the HR related genes such as RAD52 and/or knocking out the NHEJ genes KU70/80 [11,12,[15][16][17]. However, the low efficiency in simultaneous deletion of multiple genes requires further enhancing HR procedure.…”

Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris

“…We and others previously showed that overexpression of HR machinery genes such as RAD52 remarkably enhance the HR e ciency in yeast and mammalian cells [14] [16] [25,26], which however resulted in low colony numbers and brought the di culties in picking out the positive clones. One possible reason is that HR is considerably slower than NHEJ and the NHEJ can save more cells from DSBs event.…”

“…1). Recently, we and others tried to engineer the recombination machinery to improve the HR repair process by overexpressing the HR related genes such as RAD52 and/or knocking out the NHEJ genes KU70/80 [10,11,[14][15][16]]. However, the low e ciency in simultaneous deletion of multiples genes requires further enhancing HR procedure.…”