Genetic analysis of the Komagataella phaffii centromeres by a color-based plasmid stability assay

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work we designed a color-based genetic assay to investigate genetic stability in K. phaffii. Plasmids bearing each centromere and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox. Author summary The methylotrophic yeast Komagataella phaffii is considered as one of the most important platforms for the production of proteins and metabolites. We sought in this study to develop a color-based genetic system widely used in other yeasts to assess mitotically stability of vectors carrying the proposed K. phaffii centromeres. First, we constructed a K. phaffii strain (LA3) mutant for ADE2 and ADE3; this resulted in a strain that forms white colonies and when transformed with a vector (pPICH-ADE3) carrying ADE3 turns red. Next, the four K. phaffii centromeres were cloned into pPICH-ADE3 and tested in LA3 for copy number and plasmid stability. Centromeres are responsible for proper chromosome segregation during cell division, hence guaranteeing that both daughter cells receive one copy of the duplicated DNA. Our results show that three K. phaffii centromeres behaved as expected conferring extra stability to the replicative plasmids and maintaining them at low copy number. Once characterized, centromeres can be used as parts in the construction of advanced genetic manipulation tools, thus allowing the construction of strains capable of expressing large metabolic pathways for the production of complex biochemicals.


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In yeasts, adenine synthesis pathway is used as a tool for auxotrophic selection,  (Fig 1). Deletion of ADE3 in S. cerevisiae has regulatory effects in the histidine 128 synthesis pathway [30]. Consequently, ade2 ade3 strains are not only auxotrophic for 129 adenine, but also for histidine. In order to verify if this phenotype is applicable to K. 130 phaffii, we plated strains X-33, LA2 and LA3 on MD medium without supplementation, 131 comparing growth and colony color to cells plated on MD medium with adenine and 132 histidine (Fig 1). LA3 strain displayed the expected histidine auxotrophy phenotype, showing that the adenine-histidine pathways in K. phaffii and S. cerevisiae have 134 common characteristics.

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In order to assess plasmid stability, we first constructed plasmid pPICH-ADE3 136 bearing the ADE3 gene (Fig 2). When transformed with pPICH-ADE3, LA3 cells 137 should return to being red and any changes on colony color would allow a simple 138 screening of plasmid loss [26]. Although adenine auxotrophy has been explored for 139 other purposes in K. phaffii [28], this particular color-based system has not yet been 140 used for measuring plasmid stability in this yeast. 141 pPICH-ADE3 was used for cloning of all four K. phaffii centromeres. Since it 142 revealed extremely difficult to amplify entire centromeric regions we designed a 143 strategy to amplify centromeres in halves in order to reduce fragment size and to avoid 144 primer annealing inside the inverted repeats (Fig 3). Amplified fragments exhibited in 145 their ends overlapping regions that would allow recombination between each other and 146 with vector pPICH-ADE3.

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LA3 strain was individually transformed with pPICH-ADE3 and all four 148 constructed centromeric plasmids (pPICH-CEN1-4). Plasmids were verified for 149 autonomous replication by PCR analysis which confirmed their circular conformation 150 (data not shown) and by plasmid rescue in E. coli. In this case, only pPICH-CEN3 could 151 not be recovered, probably because CEN3 was the only centromere that could not be 152 cloned directly in E. coli. 153 Plasmid stability was firstly verified through colony color in non-selective 154 medium (Fig 4). When plated on YPD non-selective medium, colonies transformed with 155 pPICH-ADE3 lost their color rapidly and presented a red center with large white edges, a result consistent with plasmid instability. In contrast, strains transformed with all 157 centromeric plasmids presented a uniform red coloration throughout the colony.

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Further stability examination of the centromeric plasmids was performed by 159 growing cells in liquid YPD medium for 144 hours. After diluting and plating cultures 160 on non-selective medium, red and white colonies were counted and compared between 161 each construction (Fig 5). LA3 strain transformed with pPICH-ADE3 did not yield red 162 colonies in any growth period, indicating that the plasmid was mitotically unstable. 163 Conversely, centromeric plasmids presented a higher mitotic stability than pPICH-   peptone and 10 g L -1 NaCl, pH 7,2). When needed, agar was added to a final 218 concentration of 1,5%. When zeocin (25 µg mL -1 ) was used for bacterial antibiotic 219 selection, NaCl concentration was reduced to 5 g L -1 .

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Zeocin and geneticin, when used, were added at 100 µg mL -1 and 500 µg mL -1 ,  Polymerase. All primers used in this work are shown in Table 1.  Table 1. Primers used in this work.

Primer
Sequence Enzyme Restriction sites are underlined.