Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Recombinant protein expression in <it>Pichia pastoris </it>strains with an engineered methanol utilization pathway

Abstract Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.</p

Homologous recombination frequency (HRFb) in wt CBS7435 and <i>ku70</i> deletion strains.

<p>Over 95% homologous recombination frequencies could be reached in the <i>ku70</i> deletion strain with as little as 250 bp of homologous sequence on each side of the integration cassette. In the corresponding wild-type strain, only 16.5% homologous recombination frequency was reached with the longest (1350 bp) homologous sequence tested.</p

Strains of <i>P. pastoris</i> used and constructed during this work.

<p>The growth rates reported correspond to the maximal growth rates (^h-1) reached in minimal media during the exponential growth phase. The standard deviation reported is calculated according to the growth rates of three biological replicates. c. = complemented. BM = buffered minimal media with glucose (D), glycerol (G) or methanol (M).</p>a<p>NRRL Y-11430, ATCC 76273.</p>b<p>Centraalbureau voor Schimmelcultures.</p

Integration cassette composition and function.

<p>a) <i>KU70</i> disruption cassette based on the <i>S. cerevisiae</i> FLP recombinase system. On both sides the flipper cassette with <i>AOX1</i> promoter (<i>P_AOX1</i>), FLP recombinase (<i>FLP</i>), CYC1 terminator (<i>CYC1_TT</i>) and Zeocin™ resistance cassette are surrounded by recombinase target sequences (<i>FRT</i>) and locus specific integration sequences (5′int and 3′int). Cassette components are not drawn to scale. b) After methanol induced (<i>P_AOX1</i>) FLP production and subsequent <i>FRT</i> recognition leading to cassette excision only one <i>FRT</i> (34 bp) is left in the locus in between the 3′ and 5′ integration sequences. c) The lengths of the homologous sequences at 5′ and 3′ ends of the disruption cassettes used to compare the homologous recombination frequencies in wt and ku70 deletion strains varied from 100 bp to 1350 bp in the <i>HIS4</i> locus. Zeocin™ resistance cassette was placed in between the homologous sequences. Cassette components are not drawn to scale.</p

New shuttle vectors constructed during this study.

a<p>Promoter to regulate the expression of the gene of interest.</p>b<p>Localization of the recombinant protein. Vectors aimed for intracellular production can be used for the secretory production by adding a signal sequence.</p

Selection marker integration.

<p>Integration sites of the gene disruption cassettes in <i>P. pastoris</i> wt strains which remained autotroph after selection marker integration.</p