Additional file 2: of Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

Figure S2. Simple strategy for confirming the orientation of the insert. The forward or reverse primer used for amplifying the insert can be used together with the forward or reverse sequencing primer of the vector to confirm the correct orientation. Upon correct primer choice only the forward orientation gives a PCR fragment. The sequencing primers designed for Sanger sequencing allow sequencing of the insert from both sides. Depending on the vector, different primers should be used (e.g. when the MFalpha signal sequence or a fusion protein is present, see the primer list for all sequencing primers available)

Prozesstechnik der DME und OME-Synthese und Life Cycle Assessment

Table S1. Primers used in this study and detailed plasmid construction. Primers used for creating the S. pombe and P. pastoris plasmids are separated. Also primers for construction of the plasmids are separated from primers for sequencing and insertion of GOIs. In addition separate spreadsheets are providing information on the exact construction of the plasmids by listing the PCR products and restriction enzymes used for assembly

Synthetic Core Promoters for <i>Pichia pastoris</i>

Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for <i>P. pastoris</i>, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural <i>AOX1</i> promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields

Synthetic Core Promoters for <i>Pichia pastoris</i>

Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for <i>P. pastoris</i>, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural <i>AOX1</i> promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields

Additional file 4: of Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

Figure S3. Fluorescence measurements of fusions of HRP to eGFP. Samples are labeled in the same way as in Figure 4. eGFP fluorescence of supernatants and cell pellets of methanol induced cells were normalized per cell density (OD600)

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

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

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