One-pot synthesis of amino-alcohol using a de novo transketolase: Transaminase pathway in Pichia pastoris strain GS115

Pichia pastoris (P. pastoris) is an attractive industrial host cell due to its ability to grow up to 60% wet cell weight (WCW) by volume, a far higher level of biomass than the typical values reached by Escherichia coli (E. coli) and Saccharomyces cerevisiae. This thesis seeks to explore how the genetic tractability and high cell densities characteristic of P. pastoris can be exploited to intensify whole-cell biocatalysis. Chiral amino alcohols such as 2-amino-1,3,4-butanetriol (ABT) are key building blocks of small molecule pharmaceuticals and have previously been produced by whole-cell biocatalysis using cells engineered to overexpress a de novo enzyme pathway consisting of transketolase and transaminase. Within this work, native and foreign P. pastoris transaminases were characterized with respect to their biocatalytic potential. Genomic data mining was performed to explore the GS115 strain genome, allowing the selection of three putative Class III transaminase genes and the construction of overexpressor strains PpTAm107, PpTAm677 and PpTAm410. The well-studied ω-transaminase CV2025 from Chromobacterium violaceum was also successfully engineered to generate two strains; PpTAmCV708 for single expression of CV2025, and PpTAm-TK16 strain for CV2025 co-expression alongside a native transketolase previously characterized for L-erythrulose production. The rapid growth and high biomass characteristics of P. pastoris were successfully exploited for production of ABT by whole-cell biocatalysis. At high cell density, the best performance for the de novo pathway was obtained with the engineered PpTAm-TK16 strain, which tolerated high concentrations of substrate to achieve STY 0.57 g L-1 h-1 of ABT, 40-fold higher than levels previously achieved with E. coli for the same reaction

Codon optimisation of a Chromobacterium violaceum ω-transaminase for expression in Komagataella phaffii (Pichia pastoris)

A proprietary algorithm was applied, by a commercial service provider, to the coding DNA of <i>Chromobacterium violaceum</i> transaminase CV2025 gene, National Center for Biotechnology Information (NCBI) sequence reference WP_011135573.1, to optimise the codons used for expression in <i>Komagataella phaffii </i>(<i>Pichia pastoris</i>). The data sheet for the suggested codon optimisation is provided here, featuring 1. codon usage bias adjustment, 2. GC content adjustment, 3. restriction enzymes sites and cis-acting elements, 4. removed repeat sequences, 5. the optimized sequence and finally 6. an alignment of the starting and codon-optimized sequences

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production.

peer reviewedKomagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered

QTL2 maps to an N200D variant in <i>IRA1</i>.

(A) Detailed map of the QTL2 region in Cross 1. The upper panel shows Pp2 allele frequencies in the QTL2 region on chromosome 3, among the 30 superior and 30 inferior segregants in Cross 1, as in Fig 3A. Blue vertical bars indicate P values for biased segregation of alleles at individual SNP sites. The peak of QTL2 is 31 kb long and consists of 2 regions of 22 kb and 7 kb, each with P = 1.57 × 10−3, separated by a 2-kb region with P = 5.94 × 10−3. The lower panel shows a gene map of the 31-kb interval. IRA1 and the 8 genes colored green contain nonsynonymous SNPs in Pp2 relative to CBS7435 that are absent in Pp4. The 4 such SNP sites in IRA1 are labeled. (B) Reciprocal hemizygosity analysis of the effect of IRA1 alleles on BGL secretion in a CBS_BGL9/Pp2_BGL5 diploid. Haploid strains are included for comparison. X symbols in the cartoons indicate IRA1 alleles disrupted by insertion of a NatMX antibiotic resistance marker. Bars represent an average of 4-NP absorbance values from 3 independent cultures of control strains and at least 7 biological replicates from the reciprocally hemizygote strains. Error bars indicate standard deviation. Significant differences in BGL secretion were tested with unpaired t tests (two-tailed) and are indicated by asterisks (**, P P C) Effects of IRA1 SNP editing on BGL secretion. The edited strains were made in the haploid CBS_BGL9 background and contain individual nonsynonymous substitutions (N200D, V393L, D399N, G1466D) or a frameshift mutation (K404fs). Bars show mean 4-NP absorbance values from 3 independent cultures of control strains and at least 3 biological replicates from the edited strains. Error bars show standard deviation. Significant differences in BGL secretion between CBS_BGL9 and the other strains were tested by one-way ANOVA (Dunnet correction for multiple comparisons) and are indicated by asterisks (*, P P P S1 Data.</p

Allele frequencies at each SNP site in the selected segregant pools in Cross 1 and Cross 2.

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Uncropped version of S7 Fig.

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Evolutionary restoration of fertility in an interspecies hybrid yeast, by whole-genome duplication after a failed mating-type switch - Fig 5

<p>(A) Organization of <i>MAT</i>, <i>HML</i>, and <i>HMR</i> loci in <i>Z</i>. <i>parabailii</i> ATCC60483. The genome contains 6 <i>MAT</i>-related regions, with 1 <i>MAT</i>, 1 <i>HML</i>, and 1 <i>HMR</i> locus derived from each of the A and B parents. Pink and green backgrounds indicate sequences from the A- and B-subgenomes, respectively. The <i>MAT</i> locus in the A-subgenome (position 294 kb on chromosome 7) is intact and expressed. The <i>MAT</i> locus of the B-subgenome has been broken into 2 parts by cleavage by HO endonuclease. All 6 copies of the X repeat region (654 bp) are identical in sequence, as are all 6 copies of the Z repeat region (266 bp). Gray triangles indicate the disruption of the splicing of intron 2 in <i>MAT</i>α2 and <i>HML</i>α2 of the B-subgenome. The binding sites for primers A–F used for PCR amplification are indicated by gray arrows. (B) Sequences at the <i>MAT</i> locus breakpoint. Red, <i>MAT</i>α1-derived sequences. The HO cleavage site (CGCAGCA, giving a 4-nucleotide 3′ overhang) is highlighted in gray. Blue, the <i>GDA1-YEF1</i> intergenic region from the equivalent region of <i>Z</i>. <i>bailii</i> CLIB213^T and homologous sequences from the A-subgenome on <i>Z</i>. <i>parabailii</i> chromosomes (chrs.) 2 and 16. A 5-bp sequence (ACAAC) that became duplicated during the rearrangement is underlined. (C) Sequences of <i>MAT</i>α2 intron 2 (lowercase) from the A- and B-subgenomes. An AG-to-AC mutation (red) at the 3′ end of the intron moved the splice site by 2 bp in the B-subgenome, causing a frameshift and premature translation termination. The splice sites in both genes were identified from RNA sequencing (RNA-Seq) data.</p

SDS-PAGE analysis of BGL secretion over time in clones with and without <i>IRA1</i>^N200D.

Proteins were extracted from supernatants of 100-ml shake-flask cultures at 96 hours, 120 hours, 144 hours, and 168 hours. Comparisons are between CBS_PGAP (3 technical replicates), CBS_BGL9 (3 technical replicates), and ICs (4 independently clones in which the IRA1N200D variant was introduced by genome editing). The arrow indicates BGL, which migrates at its expected size of 120 kDa. L, molecular size standards. Due to a problem during gel loading, the sample from the fourth IC at 144 hours was split between 2 lanes. The cultures used for this experiment were the same ones used in Fig 6. (PDF)</p

Expression constructs and assessment of extracellular BGL secretion.

(A) Maps of the 2 BGL expression cassettes bearing zeocin (ZeoR) and geneticin (KanR) resistance markers. Also shown is the position and orientation of the cloned T. aurantiacus BGL gene. α-F, S. cerevisiae alpha-factor secretion signal. (B) UV imaging for qualitative assessment of extracellular BGL secretion in transformed clones of CBS7435, Pp2, and Pp4 using 4-MUG assays in 24-well microtiter plates. UV fluorescence occurs due to BGL hydrolysis of 4-MUG to 4-methylumbelliferone. (C) Line graphs showing quantitative time point assessment of BGL secretion in transformed clones of CBS7435, Pp2, and Pp4 using 4-NPG assays (3 ml cultures incubated for 96 hours). Optical absorbance at 405 nm occurs due to BGL hydrolysis of 4-NPG to 4-NP. Clones selected for use as parents in genetic crosses are marked with black rectangles. Numerical data are listed in S1 Data. BGL, β-glucosidase; 4-MUG, 4-methylumbelliferyl-β-D-glucuronide; 4-NP, 4-nitrophenol; 4-NPG, 4-nitrophenol-β-D-glucopyranoside. (PDF)</p

Effect of the <i>IRA1</i>^N200D variant on secretion of galactosidase enzymes.

(A) β-galactosidase activity of clones of IT1005 incorporating the IRA1N200D edit (green dots, 7 independently edited clones) is compared to unedited clones from the same strain (orange squares, 2 independent clones) that were transformed for CRISPR editing but failed to incorporate the IRA1N200D SNP, and to the original unedited strain IT1005 (blue triangles, 7 technical replicates). The negative control strains are CBS_pGAP (open inverted triangles, 7 technical replicates) and its IRA1N200D derivative (brown diamonds, 3 technical replicates). (B) α-galactosidase activity of clones of IT1018 incorporating the IRA1N200D edit (green dots, 11 independently edited clones) is compared to unedited clones from the same strain (orange squares, 2 independent clones) that were transformed for CRISPR editing but failed to incorporate the IRA1N200D SNP, and to the original unedited strain IT1018 (blue triangles, 7 technical replicates). The negative controls are the same as in (A). Numerical data are listed in S1 Data. (PDF)</p