Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

BACKGROUND: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. RESULTS: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. CONCLUSIONS: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Genetic determinants for enhanced glycerol growth of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae generally shows a low natural capability to utilize glycerol as the sole source of carbon, particularly when synthetic medium is used and complex supplements are omitted. Nevertheless, wild type isolates have been identified that show a moderate growth under these conditions. In the current study we made use of intraspecies diversity to identify targets suitable for reverse metabolic engineering of the non-growing laboratory strain CEN.PK113-1A. A genome-wide genetic mapping experiment using pooled-segregant whole-genome sequence analysis was conducted, and one major and several minor genetic loci were identified responsible for the superior glycerol growth phenotype of the previously selected S. cerevisiae strain CBS 6412-13A. Downscaling of the major locus by fine-mapping and reciprocal hemizygosity analysis allowed the parallel identification of two superior alleles (UBR2CBS 6412-13A and SSK1CBS 6412-13A). These alleles together with the previously identified GUT1CBS 6412-13A allele were used to replace the corresponding alleles in the strain CEN.PK113-1A. In this way, glycerol growth could be established reaching a maximum specific growth rate of 0.08h(-1). Further improvement to a maximum specific growth rate of 0.11h(-1) could be achieved by heterologous expression of the glycerol facilitator FPS1 from Cyberlindnera jadinii.

A novel methanol-free Pichia pastoris system for recombinant protein expression.

BACKGROUND: As one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (P AOX1 ) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in P AOX1 activation or repression by different carbon sources may be promising targets. RESULTS: We identified two kinase mutants: Δgut1 and Δdak, both of which showed strong alcohol oxidase activity under non-methanol carbon sources. Based on these two kinases, we constructed two methanol-free expression systems: Δgut1-HpGCY1-glycerol (P AOX1 induced by glycerol) and Δdak-DHA (P AOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Δdak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Δdak-DHA system was generally better than the constitutive GAP promoter, and reached 50-60 % of the traditional methanol induced system. CONCLUSIONS: We successfully constructed a novel methanol-free expression system Δdak-DHA. This modified expression platform preserved the favorable regulatable nature of P AOX1 , providing a potential alternative to the traditional system.

Submucosal Injection of the RNA Oligonucleotide GUT-1 in Active Ulcerative Colitis Patients: A Randomized, Double-Blind, Placebo-Controlled Phase 2a Induction Trial.

BACKGROUND AND AIMS: Carbohydrate sulfotransferase 15 [CHST15] biosynthesizes sulphated matrix glycosaminoglycans and is implicated in intestinal inflammation and fibrosis. Here, we evaluate the efficacy and safety of the double-stranded RNA oligonucleotide GUT-1, a specific blocker of CHST15, as induction therapy in patients with ulcerative colitis [UC]. METHODS: In this randomized, double-blind, placebo-controlled, phase 2a study, we enrolled endoscopically active UC patients, refractory to conventional therapy, in five hospital centres across Germany. Patients were randomized 1:1:1 using a block randomized technique to receive a single dosing of 25 nM GUT-1, 250 nM GUT-1, or placebo by endoscopic submucosal injections. The primary outcome measure was improvement of endoscopic lesions at weeks 2 or 4. The secondary outcome measures included clinical and histological responses. Safety was assessed in all patients who received treatment. RESULTS: Twenty-eight patients were screened, 24 were randomized, and 21 were evaluated. Endoscopic improvement at weeks 2 or 4 was achieved by 71.4% in the GUT-1 250 nM, 0% in the GUT-1 25 nM, and 28.6% in the placebo group. Clinical remission was shown by 57.1% in the GUT-1 250 nM, 0% in the GUT-1 25 nM, and 14.3% in the placebo groups. Histological improvement was shown by 42.9% in the GUT-1 250 nM, 0% in the GUT-1 25 nM, and 0% in the placebo groups. GUT-1 250 nM reduced CHST15 expression significantly and suppressed mucosal inflammation and fibrosis. GUT-1 application was well tolerated. CONCLUSION: Single dosing by submucosal injection of GUT-1 repressed CHST15 mucosal expression and may represent a novel induction therapy by modulating tissue remodelling in UC.