BackgroundThe methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production.ResultsThe metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source.The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates.ConclusionsOverall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.

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The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.

Pichia (syn. Komagataella) pastoris is a widely used yeast platform for heterologous protein production. Expression cassettes are usually stably integrated into the genome of this host via homologous recombination. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden, particularly for secreted proteins. We have constructed a series of strains harboring different copy numbers of a Rhizopus oryzae lipase gene (ROL), aiming to find the optimum gene dosage for secreted Rol production. In order to accurately determine ROL gene dosage, we implemented a novel protocol based on droplet digital PCR (ddPCR), and cross validated it with conventional real-time PCR. Gene copy number determination based on ddPCR allowed for an accurate ranking of transformants according to their ROL gene dosage. Results indicated that ddPCR was particularly superior at lower gene dosages (one to five copies) over quantitative real-time PCR (qPCR). This facilitated the determination of the optimal ROL gene dosage as low as two copies. The ranking of ROL gene dosage versus Rol yield was consistent at both small scale and bioreactor chemostat cultures, thereby easing clone characterization in terms of gene dosage dependent physiological effects, which could be discriminated even among strains differing by only one ROL copy. A selected two-copy strain showed twofold increase in Rol specific production in a chemostat culture over the single copy strain. Conversely, strains harboring more than two copies of the ROL gene showed decreased product and biomass yields, as well as altered substrate consumption specific rates, compared to the reference (one-copy) strain. Biotechnol. Bioeng. 2016;113: 1542-1551. © 2015 Wiley Periodicals, Inc.

A cRiSpR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12 elena cámara, ibai Lenitz & Yvonne nygård * Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to ~ 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains. The yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms for industrial applications ranging from wine and beer fermentations to the production of biofuels and high-value metabolites 1,2. However, some of the current production processes are compromised by low yields and productivities, thus further optimization is required 3. In particular, the production of second-generation bioethanol and other biochemicals from lignocellulosic biomass, which provides an alternative to oil-based chemicals, suffers from sub-optimal productivity 4. During the hydrolysis of the raw material, inhibitory compounds (e.g. organic acids and aromatic aldehydes) are formed or released, compromising the microbial performance 5. While quite some work has been done on elucidating genes required for tolerance, much less work has been done on improving tolerance towards stress by altering expression of genes 6. Some previous studies demonstrated deletion 7-9 or overexpression 10-14 of endogenous genes to improve tolerance of S. cerevisiae towards inhibitors commonly found in lignocellulosic hydrolysates. However, most of the published work focuses on improving the tolerance of laboratory yeast strains that generally have weaker tolerance to stress 6 , while translation of beneficial modifications to more robust, industrial strains often is very challenging. The choice of yeast strain to be engineered is crucial for the successful implementation of the engineered phenotype in an industrial production process 15. Yeast strains used in industrial processes tend to be genetically diverse, since they usually arise from hybridization between different species 16. Hence...