Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P AOX1 ) and/or the constitutive GAP promoter (P GAP ). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P GAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH outperformed P GAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that PDH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for PDH under different feeding and osmolarity conditions. In all of them the PDH controlled process outperformed the P GAP one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific productivity than P GAP fed-batch at low μ. Compared to the optimum approach for P GAP -based process, the best PDH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, PDH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy.


Cloning of the truncated promoter sequences
The different promoter truncations (purified PCR amplicons) were then cloned into an entry plasmid by isothermal assembly following Gibson (figure 1) (Gibson, 2009;Gibson et al., 2009). The entry plasmid contained a 1100 bp homologous region to the 3'UTR of the K. phaffii ARG4 gene to ensure comparability of the different constructs by targeted integration, eGFP as a reporter gene and the AOX1 transcription terminator for eGFP transcription termination. Additionally, a zeocin TM resistance cassette was present to enable selection and a pUC origin of replication for plasmid maintenance and propagation in E. coli.
For the assembly, 100 ng of the linear entry plasmid (linearized between 3'UTR of ARG4 gene and eGFP coding sequence) were mixed with 50-80 ng of the PCR amplicon (promoter truncation) in a total volume of 5 μL. The DNA was mixed with 15 μL of self-made master mix and incubated at 50°C for one hour. After assembly, 2.5 µL of the reaction mixture was introduced into E. coli for propagation of newly assembled plasmids, transformants were selected on LB plates supplemented with an appropriate amount of Zeocin. Correct assembly of the plasmids was verified by Sanger sequencing (service provided by Microsynth AG).

K. phaffii transformation with the truncated promoter sequences
Plasmids were then linearized by SmiI and cells of the K. phaffii strain BSY11dKU70 were transformed with 1 µg linearized DNA. Preparation and transformation of electrocompetent K. phaffii cells was performed according to Lin-Cereghino et al. (2005). Selection was done on zeocin containing YPD agar plates. Figure S1: Plasmid Map of the screening plasmid

Cultivation and eGFP measurement of the truncated promoter clones
Seven clones per each construction were then cultivated in 96-well plates for 6 days. Starting volume was 250 µL buffered minimal media with either 1% (w/v) glucose (BMD1) or 1% (w/v) glycerol (BMG1). After each 24 h 40 µL of culture were discarded, 10 µL were used for eGFP and cell density measurement in a plate reader (eGFP: 488 nm absorption, 507 nm emission; cell density: 600 nm), and 100 µL of fresh BMD1 or BMG1 were added to the culture to be as near as possible to a continuous cultivation as it is possible employing deep-well plates.
Supplementary Table S2: List of primers used for qPCR to determine gene dosage of PDH-C by amplifying the Zeocine gene (Zeo R ) in the expression cassette and Arginine gene (ARG4) as a housekeeping (Krainer et al., 2012).

Supplementary Figures
Supplementary Figure S2. SDS-PAGE comparison of all fed-batches performed with PDH-C, GAP-C and PDF-C. MM corresponds to the molecular marker Precision Plus Protein™ All Blue Prestained Protein Standards. B means the sample taken at the end of the batch phase, G at the end of the exponential growth-phase and I after applying the pseudo-starving conditions, while the numbers represent samples.
Supplementary Figure S3. SDS-PAGE comparison of osmotic shock fed-batch with simple pseudostarving fed-batches. MM corresponds to the molecular marker Precision Plus Protein™ All Blue Prestained Protein Standards. B means the sample taken at the end of the batch phase, FB at the end of the exponential growth-phase, OS after applying the osmotic shock and S1 or S5 represent de the samples 1 and 5 from the induction phase.
Supplementary Figure S4. Expression levels of the different PDH promoter truncations in raw fluorescence units normalized by cell density. Cells were cultivated in biological 7-fold replicates which represent the standard deviation, taking samples after 58, 60 and 132 h of cultivating with glucose as a sole carbon source. As controls, the same construct was used with PGAP and PDC instead of PDH.-