TYPE Original Research PUBLISHED 23 March 2023 DOI 10.3389/fbioe.2023.1130583 Enabling growth-decoupled OPEN ACCESS Komagataella phaffii recombinant EDITED BY Wan Abd Al Qadr Imad Wan-Mohtar, University of Malaya, Malaysia protein production based on the REVIEWED BY Mark Blenner, methanol-free PDH promoter University of Delaware, United States Suchada Chanprateep Napathorn, Chulalongkorn University, Thailand Núria Bernat-Camps 1,2, Katharina Ebner 3, Pinar Calik, Veronika Schusterbauer 3, Jasmin Elgin Fischer 3, Middle East Technical University, Türkiye Miguel Angel Nieto-Taype 1, Francisco Valero 1,2, Anton Glieder 3 *CORRESPONDENCE Xavier Garcia-Ortega, and Xavier Garcia-Ortega 1,2* xavier.garcia@uab.cat 1 Department of Chemical, Biological, and Environmental Engineering, School of Engineering, Universitat SPECIALTY SECTION Autònoma de Barcelona, Bellaterra, Spain, 2Austrian Centre of Industrial Biotechnology (ACIB), Graz, This article was submitted to Industrial Austria, 3Bisy GmbH, Hofstaetten, Austria Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology RECEIVED 23 December 2022 The current transition towards the circular bioeconomy requires a rational ACCEPTED 01 March 2023 development of biorefineries to sustainably fulfill the present demands. The use of PUBLISHED 23 March 2023 Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability CITATION to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, Bernat-Camps N, Ebner K, while producing high- and low-added value products. Recombinant protein Schusterbauer V, Fischer JE, Nieto-Taype MA, Valero F, Glieder A and production (RPP) using K. phaffii has often been driven either by the methanol Garcia-Ortega X (2023), Enabling induced AOX1 promoter (PAOX1) and/or the constitutive GAP promoter (PGAP). In growth-decoupled Komagataella phaffii the last years, strong efforts have been focused on developing novel expression recombinant protein production based on the methanol-free PDH promoter. systems that expand the toolbox variety of K. phaffii to efficiently produce diverse Front. Bioeng. Biotechnol. 11:1130583. proteins that requires different strategies. In this work, a study was conducted towards doi: 10.3389/fbioe.2023.1130583 the development of methanol-free expression system based on a heat-shock gene COPYRIGHT promoter (PDH) using glycerol as sole carbon source. Using this promoter, the © 2023 Bernat-Camps, Ebner, Schusterbauer, Fischer, Nieto-Taype, recombinant expression is strongly induced in carbon-starving conditions. The Valero, Glieder and Garcia-Ortega. This is classical PGAP was used as a benchmark, taking for both strains the lipase B from an open-access article distributed under Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH the terms of the Creative Commons Attribution License (CC BY). The use, outperformed PGAP controlled expression in shake-flask cultivations when using a distribution or reproduction in other slow-release continuous feeding technology, confirming that PDH is induced under forums is permitted, provided the original pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. author(s) and the copyright owner(s) are credited and that the original publication Several optimization rounds were carried out for PDH under different feeding and in this journal is cited, in accordance with osmolarity conditions. In all of them the PDH controlled process outperformed the PGAP accepted academic practice. No use, one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific distribution or reproduction is permitted which does not comply with these terms. productivity than PGAP fed-batch at low μ. Compared to the optimum approach for PGAP-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. KEYWORDS Komagataella phaffii, PDH, recombinant protein production, methanol-free expression system, growth-decoupled bioprocess, promoter regulation, Pichia pastoris Frontiers in Bioengineering and Biotechnology 01 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 1 Introduction related to methanol metabolization usually cause an increase in the operational costs (Çalik et al., 2015). Hence, in the last years the Currently, the circular bioeconomy implementation is usually focus has shifted towards studying new expression systems that considered one of the most promising solutions to address the twin avoid the use of methanol, but still achieving high titers and challenges of climate change and growing population (Ubando et al., productivities (Juturu and Wu, 2018; Yang and Zhang, 2018). 2020). Ideally, the biorefinery concept enables sustainable economic An interesting approach for identifying novel methanol-free growth by facilitating the production of a broad spectrum of expression systems is to move away from MUT related promoters, products from different renewable feedstocks using physico- for example by choosing promoters from other metabolic pathways. chemical and biological procedures (Areniello et al., 2022). To This would be the case of the glyceraldehyde-3-phosphate promoter develop this circular network, the ability of microorganisms to (PGAP) and the phosphoglycerate kinase promoter (PPGK), which are convert raw substrates into small molecules and proteins is involved in the glycolysis and gluconeogenesis pathway, exploited via fermentation processes, using either wild-type or respectively. Although both are constitutive, PPGK presents a engineered organisms (Cherubini, 2010). However, many rather weak expression profile, while PGAP is considered a strong interesting feedstocks, such as lignocellulosic biomass, usually promoter that can reach similar expression levels as PAOX1 (Vogl and require enzymatic pre-treatment to become bioaccessible for the Glieder, 2013). The use of PGAP-based expression systems also microorganism (Tamburino et al., 2022). makes fed-batch cultivations easier, since no transition phase is In the last decades, Komagataella phaffii, a yeast formerly known required to shift the carbon source, making it a commonly used as Pichia pastoris, has become widely used as a cell factory for alternative to the PAOX1 bioprocess (Yang and Zhang, 2018). recombinant protein production (RPP) and metabolites (Juturu and However, strong and constitutive expression is not always Wu, 2018). Interestingly, K. phaffii is an ideal platform to integrate desired, especially when protein folding is the limiting step or in a biorefinery, at both the feedstock pre-treatment stage and RPP can generate cell toxicity (Vogl and Glieder, 2013). Another production fermentation stage. In addition to the exceptional promising promoter identified from a metabolic pathway is the ability to metabolize methanol, K. phaffii is used to produce promoter of the alcohol dehydrogenase 2 (PADH2), whose gene is high-value added proteins using different cheap substrates, responsible for ethanol consumption (Karaoglan et al., 2016b). including glycerol derived from biodiesel industry as the sole Upon induction with ethanol, PADH2-based strain achieved carbon source (Potvin et al., 2012). Alternatively, K. phaffii is similar specific productivity than the PAOX1-based strain when also a suitable cell chassis for the synthesis of low-value producing a xylanase under standard cultivation conditions commodity enzymes, such as cellulases for the upstream (Karaoglan et al., 2016a). Due to its potential to replace the feedstock transformation of lignocellulosic biomass (Bernardi historical K. paffii promoters, several engineered variants have et al., 2019; Yang et al., 2019). To facilitate this ubiquitous been generated by modifying or replacing the regulatory regions presence of K. phaffii in biorefinery, a wide range of molecular of PADH2, achieving up to 4-fold increases in the product to biomass tools are needed to meet each target protein optimal expression yield (Ergün et al., 2019; Erden-Karaoğlan et al., 2022). Although strategy. Among those tools investigated over the past years, ethanol is less hazardous than methanol, these processes present the promoter selection, which has a major impact in both the same disadvantage of having longer fed-batch cultivations, due to process development and final product obtained, has generated the adaptation phase to the new carbon source and the slower relevant outcomes (Vogl and Glieder, 2013; Yang and Zhang, specific growth rates (Potvin et al., 2016; Erden-Karaoğlan et al., 2018; Vogl et al., 2020). 2022). Due to its ability to grow on methanol as sole carbon source, K. Another option that avoids the use of methanol and has gained phaffii owns a set of strong and inducible promoters that regulates great attention is to develop methanol-free PAOX1-based systems. tightly the methanol utilization (MUT) pathway. Among them, the This is achieved by triggering the methanol activating pathway or alcohol oxidase 1 promoter (PAOX1) has been widely used for driving inactivating the catabolite repression pathway (Yang and Zhang, the expression of numerous recombinant proteins (Vogl and 2018). Based on this pathway engineering approach, the Glieder, 2013). In terms of regulation, PAOX1 is tightly repressed development of several methanol-independent processes has been when K. phaffii is grown on glucose, glycerol or ethanol, and slightly reported in the literature, where transcriptional repressors or genes de-repressed upon its depletion. Full induction is only achieved after involved in PAOX1 regulation were knocked out and/or methanol addition, which allows obtaining high product titers and transcriptional activators were overexpressed (Shen et al., 2016; yields (Ahmad et al., 2014). This inducible regulation enables the Wang et al., 2017; Vogl et al., 2018; Dalvie et al., 2022). uncoupling of growth and protein production, by using glycerol as Interestingly, the producer system designed by Wang et al. the carbon source in the batch phase and then switching to (2017) (MF1), which was repressors-deficient together with an methanol, which induces the target protein expression (Looser overexpression of an activator gene, reached 58.6% of the insulin et al., 2015). This strategy avoids cell burden due to protein precursor amount produced with wild-type (WT) PAOX1 system, but overexpression during the biomass generation phase, being avoiding the use of methanol. This was the highest activation especially suitable for the production of toxic proteins (Yang and achieved by a PAOX1-based system without methanol induction Zhang, 2018). Although PAOX1-based expression is widespread for (Vogl et al., 2018). producing efficiently recombinant proteins; the use of methanol, Alternatively, related methylotrophic yeast species are a rich especially for biopharmaceuticals, is often avoided at industrial scale source for investigating novel promoters (Stadlmayr et al., 2010; due to storage and handling costs (Karbalaei et al., 2020). Vogl et al., 2020). PHpFMD, as an example, is an orthologous Additionally, the higher heat production and O2 requirements promoter from Hansenula polymorpha, which shows derepressed Frontiers in Bioengineering and Biotechnology 02 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 regulation when growing on either glycerol or glucose, and and productivities. It can be also found as a commercial variant additionally is further inducible by methanol. According to the named PDC (Vogl et al., 2016; Fischer et al., 2019). literature, it is the strongest promoter so far reported in K. phaffii Although the number of available promoters for RPP in K. when grown on methanol (Vogl et al., 2020), which served to phaffii is becoming wider, there are still few options that meet the generate a commercial variant named PDF. Recently, the PDF- current trend of developing methanol-independent bioprocess that based system surpassed the specific production rate (qp) of the are not growth-coupled. In this study, the aim was to identify novel methanol-free benchmark PGAP-based system by the factor of nine, methanol-free and tightly regulated promoters in K. phaffii, when expressing Candida antarctica lipase B (CalB) in chemostat specifically under strict carbon-limiting conditions and without cultivations (Fischer et al., 2019; Garrigós-Martínez et al., 2021). adding any inducer. Using RNA-Seq technology, several genes All the expression systems mentioned above were engineered or were observed to be highly expressed when applying low glycerol identified from known highly expressed genes, either from K. phaffii feeding, which allow cell maintenance but no biomass growth. From or other related yeasts. Currently, the emerging systems biology the different candidates identified, the promoter region of heat shock tools can also ease the identification of novel expression systems. In protein 12 gene (HSP12) was selected as promising to develop a particular, transcriptomics is a powerful method for identifying novel expression system. This family of proteins, which are upregulated genes under specific conditions (Vogl and Glieder, upregulated under general stress conditions, has been extensively 2013). This way, the pursuit of novel methanol-free and studied in fungi (Tiwari et al., 2015). Specifically in S. cerevisiae, differently regulated expression systems can be rationally HSP12 is tightly repressed by glucose, being expressed in the developed. The first genome-wide promoter study used publicly stationary phase but not when growing (de Groot et al., 2000; available data obtained from the heterologous microarray Karreman and Lindsey, 2005). In this work, the promoter region hybridization of K. phaffii cDNA to a Saccharomyces cerevisiae of HSP12 in K. phaffii was identified and named as PDH. To study its specific microarray, from fed-batch cultivations on glucose, regulation and potential for RPP, the industrially relevant enzyme glycerol or methanol. Together with already reported promoters, CalB was used as a reporter protein. The performance of PDH-based PTHI11, which regulates the expression of a protein involved in expression system in different cultivation set-ups was compared to thiamine precursor synthesis, was isolated. The activity of this the commonly used PGAP and the strong and commercial PDF and novel promoter was found to be uninfluenced by any carbon or PUPP, all considered as methanol-free benchmarks. nitrogen source, but regulated by the thiamine availability This work will contribute to enlarge the Pichia toolbox by (Stadlmayr et al., 2010). The emergence of whole genome providing more alternatives for the efficient production of sequencing opened the doors for K. phaffii specific transcriptome different kind of proteins. On balance, this can promote the tools. This included specific K. phaffii microarray chips (Graf et al., transition towards the circular bioeconomy, thus significantly 2008) and whole genome assemblies of multiple K. phaffii strains, decreasing our carbon footprint. which build the basis for RNA-Seq based transcriptome studies (De Schutter et al., 2009; Mattanovich et al., 2009; Küberl et al., 2011; Liang et al., 2012). 2 Materials and methods Using RNA-Seq the genome annotation of K. phaffii was further improved and gene expression in glycerol or methanol chemostat 2.1 Glycerol ® release measurement from cultivations was analyzed (Liang et al., 2012). From this data, a FeedBeads methanol-free strong and constitutive promoter called PGCW14 was identified, which natively regulates the expression of a potential This work has required to run carbon-limited cultures in shake- glycosyl phosphatidyl inositol (GPI)-anchored protein (Liang et al., flask cultivations, which has been performed using the glycerol- 2013). Due to the great performance that PGCW14 exhibited, a releasing FeedBeads® supplied by Kuhner (SMFB12001, Kuhner commercial variant called PUPP was generated and characterized, Shaker, Basel, Switzerland). This slow-release substrate presenting up to 9-fold more qp than the PGAP-based strain technology is based on silicone matrix discs with the substrate producing CalB in glycerol-based chemostat cultivations (glycerol or glucose) embedded. They ensure that this technology (Garrigós-Martínez et al., 2021). In the work described by reduces the negative influences of O2 limitation, by-products Prielhofer et al. (2013), a rational approach was reported to production and pH-shifts, while also improving the product yields. identify novel promoters inducible under glucose-limiting A kinetic characterization of the glycerol released by conditions. In this case, the methodology was based on K. phaffii 3 FeedBeads® was performed under standard cultivation specific microarray hybridization of mRNA samples from a glucose conditions, being 25°C and 180 rpm in an orbital shaker (Infors, chemostat cultivation. The promoter of a high affinity glucose Switzerland). The study was run per triplicate in 500 mL baffled transporter (PGTH1) was successfully identified, which is repressed shake-flasks with 50 mL buffered minimal media without carbon on glycerol and highly induced in glucose-limiting conditions source (BM, 200 mM potassium phosphate, 1.34% YNB, pH 6 or (Prielhofer et al., 2013). Finally, in a genome-wide transcriptional pH 3). First, the effect of pH was evaluated by incubating three response study to different carbon sources, the promoter involved in FeedBeads® in BM shake-flasks at pH 6 and pH 3 for 72 h. Then, the reactive oxygen species defense, named PCAT1, was identified for kinetic characterization of 3 Feedbeads® was performed in BM being strongly induced by methanol and oleic acid, reaching similar pH 6 by monitoring for 120 h the glycerol release under the expression levels as PAOX1. Interestingly, this promoter also presents same incubating conditions. Several samples were taken over a tight repression and de-repression on glucose obtaining high titers time to determine glycerol concentration by HPLC. Frontiers in Bioengineering and Biotechnology 03 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 2.2 RNA sequencing and analysis as eGFP was expressed intracellularly. As a control the same construct was used with a constitutive promoter (PGAP) A commercially available WT K. phaffii strain BSYBG10 (bisy (Waterham et al., 1997) and a carbon source repressed promoter GmbH, Hofstaetten/Raab, Austria) was cultivated in 25 mL minimal PCAT1 (Vogl et al., 2016) expressing also the eGFP. Cultivations were media (BM, 200 mM potassium phosphate pH 6, 1.34% YNB, 4 × done in minimal media containing 1% glycerol or glucose as sole 10−5% biotin) with 0.25% glycerol as carbon source in 250 mL carbon source (BMG or BMD, 200 mM potassium phosphate pH 6, baffled shake flasks for 9 h (28°C, 130 rpm, 80% humidity) until 1.34% YNB, 4 × 10−5% biotin and 1% glycerol or glucose). the initial glycerol was depleted. Afterwards, two glycerol FeedBeads® (SMFB12001, Kuhner Shaker, Basel, Switzerland) were added, followed by the addition of 25 mL fresh minimal 2.4 CalB strain construction, screening and media supplemented with different inductors (xylitol, galactose, gene dosage determination xylose, sorbitol) in duplicates to an end concentration of 0.25% in the media. In addition, one uninduced control culture was The parental strain BSYBG11 (aox1-/Muts), a single colony included as reference, only adding BM without any carbon streak out of K. phaffii strain BG11 (BioGrammatics Inc.) source. After 3 hours, samples from the biological duplicates were deposited at bisy GmbH (Hofstaetten, Austria), was transformed taken and centrifuged at 5000xg. The cell pellet was frozen at −80°C with an expression vector containing the CalB gene under control of and sent for RNA isolation and sequencing at BioGrammatics Inc. the PDH promoter region. The procedure followed the same (Carlbad, CA, United States). Total RNA extraction, as well as approach as Garrigós-Martínez et al. (2021). Briefly, the quality control and library preparation were performed according recombinant vector was based on pPpT4_Alplha_S vector. To to BioGrammatics standards. Poly-A enriched total RNA was avoid multi copy expression cassette integration, and thus also sequenced as 50 bp single reads on an Illumina sequencer. Reads avoiding the gene dosage effect on the comparison, low amounts were mapped to the K. phaffii CBS7435 reference genome of linearized plasmid DNA (<1 µg of DNA) were used for (Sturmberger et al., 2016) and read counts were generated using transformation. the STAR aligner (Dobin et al., 2013). Read and mapping quality was Several candidate clones were screened using glycerol as carbon determined using fastqc (Andrews, 2010) and Qualimap (García- source in DWP using BMG (200 mM potassium phosphate pH 6, Alcalde et al., 2012). To compare expression strength between genes 1.34% YNB, 4 × 10−5% biotin and 1% glycerol), as described by and samples, the transcripts per million (TPM) were calculated in Garrigós-Martínez et al. (2021). Seven clones displaying average the R/Bioconductor statistical environment (Gentleman et al., 2004; performance were selected and further rescreened in DWP in Robinson et al., 2010) using the edgeR package (Robinson et al., biological replicates, in order to validate the previous results. From 2010). Genes were subsequently ordered by their median, mean and these results, three clones were selected to check the gene copy number. maximum TPM to identify highly expressed genes on all conditions. The gene copy/dosage number was determined using qPCR, following the method described by Abad et al. (2010). The protocol is described by Krainer et al. (2012) and based on the amplification of 2.3 Identification of the promoter sequence ™ the Zeocin resistance-mediating gene Zeocin®, located on the based on eGFP expression integrated expression cassette, and the house-keeping gene Arg4. Therefore, the copy number of CalB gene was indirectly determined To identify the promoter region, 1,000 bp upstream of the HSP12 by the copy number verification of Zeocin®. gene in K. phaffii were chosen to test for promoter activity and ordered as Accordingly, and including the producer clones previously synthetic DNA at IDT (Coralville, IA, United States). Subsequently, 50 bp constructed in the research group and described by Garrigós- truncations were made down to 97 bp residual length to determine the Martínez et al. (2021), a set of 4 isogenic strains producing the CalB minimal length of the promoter region capable to drive protein enzyme were used in the present study: GAP-C, PDF-C, UPP-C, and expression. Four versions were not clonable in first instance and were PDH-C. All the strains should only differ in the promoter used to therefore discarded (PDH2, 9, 10, 17). The 14 cloned constructs consisted regulate the CalB expression. of the respective promoter, eGFP as reporter gene, the AOX1 transcription terminator and a 1,100 bp homologous region to the 3′UTR of the K. phaffii ARG4 gene, which was used to target the 2.5 Shake-flask cultivations for CalB genome integration. The plasmid map and the truncated sequences can producer clones be found in the Supplementary File S1: Supplementary Figure S1; Supplementary Table S2, as well as transformation, cloning and The strains PDH-C, GAP-C, UPP-C and PDF-C were used for cultivating protocol (Supplementary File S1). To facilitate integration shake-flask cultivations, using 500 mL baffled flasks with 50 mL of into the right locus, the K. phaffii strain BSY11dKU70 was used (aox1-/ buffered minimal glycerol media (BMG, 200 mM potassium Muts), which lacks the ability of non-homologous end-joining and phosphate pH 6, 1.34% YNB, 4 × 10−5% biotin and 1% glycerol). therefore ensures targeted integration with over 90% efficiency Shake-flasks were inoculated at an initial OD600 of 0.2 from an (Näätsaari et al., 2012). overnight pre-culture (growing in YPG (1% yeast extract, 2% The screening cultures were performed in a semi-continuous peptone and 2% glycerol) at 25°C and 180 rpm). After 20 h of manner on 96-deep well plate (DWP) format for 6 days while taking incubation at 25°C and 180 rpm, total glycerol depletion was samples every 24 h for the first 4 days and a last sample after 132 h. confirmed by HPLC and three glycerol FeedBeads® (SMFB12001, Here, the fluorescence measured and normalized to the cell density Kuhner Shaker, Basel, Switzerland) were added to start the feeding Frontiers in Bioengineering and Biotechnology 04 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 phase. The cultivations were run per triplicate and lasted for 72 h 2.6.3 Osmotic shock in fed-batch and several samples were taken over time for enzyme analysis. Two fed-batch cultivations with PDH-C were performed using The shake-flask cultures to test osmotic shock effect were the PS approach and two different osmotic conditions: 0.3 and 0.6 M carried out only with PDH-C strain at three different KCl KCl. After the biomass generation phase, being around 70 g L-1 concentrations: 0 M, 0.3 M, and 0.6 M. The cultivation DCW, the appropriate volume of a 4 M KCl solution was added protocol used was the same previously described in this to achieve the target osmolarities of 0.3 and 0.6 M. Then, the section, but an osmotic shock was induced after 24 h by constant feeding rate was started to reproduce PS conditions. adding 10 mL of water, 2.4 M KCl or 3.6 M KCl to get 0 M, 0.3 M and 0.6 M KCl in the shake-flasks, respectively. Three FeedBeads® were as well used for the feeding phase, running each 2.7 Biomass determination osmolar condition per triplicate. For shake-flask cultivations, cell growth was monitored by measuring per triplicate the optical density at 600 nm (OD600) in 2.6 Fed-batch cultivations a DR 3900 HACH Spectrophotometer (Hach Company, Ames, IA, United States). During fed-batch cultivations, biomass All fed-batch cultivations were conducted in a 5 L Biostat B concentration was measured in quadruplicates in terms of dry fermenter (Sartorius Stedim, Goettingen, Germany) starting with an cell weight (DCW), as described elsewhere (Cos et al., 2005). The initial volume of 2 L. The media composition was the same as relative standard deviation (RSD) in all measurements was lower described previosuly (Gasset et al., 2022), but with glycerol instead of than 5%. glucose in the fed-batch feeding solution. For all cultivations, temperature was controlled at 25°C, pH permanently adjusted to 5 with NH4OH 15% v/v and the airflow was 2 L min-1. Dissolved 2.8 Osmotic stress analyses oxygen (DO) was kept above 35% by stirring speed cascade (600–1,200 rpm) and by O2 enrichment if needed, always The actual osmolarity in the shake-flask and fed-batch maintaining 2 L min-1 of total gas inlet flow. The software Eve® supernatants of the osmotic shock studies were determined (INFORS HT, Bottmingen, Switzerland) was used to monitor and measuring the conductivity with a Cond 8 conductimeter (XS control the cultivations. Instruments, Carpi, Italy). 2.6.1 Fed-batch characterization PDH-C, GAP-C and PDF-C strains were cultivated under 2.9 Quantification of carbon source and by- carbon-limiting conditions at different specific growth rates (μ). products After depletion of all glycerol contained in the batch media, an exponential pre-programmed feeding rate based on the biomass HPLC (Dionex Ultimate 3,000, Dionex, Sunnyvale, CA, mass balance was started to maintain a constant μ (being 0.025 h−1 United States) with an ionic exchange column (ICSep ICE-COR- for PDH-C, 0.025 h 1 or 0.15 h 1 for GAP-C and 0.05 h−1 for PDF-C). EGEL 87 H3, Transgenomic Inc., Omaha, NE, United States) was All fermentations were stopped when the goal dry cell weight used to quantify the concentration of glycerol and other metabolites (DCW) of 80 g L−1 was achieved. More details about the fed- in the fed-batch cultures and the FeedBeads® kinetic characterization batch strategy are described in previous works of the group samples. More details of the column and software used are described (Garcia-Ortega et al., 2013). elsewhere (Jordà et al., 2014). RSD was below 1%. 2.6.2 Fed-batch optimization Protein expression from the strain PDH-C was induced by 2.10 Off-gas analyses pseudo-starving conditions, following two strategies: single pseudo-starving (PS) and three stages of pseudo-starving (3- A BlueInOne FERM gas analyser (BlueSens, Herten, Germany) PS). After the batch phase of the PS approach, a fast biomass was used for monitoring CO2 and O2 molar fraction and absolute growth fed-batch (constant μ of 0.15 h−1) was started to reach a humidity from all fed-batches exhaust gas. To get more accurate goal biomass concentration of 70 g L−1 of dry cell weight (DCW). measurements, the gas analyzer was re-calibrated in each fed-batch. Then, a pseudo-starving phase was initiated by adding a The data recorded were used to calculate key respirometric constant glycerol feeding rate to reach the same overall parameters: oxygen uptake rate (OUR), carbon dioxide evolution specific glycerol uptake rate (qS) provided by three rate (CER), their corresponding specific rates (qO2 and qCO2) and FeedBeads® (0.005 g gX −1 h−1), which is equivalent to an initial respiratory quotient (RQ). RSD was less than 5% in all cases. µ of 0.003 h −1. In contrast, several cycles of growing—induction phases were performed in the 3-PS approach. Every biomass generating phase was conducted at a μ of 0.15 h−1, stopping at the 2.11 Enzymatic analyses target biomass concentration of 40, 60, and 80 g L−1 of DCW, which were followed by a 24 h period of the induction Lipolytic activity of secreted CalB was determined using a conditions. These induction conditions were designed to p-nitrophenyl butyrate (pNPB)-based assay (2635–84–9, Merck, achieve a qS of 0.005 g gX−1 h −1 with a constant feeding rate. Darmstadt, Germany). The reaction buffer was as follows: Frontiers in Bioengineering and Biotechnology 05 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 300 mM Tris-HCl pH 7 containing 4.83 mM p-NPB and 0.93% acetone. After pre-heating the buffer at 30°C, 900 μL of reaction buffer were mixed with 100 μL of culture supernatant. P-nitrophenol color development at 405 nm was monitored in a Specord 200 Plus spectrophotometer (Analytik Jena GmbH, Jena, Germany) at 30°C for 2 min. One activity unit was defined as the amount of enzyme needed to release 1 μmol p-nitrophenol · min-1 under assay conditions. RSD was below 4%. Further protein analyses of fed-batch supernatant samples were performed by SDS-PAGE. For sample preparation, 15 µL of supernatant were mixed with 5 µL of loading buffer (Laemmli buffer 4x + β- mercaptoethanol in 10:1 relation, Biorad, Hercules, CA, USA) and incubated at 95°C for 5 min. After cooling down, 15 µL were loaded FIGURE 1 into 4%–15% Mini-PROTEAN TGX Precast Protein Gels (Biorad, ® Characterization in shake-flask of three FeedBeads releasing Hercules, CA, United States) and run at 120 V for about 80 min. glycerol at 25°C in BM. The dots indicate the released glycerol amount measured by HPLC, while the line describes the release kinetic. The The visualization and analyses of the gel was performed using Image error bars indicate the standard deviation between triplicates. Lab (Biorad, Hercules, CA, United States). 2.12 Consistency test and data reconciliation non-linear kinetic typical profile drug release. Ritger and Peppas The biomass elemental composition of K. phaffii growing on (Ritger and Peppas, 1987) developed a simple relationship that glycerol at different growth rates was previously determined by describes the solute release from discs, which was adapted to: Tomàs-Gamisans et al. (2018). For fed-batch characterization, yields and rates were calculated using equation based on mass balances, that Mt  ktn can be found elsewhere (Garcia-Ortega et al., 2016; Ponte et al., 2016). In where Mt is the amount of solute released (mg), t is the release time all cases, carbon and electron data recovery was above 90%. To further in hours, k is a constant and n is the diffusional exponent, which is validate the results, measurement consistency was checked and characteristic of the release mechanism. Using this formula, the reconciliation procedures were applied, constraining by carbon and kinetic model obtained to explain the mg of glycerol released by electron balances (Ponte et al., 2016). The confidence level reached in 3 FeedBeads® over hours was: the statistical consistency test was 95% for all fed-batch characterization. Mt  5.13t0.65 3 Results and discussion which fully describes the release kinetic with an r2 of 0.999 and a standard error of 1.132 (Figure 1). ® 3.1 FeedBeads release kinetic According to the diffusional exponent (0.65), the FeedBeads® characterization release mechanism is due to diffusion and erosion of the matrix (Padmaa Paarakh et al., 2018). The glycerol release kinetic When developing and implementing new process strategies for determined for the glycerol FeedBeads® was in the same order as RPP, a significant operational gap still exists between early-stage previously found for the glucose FeedBeads®, being 1,34 t0,77 and cultivations in shake-flask or DWP and fed-batch processes in 1,63 t0,74 mg of glucose per hour and one FeedBeads® (Prielhofer fermenters. However, working under process-relevant conditions et al., 2015; Halka et al., 2018). is crucial when performing small scale cultivations (Looser et al., Taking this glycerol release kinetic, the growth parameters of a 2015). The so-called FeedBeads® developed by Kuhner-shaker standard K. phaffii shake-flask cultivation can be predicted assuming GmbH (Germany) would bridge this gap, since shake-flask that all the glycerol released by the FeedBeads® is immediately cultivations can be fed under fed-batch-like conditions. To study consumed. Therefore, when adding 3 FeedBeads® after a batch the conditions this technology provides, a characterization of phase conducted with 1% BMG, the specific growth rate (µ) glycerol release FeedBeads® was carried out in shake-flasks at would be 0.003 h-1 and the specific substrate uptake rate (qs) standard cultivation conditions. 0.005 gS gX−1 h−1. Accordingly to the usual µ used in fed-batch Based on the product specifications, three FeedBeads® were used to cultures, which is in the range from 0.025 h-1 to 0.15 h-1, the use characterize the release kinetic. First, since K. phaffii acidifies the media of 3 FeedBeads® would allow severe carbon-limiting conditions. when growing, the glycerol release was tested at pH 6 (buffered minimal media standard) and 3 (acidic pH) at 25 °C. No differences were detected after 72 h of releasing (data not shown) between the two pH values, 3.2 Identification of a novel promoter based suggesting that the kinetic is not pH-dependent. on RNA sequencing In order to characterize glycerol release kinetic, a new experiment was performed with BM at pH 6°C and 25 °C for In order to identify a novel promoter by seeking highly 120 h. As can be seen in Figure 1, glycerol release followed a expressed genes under specific conditions, the use of RNA-seq Frontiers in Bioengineering and Biotechnology 06 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 while also adding appropriate overhangs to the eGFP expression vector for Gibson Cloning (Gibson et al., 2009). An overview of all truncations is shown in Figure 2A. As reporter gene, the intracellularly expressed fluorescence protein eGFP was used. The vector also contained 5′ and 3’ homologous regions to the ARG4 locus in K. phaffii to minimize ectopic genomic integration and make the results more comparable. The eGFP expression levels obtained growing on glycerol (Figure 2B) and glucose (Supplementary File S1: Supplementary Figure S4) were rather similar. The shortest variant of the putative promoter sequence still showing the same expression level as PDH1 (1,000 bp) was PDH13 (Figure 2B) with only 352 bp, whose promoter sequence will be referred for now on as P DH . 3.3 Strain generation, screening, and gene dosage determination To study the performance of the novel isolated PDH promoter controlling CalB expression and compare the results with other new expression systems previously reported by Garrigós- Martínez et al. (2021), the same cloning approach was followed to generate an isogenic PDH-based CalB producer clone. Due to the high clonal variability that K. phaffii presents, one of the critical steps in bioprocess development is selecting an appropriate producing clone (Looser et al., 2015). Since the aim was to isolate a clone with a single expression FIGURE 2 cassette integrated in the genome, a high-throughput screen of (A) Schematic representation of 14 PDH promoter truncations for identification of the essential promoter region with the reporter 32 clones was performed in DWP using glycerol as sole carbon protein eGFP. (B) Expression levels of the different PDH promoter source, with the scope of selecting an averagely producing clone. truncations in raw fluorescence units normalized by cell density. Seven putative single-copy integration transformants were Cells were cultivated in biological 7-fold replicates which represent the standard deviation, taking samples after 48, 60, and 132 h of rescreened in biological triplicates and the previous results cultivating with glycerol as sole carbon source. As controls, the same were validated. The single cassette integration of three of these construct was used with PGAP and PDC instead of PDH. candidate clones was determined by qPCR (Supplementary File S1: Supplementary Tables S2, S3). Therefore, a confirmed single copy clone with an average CalB production was selected for characterizing its regulation and performance, being named was considered more suitable than microarrays since it offers a PDH-C. higher dynamic range. Furthermore, RNA-seq is not restricted to known genes and the resulting gene expression measures are highly reproducible (Nagalakshmi et al., 2008). Therefore, with the aim of 3.4 Shake-flask cultivations, first insights finding a methanol-free and tightly regulated promoter under into PDH-C system glycerol restriction, RNA from a WT K. phaffii cultivation under glycerol de-repressed conditions was isolated to perform RNAseq Since the regulation of HSP12 in S. cerevisiae is related to analyses (The transcriptome data was made available under ENA stress damage, temperature and pH stressing conditions were project PRJEB58889). applied to preliminary deep-well plate cultivations with PDH-C The gene that showed the highest mean, median as well as to induce CalB expression (Praekelt and Meacock, 1990). maximum transcripts per million (TPM), was the However, no positive or inducing effect was observed ACIB2EUKG772368, a homologue to the heat shock protein compared to standard cultivation conditions (unpublished 9/12 from S. cerevisiae (Supplementary File S2: Supplementary data). As reported in S. cerevisiae, HSP12 is repressed in the Table S4), hereafter called HSP12 in accordance with its exponential phase and activated when the cells enter stationary homologue in S. cerevisiae. As this gene was highly expressed phase (de Groot et al., 2000). Therefore, new culture strategies in all the tested conditions and was previously reported to be were designed with the aim to validate the potential of PDH for highly expressed under stress conditions, its promoter was controlling the target recombinant expression in growth considered an interesting candidate to develop an alternative decoupled conditions. To do so, PDH-C was cultivated in expression system based on it. To examine the promoter region, shake-flask until total glycerol depletion was confirmed. Then, 1,000 bp upstream of the start codon were ordered as synthetic three glycerol FeedBeads® were added to reach the desired DNA. Systematic truncations on the 5′ end were made by PCR, stationary state by applying severe carbon-limiting conditions. Frontiers in Bioengineering and Biotechnology 07 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 FIGURE 3 ® Shake-flask cultivations of CalB producing strains using three glycerol FeedBeads (addition time is indicated by red dotted lines). Time-course evolution of OD600 (dotted line) and lipolytic activity (solid line). (A) PDH-C performance is compared to the benchmarks GAP-C, UPP-C and PDF-C. (B) Omotic shock cultivations of PDH-C at low, middle and high osmolarities. The red line also indicates the 10 mL addition of water or KCl solution. The error bars indicate the standard deviation between biological triplicates. The benchmark strains GAP-C, UPP-C and PDF-C were these conditions allowed to induce CalB production, suggesting that cultivated under the same conditions. PDH is tightly repressed by glycerol, observing no induction in the For all the strains compared, the biomass in terms OD600 was growing phase. These results are coherent with the previously rather constant after the addition of FeedBeads® (Figure 3A). The published studies in S. cerevisiae using glucose as carbon source average specific growth rate (µ) during the feeding phase was around (de Groot et al., 2000). Moreover, after addition of the FeedBeads® 0.003 h-1 for all strains, while the average specific substrate uptake the cultivation entered the so-called pseudo-starving condition in rate (qs) was 0.005 gS gX−1 h−1. Regarding CalB production by PDH- which the performance of PDH-C even surpassed the performance C (Figure 3A), almost no lipolytic activity was detected in the of the constitutive promoter-based UPP-C and GAP-C. CalB supernatant when cells were growing exponentially in carbon- activity in UPP-C and GAP-C supernatants was virtually excess conditions (batch phase). In contrast, CalB production constant after the addition of FeedBeads®, since both are growth- rose sharply when adding the FeedBeads®, achieving 5.2-fold and related expression systems (Garrigós-Martínez et al., 2021). In 1.5-fold more lipolytic activity (normalized by OD600) than GAP-C contrast, for PDF-C containing the strong and tunable promoter and UPP-C, respectively. PDF-C instead, showed 2.3-fold more PDF (Garrigós-Martínez et al., 2021), the lipolytic activity in the lipolytic activity than PDH-C. supernatant increased markedly over time, outperforming PDH-C The shake-flask results confirmed that three FeedBeads® in terms of CalB production (Figure 3A). Despite this, the PDH- provided enough glycerol for cell maintenance and CalB based expression system was considered valuable for further studies production, but not for growth, achieving the desired stationary in more scalable cultures, such as bioreactors, due to its non-growth- phase conditions from now on called pseudo-starving. In PDH-C, coupled performance. Frontiers in Bioengineering and Biotechnology 08 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 FIGURE 4 Biomass and CalB titer time-course of carbon-limiting fed-batch cultivations with PDH-C at low µ (0.025 h-1) and GAP-C at high (0.15 h-1) and low µ (0.025 h-1). The errors bars represent the standard deviation of the measurements. TABLE 1 Physiological and product-related parameters from carbon-limiting respectively, which will be named for now on low, middle and high fed-batch cultivations with PDH-C at low µ (0.025 h-1) and GAP-C at high osmolarity (Dragosits et al., 2010). (0.15 h-1) and low µ (0.025 h-1). All parameters are calculated from the feeding phase. After the pulses and FeedBeads® addition the OD600 was again rather constant (Figure 3B). As expected, CalB production was GAP-C GAP-C PDH-C triggered just after starting the induction phase for all three 0.025 0.15 0.025 cultivations (Figure 3B). Compared with low osmolarity, a 35% Real μ (h−1) 0.025 0.15 0.025 increase in lipolytic activity was achieved at middle osmolarity, while a decrease of 25% was observed for high osmolarity. According to qs (gS gx−1 h−1) 0.046 0.25 0.044 Dragosits et al., 2010, high osmolarity upregulates the unfolded- Yx/s (gx−1 gS−1) 0.54 0.61 0.58 protein response (UPR) and the expression of general stress related RQ 0.72 0.68 0.70 proteins in WT K. phaffii strains. Nevertheless, in the cited article these changes were lower and even undetectable in the producing Titer (kAU) 18.58 11.70 26.82 strain (a recombinant antibody fragment under PGAP control), since Yp/x (AU gx ) −1 100.45 61.28 185.22 the stress responses were already induced due to protein over- −1 -1 expression. When growing PDH-C under middle and high qp (AU gx h ) 2.45 9.44 4.59 osmolarity conditions, it could be hypothesized that PDH was already close to being fully activated by pseudo-starving conditions and protein over-expression, which might explain the As recently published, HSP12 belongs to a protein family whose low CalB increase and decrease after osmotic shock. Nonetheless, role is to repair stress-related damage, being one of the mediators of since it was considered interesting to study these cultivation hyperosmotic stress adaptation in K. phaffii (Wang et al., 2020). conditions in a more controlled environment such as a With the aim to further enhance the production performance, new bioreactor, further fed-batch cultures were also performed and shake-flask cultivations with the PDH-C were carried out at reported in the present work. different osmolarities to induce osmotic stress. The desired osmolarities were reached by adding the same volume of different concentrations of KCl solution. This osmolyte was chosen 3.5 Fed-batch characterization in carbon- since it was already present in the bioreactor culture medium. Cell limiting conditions growth was clearly affected when KCl was added at a concentration of 0.4 M, 0.6 M and 1.2 M at the beginning of the cultivation, with the Currently, fed-batch cultivation is the most widely used maximum growth rate (µmax) markedly reduced at the highest operational mode for RPP, since it allows to reach higher cell osmolarity (data not shown). Therefore, it was decided to not densities and product titers in a controlled environment. More proceed experiments with a KCl concentration of 1.2 M and add a specifically, a pseudo-stationary state is achieved when pulse of water or KCl to each cultivation after the batch phase, to get a implementing an exponential feeding profile that controls and final KCl concentration of 0 M, 0.3 M and 0.6 M. These concentrations maintains a constant µ, working under carbon-limiting resulted in a cultivation osmolarity of 24.4, 59.2, and 88.4 mS/cm, conditions (García-Ortega et al., 2019). To characterize PDH-C Frontiers in Bioengineering and Biotechnology 09 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 FIGURE 5 Optimization of PDH-C fed-batch cultivations through pseudo-starving conditions compared to the benchmark cultivation of GAP-C at low µ (0.025 h-1). (A) Biomass and CalB titer evolution over time of PDH-C optimized cultivations (PS and 3-PS) and at low µ (0.025 h-1). (B) Product to substrate yield (YP/S) of the PDH-C optimized cultivations compared to PDH-C and GAP-C at low µ (0.025 h-1). The errors bars represent the standard deviation of the measurements. performance under these controlled conditions and according to the In relation to recombinant protein production, CalB levels shake-flask results, a fed-batch cultivation was carried out at a low increased exponentially over time from the very beginning of the constant µ of 0.025 h−1. As a benchmark, two fed-batch cultivations culture for both GAP-C cultivations, due to the growth-coupled at high (0.15 h−1) and low (0.025 h−1) µ were performed with GAP- nature of the expression system (Türkanoğlu Özçelik et al., 2019). In C, considering the high µ cultivation as the optimal strategy for PGAP contrast, CalB titer in PDH-C was almost negligible after the batch in terms of productivity (Nieto-Taype et al., 2020). phase but rapidly increased in the feeding phase following a non- As expected, biomass production increased exponentially exponential profile. At the end of the cultivations, PDH-C at low μ over time and in agreement with the set-point µ (Figure 4), surpassed GAP-C at low and high μ, presenting 1.4- and 2.3-fold reaching a DCW of almost 80 g L−1 in all fed-batch cultivations. titer increases, respectively. Nevertheless, GAP-C cultivation at high As can be observed in Table 1, GAP-C at low μ and PDH-C µ presented 2.1-fold higher specific production rate (qp) than PDH- presented the same qS, while GAP-C at high μ had a greater one in C at low µ due to the shorter cultivation time (Table 1). On the other agreement with the target μ. Moreover, all key parameters that hand, in terms of operational requirements this higher-growth are related to the physiological state of the cultivation were GAP-C cultivation should be considered less advantageous in consistent with the ones obtained in chemostat cultivations comparison with PDH-C cultivation performed at low µ, since performed with GAP-C and the other isogenic clones the faster the growth rate the higher the cooling and oxygen producing CalB, such as PDF-C and UPP-C (Garrigós- demands. For both mentioned cases, however, those operational Martínez et al., 2021). These similarities suggested that CalB requirements would still be far from the demanding methanol-based production with the new promoter PDH did not alter the bioprocesses. On balance, although PDH-C results were promising physiological state of the cultivation. as starting point, the enhancement in CalB production in relation to Frontiers in Bioengineering and Biotechnology 10 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 TABLE 2 Comparison of key performance indicators of PDH-C standard and optimized fed-batch cultivations with the benchmarks GAP-C and PDF-C fed-batch cultivations. All the parameters are calculated from the feeding phase. GAP-C GAP-C PDH-C PDH-C PDH-C PDF-C PDH-C PDH-C 0.025 0.15 0.025 PS 3-PS 0.05 PS-middle PS-high Real μ (h−1) 0.025 0.15 0.025 - - 0.052 - - Specific productivity (AU gX−1 h−1) 1.25 5.03 2.09 4.53 2.68 14.42 3.85 3.37 Volumetric productivity (AU L−1 h−1) 92.67 361.44 151.66 294.21 202.78 1,056.39 272.29 182.32 Yp/s (AU gS−1) 49.54 35.55 84.03 105.11 119.54 298.81 94.13 72.36 Titer (kAU) 18.58 11.46 26.82 26.48 41.82 90.67 25.52 19.65 −1 Lipolytic activity (AU mL ) 6.69 4.35 9.82 9.97 15.04 33.36 9.17 6.19 setting a constant glycerol feeding rate to achieve a qs of 0.005 gS gX−1 h−1, which means an initial μ of 0.003 h-1. The PDH-C biomass and CalB production profiles conducting PS and 3-PS approaches are compared to PDH-C fed-batch at low µ (FB-0.025) in Figure 5A. In terms of cell growth, PS and 3-PS kept the biomass constant around the selected DCW in the induction phases, following the desired profile observed in shake-flasks when adding the FeedBeads®. As expected, in the PS approach CalB production slightly increased as the biomass grew but boosted when the induction was applied, verifying that pseudo-starving conditions were successfully implemented in this approach. Interestingly, during the first FIGURE 6 Optimization of PDH-C fed-batch cultivations through osmotic 24 h of induction the production increased rather linearly over shock compared to the benchmark cultivation of PDH-C PS. Biomass time, reaching a similar titer as PDH-0.025 at the same DCW. and CalB titer evolution over time are represented of PDH-C After that, the curve started to flatten similar to the behavior cultivations at high, middle and low (PS) osmolarity. The errors bars represent the standard deviation of the measurements. observed in shake-flasks. K. phaffii has been demonstrated to have a better adaptation to severe carbon starvation conditions than S. cerevisiae, showing a high viability and low maintenance requirements at near-zero specific growth rates (Rebnegger et al., 2016). During all PS cultivation, high cell viability was observed GAP-C was much lower than in shake-flasks cultivations, suggesting according to the physiological parameters and the lack of visible that there was room for improvement in the operational strategy. cell lysis in SDS-PAGE (Supplementary File S1: Supplementary Figure S2), indicating that the stop of CalB production at the end of the cultivation was not caused by cell lysis. Additionally, the 3.6 Fed-batch optimization by pseudo- absence of lower molecular weight bands on SDS-PAGE, which starving would indicate CalB proteolysis, suggests that no active host proteases were present. Moreover, this dynamic expression was When RPP bioprocess is optimized, the usual Key also observed in S. cerevisiae after applying different stressing Performance Indicators (KPI) to maximize are the product to conditions to activate Phsp12, being followed by GFP fluorescence biomass yield (YP/S), the volumetric productivity (QP) and the (Karreman and Lindsey, 2005; Xiong et al., 2018) and HSP12 final product titer. Prioritizing one or another ultimately will relative transcript levels (Chowdhary et al., 2017; Ren et al., always depend on the product application. Most certainly, 2022). In accordance, PDH activation in K. phaffii also seemed to biomass amount will greatly affect the production, but decrease over time, although the stressing conditions were considering that in the end it is usually a waste by-product, maintained. the maximum amount reachable should be limited into range of These observations inspired the 3-PS design, in which we between 80–100 g L-1 DCW (García-Ortega et al., 2019). reduced the induction phases to 24 h and then reset the likely Accordingly, a PDH-C fed-batch optimization process was non-producing state of the cultivation by starting a new biomass carried out by mimicking the pseudo-starving conditions production phase. As a result, the CalB titer successfully increased achieved in shake-flask cultivations, performing a single almost linearly in the first two induction phases of the 3-PS induction phase (PS) or three at different biomass cultivation. However, during the third induction phase the concentrations (3-PS). For all the strategies, the mentioned production ceased after 12 h. Although the PS-3 cultivation biomass concentration limit was never exceeded. The achieved 1.3-fold more titer than the PS cultivation, the induction through pseudo-starving was implemented by investment of induction time was 2.5-fold higher, making the Frontiers in Bioengineering and Biotechnology 11 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 bioprocess more complex and time-demanding. Therefore, PS was processing costs (García-Ortega et al., 2019). Comparing the the strategy chosen for further experiments. achieved Qp with the benchmarks, PS cultivation showed a 3.6- fold increase and a 3.2-fold decrease in relation to GAP-C 0.025 and PDF-C 0.05, respectively. Regarding GAP-C 0.15, the final titer was 3.7 Fed-batch optimization by osmotic 2.3-fold higher in PS cultivation while Qp was quite similar. shock Furthermore, regarding the osmotic shock approaches, PS-high and PS-middle cultivations showed lower Qp in reference to PS, With the goal to further boost the RPP using the PDH-based as expected. All these Qp differences are also applicable to Qv, expression system, also osmotic shock was applied to the PS approach although the final biomass was slightly different in each process. to increase PDH activation. Again, shake-flask conditions were reproduced In general, using glycerol as a carbon source is economically in the bioreactor, performing a pulse of KCl solution at the beginning of more attractive than glucose or methanol. Moreover, crude glycerol the induction phase to get a middle and high osmolarity of 0.3 and 0.6 M, from biodiesel industry can be used for K. phaffii fermentations with respectively. The conductivities measured during the induction phase was no extra refinements needed, promoting the circular bioeconomy 45.5 and 82.4 mS/cm, respectively, which were in line with measurements (Potvin et al., 2012; Robert et al., 2017). Beside using a cheaper of shake-flask cultivations. The results of PS fed-batch cultivation at carbon-source, reducing the amounts needed for the process can middle (PS-middle) and high (PS-high) osmolarity were compared to the have a great impact on the production costs. Therefore, the product standard PS cultivation (16.5 mS/cm), in terms of total biomass and CalB to substrate yield (YP/S) is usually also a crucial parameter to produced. maximize, especially for low added-value products. In PDH-C PS Practically, no differences were observed in total biomass and CalB cultivation strategy, the overall YP/S was 2.1-fold higher and 2.8-fold titer during the biomass production phase of all three fed-batch lower than GAP-C 0.025 and PDF-C 0.05, respectively (Table 2). cultivations (Figure 6), exhibiting the high reproducibility of the Interestingly, the growth-coupled regulation of GAP-C was clearly platform used. Unfortunately, no remarkable differences in CalB titer observed in Figure 5B, where the evolution of the accumulated YP/S were observed between PS and PS-middle cultivations, while the over time was kept constant. In contrast, the linear trend in PDH-C performance of PS-high cultivation suggested that the high showed different slopes depending on the feeding strategy applied osmolarity caused a detrimental effect. In fact, when the pulse of (Figure 5B). When starting the fed-batch phase at a constant µ of KCL solution was added to PS-high cultivation, a slight decrease in 0.025 h−1 (PDH-C 0.025), a marked increase in YP/S was observed in total biomass was observed (10%) and a markedly lower final CalB the first 16 h, which then decreased linearly over time. In PDH-C PS, production was achieved. However, no evident cell lyses in PS-high the stress by pseudo-starving was initiated once the biomass was cultivation was detected by SDS-PAGE (Supplementary File S1: grown to 70 g L−1, which resulted in a YP/S linear increase for 30 h Supplementary Figure S3) although the foaming observed right after before starting to saturate. Since this saturation was already observed the KCl solution pulse. Moreover, the lower lipolytic activity of PS-high in CalB titer, the pseudo-starving application in PDH-C 3-PS was cultivation was consistent with protein band intensity in the SDS-PAGE. performed for 24 h at 40, 60, and 80 g L−1 of DCW. As in CalB titer, The improvement in CalB production observed in shake-flask the YP/S increased linearly over time in each induction phase except scale when cultivating PDH-C at middle osmolarity could not be for the third one, which stopped after 12 h of induction. reproduced in a bench-top bioreactor, since none of the osmolarity The slopes achieved in the different induction phases of PS and strategies tested could further boost the target RPP. Therefore, 3-PS suggested that the lower the biomass is, the higher CalB is applying two stresses at the same time, pseudo-starving and produced. This statement would agree with the high activation osmotic shock, apparently did not have a synergetic effect for the observed in PDH-C 0.025 when the biomass was 22 g L−1, PDH activation. activation that did not stand over time since the cultivation was actually growing. However, the shift from growing at µmax to 0.025 h−1 was most likely enough to activate PDH at the 3.8 PDH-C, a promising platform for beginning. Therefore, this process parameter not only shows the growth-decoupled protein expression growth-decoupled regulation of PDH and its transient activation, but approaches also the impact that the initial biomass can have. Although biomass can be used as single-cell protein (SCP) for food industry (Jach et al., Considering the scalability of the fed-batch process and from an 2022), working at low cell densities but achieving high titers makes operational point of view, one of the most important features to PDH-based expression system more advantageous for industrial scale minimize are heat production and O2 consumption. While the KPI bioprocess, since problems related to mixing time could be to maximize are usually the productivities and/or the final titer, minimized (Junker, 2004). depending on the market of the target protein. For PDH-C, the best Based on the results of all PDH-C cultivations, one could theorize optimization approach for high productivities was PS cultivation, that this expression system is highly repressed in the presence of glycerol. since it allowed to achieve the highest specific (Qp) and volumetric In S. cerevisiae though, there has been evidence that glucose-6-phosphate (Qv) productivity (Table 2) while keeping the process relatively formation is essential for HSP12 repression, however the signal simple. On the other hand, 3-PS cultivation achieved the highest transmission pathway is still unknown (de Groot et al., 2000). When final titer comparing to all GAP-C and PDH-C cultivations, which growing K. phaffii in chemostat cultivations with glycerol as a carbon meant 2.3 times more than the best GAP-C production source, an increase in the metabolic flux distribution through performance. This parameter is critical for high value-added gluconeogenesis, and therefore glucose-6-phosphate formation, was products since high titers allow to reduce the downstream detected as the μ increased (Tomàs-Gamisans et al., 2019). Although Frontiers in Bioengineering and Biotechnology 12 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 really low μ were not studied, it is quite plausible that PDH regulation also In shake-flask cultivations, pseudo-starving conditions were depends on glucose-6-phosphate formation, which might be inexistent achieved by employing the feeding technology FeedBeads ®, which at pseudo-starving conditions. Consequently, this regulation provides slowly release a defined amount of glycerol over time. Using this not only a growth-decoupled expression system, but also a slightly strategy, PDH-C surpassed the benchmark producing clones tunable one depending on the μ selected. GAP-C and UPP-C, but not PDF-C. To further investigate the Despite the promising results described, the heterologous PDF is still potential of PDH-C with the aim to optimize the process, a set of the strongest promoter reported in K. phaffii so far, presenting a fed-batch cultivations were successfully conducted under growth-dependent regulation with its maximum at medium µ (Vogl standard carbon-limiting conditions and with different feeding et al., 2020; Garrigós-Martínez et al., 2021). Nevertheless, both approaches. In terms of physiological parameters, no obvious expression systems showed a similar protein quality with no CalB differences could be observed between the set of CalB producer degradation (Supplementary File S1: Supplementary Figure S2). Non- strains based on different promoters, although the titers achieved etheless, PDH can tightly repress the recombinant expression during were notably different, suggesting that CalB production does not biomass growth. This regulation represents an advantage over PDF, since affect the cell fitness. protein folding might be facilitated due to the low metabolic burden that Due to the natural growth-decoupled regulation of PDH, switching protein overexpression and cell maintenance can cause to the cell between high and low feeding rates was a successful and relatively machinery (Wu et al., 2012). Besides, having detached growth and simple strategy to boost the CalB production. As a result, a single cycle production phases represents a better choice to produce toxic proteins of biomass growth and expression induction through pseudo-starving that have detrimental effects on growth. This is the case of the widely allowed achieving the highest productivities for PDH-C clone. In studied Rizhopus oryzae lipase, which cannot be constitutively particular, this approach obtained 3.6-fold higher Qp than the GAP- expressed but has been efficiently produced in methanol-induced C fed-batch cultivation at low µ, while it was really close to the optimal expressions systems (López-Fernández et al., 2021). GAP-C fed-batch cultivation. On the other hand, three biomass- As an example of this growth-production detachment, while induction cycles with PDH-C provided the highest titer compared GAP-C and PDF-C presented respectively the 15.4% and 7.7% of the with all GAP-C cultivations, being in the range of 2.3- to 3.6-fold times total final CalB activity at the end of the batch phase, PDH-C higher. produced only the 4.8%. Moreover, some high-value added products When applying pseudo-starving conditions together with require a moderate expression to produce functional proteins that osmotic shock to PDH-C, neither in shake-flask nor in fed- meet the pharmaceutical grade (Juturu and Wu, 2018). Therefore, batch cultivations a remarkable increase in protein production although PDH is not as strong as PDF, its growth-decoupled was achieved, despite PDH being natively related to stress-damage regulation might enable a more efficient production of certain repair. Therefore, it was conjectured that the majority of stress recombinant proteins, while still implementing a methanol- mechanisms were already activated by the protein over- independent approach. In fact, this turn-on/off regulation expression and carbon source deprivation. controlled by simply minimizing the glycerol feeding rate must Exploiting the derepressed regulation of PDH, growth- be especially highlighted, since it represents an operational decoupled fed-batch cultivation strategies were successfully advantage over carbon-controlled inducible promoters that implemented, achieving a higher production of CalB than require a transition phase. Consequently, PDH expands the Pichia GAP-C. Although PDF-C still showed better results in terms toolbox to efficiently produce recombinant proteins, an alternative of final titer and overall productivities, the novel promoter PDH option especially for the low added-value products that are essential presents an innovative and promising alternative to methanol- to promote the transition towards a circular bioeconomy. based bioprocesses, especially for toxic, low- and high-value added products. In addition, PDH is operationally more advantageous than other methanol-free promoters since it can 4 Conclusion be tightly controlled only by adjusting the feeding rate. To sum up, this expression system increases further the versatility of K. The more popular K. phaffii becomes as a cell factory for RPP the phaffii and expands its toolbox for RPP, being a step forward to more expression systems with a diverse regulation are required by the achieve the circular bioeconomy transition. industry, which is currently critical for establishing a circular bioeconomy. Accordingly, in this study a methanol-free and growth- decoupled expression system based on the new promoter PDH has been Data availability statement identified and studied under process-relevant conditions, from shake- flask to benchtop fed-batch cultivations. PDH was identified as the main The transcriptome data presented in the study are deposited in regulatory sequence of the endogenous HSP12 gene, which presumably the European Nucleotide Archive (ENA) Database repository, under codes for heat-shock protein 12. From the results of this study, it can be accession number PRJEB58889. stated that PDH is highly and transiently induced upon carbon source limitation, the so-called pseudo-starving conditions, which corresponds well to the native function of HSP12 in S. cerevisiae (de Groot et al., Author contributions 2000). Considering that under almost total carbon-source starvation, the CalB expression is transiently boosted, we hypothesize that glucose- KE and VS: performed shake-flask cultures for 6-phosphate formation, a metabolite whose flux is reduced at lower transcriptomic analyses and its interpretation. KE and JF: growth rates, is essential to tightly repress PDH. performed promoter truncations and shake-flask cultures for Frontiers in Bioengineering and Biotechnology 13 frontiersin.org Bernat-Camps et al. 10.3389/fbioe.2023.1130583 PDH-based GFP expression tests. MN-T and KE: constructed Conflict of interest PDH-based CalB expression strain and performed preliminary studies of expression. NB-C: performed FeedBeads analyses, Authors KE, VS, JF, and AG and XGO were employed by the FeedBeads-based shake-flask cultures for CalB expression company Bisy GmbH. The authors of Bisy GmbH declare that they determination, fed-batch cultures and prepared the draft have interests to commercialize the promoters used for this manuscript. NB-C, AG, FV, and XG-O discussed the overall recombinant protein production study. The authors of UAB results and participated in the manuscript preparation for declare no competing interest. submission. All the authors contributed to editing the draft The remaining author declares that the research was conducted and approved the final manuscript. in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding Publisher’s note This work was supported by the Austrian Federal Ministry of Climate Protection (BMK), the Federal Ministry of Economic Affairs All claims expressed in this article are solely those of the authors and (BMDW), the Styrian Business Promotion Agency (SFG), the do not necessarily represent those of their affiliated organizations, or Standortagentur Tirol, the Government of Lower Austria and the those of the publisher, the editors and the reviewers. Any product that Vienna Business Agency in the framework of COMET-Funding may be evaluated in this article, or claim that may be made by its Program, managed by the Austrian Research Promotion Agency FFG. manufacturer, is not guaranteed or endorsed by the publisher. Acknowledgments Supplementary material We would like to acknowledge the Austrian Centre of Industrial The Supplementary Material for this article can be found online Biotechnology (acib) for funding the project through the COMET at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1130583/ program, including NB-C scholarship. full#supplementary-material References Abad, S., Kitz, K., Hörmann, A., Schreiner, U., Hartner, F. S., and Glieder, A. (2010). De Schutter, K., Lin, Y. C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., Real-time PCR-based determination of gene copy numbers in Pichia pastoris. et al. (2009). Genome sequence of the recombinant protein production host Pichia Biotechnol. 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