2. Research Material and Methods
3.8 Optimal Time for Harvesting
The calculation of specific productivities (Figure 12D) also gives some addi- tional insights in regard to timing. rµ for both BR5 and BR6 plateaued after a certain point, which was noticeably earlier than rv plateaued. This means that, on average, the productivity of individual cells is starting to slow down, possibly because the oldest cells are reaching the end of their productive lifespan. Meanwhile, new cell copies continue to maintain a low production rate which is indicated as a production rate that is directly proportional to the rate of increase in biomass, i.e., specific productivity remains constant.
This has important implications regarding optimization of timing. Here, I propose two options for harvesting depending on time constraints and the desired yield (Figure 13). If the aim is to minimize duration while also not significantly reducing protein yield in the high gas flow bioreactor, the dura- tion of the methanol feeding could be shortened to as little as 1.5 days, mean- ing that harvesting could already be done after 60 hours (High gas flow point A). Meanwhile, the low gas flow bioreactor seems to require approximately 2.5 days in the methanol phase to reach the plateau in specific productivity and thus the earliest harvesting time point is after approximately 96 hours (Low gas flow point A).
If the aim is to maximize yield in turn, it might make more sense to wait until the volumetric productivity stops increasing, as opposed to specific produc- tivity. Given the available data, this means that with high gas flow, the harvest should be done after approximately 108 hours (High gas flow point B) and with low gas flow, after approximately 120 hours (Low gas flow point B), as long as cell growth remains normal during this time. It should be noted that, given the apparent continued increase in growth at the point of harvest, BR5 and BR6 could have been run for even longer. This means that additional runs would be needed to confirm these insights into the typical maximum duration of fermentations.
43
Figure 13 | Potential harvesting time points depending on gas flow and tim- ing criteria. Point A: The time at which specific productivity plateaus, the earliest harvesting point; Point B) The time at which volumetric productiv- ity plateaus, the latest harvesting point.
This shows that when optimizing duration and using the high gas flow biore- actor, the total duration of the production could be halved. However, in ad- dition to a considerable penalty to protein yield (in this case approximately one third of total yield) the nature of the workweek should be considered when shortening the duration to avoid laborious downstream processing steps during weekends. In some cases, it might be optimal to extend the du- ration over the weekend, for increased yield and to ensure only monitoring of the process is required during the weekend. That being said, the two sce- narios outlined here both have their advantages and the ideal duration is likely to be somewhere between these two time points. On an additional note, there is also a third scenario which is to wait until protein production stops completely, but due to the nature of diminishing returns of a batch biopro- cess, this wastes an unnecessary amount of time in exchange for a marginal increase in yield.
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4 Conclusions
To summarize, P. pastoris is a suitable expression system for bioreactor scale production of PcaLOOLs. Here, an HCDF approach was used with a metha- nol-limited fed-batch feeding strategy to successfully produce yields compa- rable to literature values. The methanol feeding rate was changed step-wise based on DO values. In many of the runs, controlling DO with methanol alone was not sufficient since the oxygen demand of the cells frequently surpassed the aeration capabilities of the bioreactor system, even at low methanol feed- ing rates. While this did bring some inconsistency to the runs, since DO could not be kept constant across them, the high yields suggest that a consistent DO of above 35% is less important for maximizing yield than the cell densities achieved and the amount of methanol fed. Therefore, in a situation where DO begins to drop below the set threshold, it might be worth to continue metha- nol feeding at a lower DO level in order to maximize yield as opposed to stop- ping methanol feeding altogether and wait for DO to return to normal. As long as DO is above critical levels and cell density continues to increase, the cells appear to be healthy enough to produce protein at a sufficient rate.
The volumetric yields of protein are directly correlated with the amount of methanol fed. Therefore, while an optimal DO set point has been previously specified [3], the yields across these runs were more dependent on how low one dared to go in terms of DO levels. However, since a low DO level is still likely to affect the specific productivity of the cells, the simplest way to max- imize yield in the future is to improve the efficiency of oxygen transfer. This was seen in the 7L bioreactor, which was capable of gas flow rates more than three times that of the 5L bioreactor. As a result, the specific growth rate in the methanol phase almost doubled and the yield increased by 50%, when comparing BR6 and BR5. Another way to further increase oxygen transfer efficiency is the injection of pure oxygen into the gas flow. However, this poses some safety risks and is unlikely to be cost-effective.
A major challenge that was not fully solved during this thesis was suitable downstream processing. The first three productions used downstream pro- cessing that worked well in terms of final yields but was poorly scalable due to the extensive centrifugation and depth filtration required. The second method which was used for the last three productions, used a crossflow fil- tration system with a microfiltration step to remove cells, followed by an ul- trafiltration step for concentration. In the first step, extensive fouling of the microfiltration membrane was experienced, while in the second step, the pro- tein was spoiled due to reasons that are not fully clear, but the most likely explanation was salt contamination from the membrane. As described in sec- tion 3.6, membrane fouling could be solved by improved system parameters,
45
while the issues experienced during UF concentration could be solved by in- stalling a new membrane altogether.
Due to the two different harvesting methods used, yield results are also di- vided into two categories. Across the first three runs, which gave the most objectively accurate final yields, BR3 reached the highest yield at 1142.42 ± 42.92 mg which was a substantial increase from the two first runs with yields of 274.82 ± 7.12 mg and 178.21 ± 1.66 mg from BR1 and BR2 respectively.
The main reasons for this increase were a higher pH and a higher amount of methanol fed. The values for the last three runs were estimated by quantify- ing SDS-PAGE bands and were therefore less comparable to the first three.
However, across these runs, the highest yield was achieved in BR6 at 2013.20
± 849.60 mg, followed by BR5 at 1352.91 ± 407.32 mg and BR4 at 382.15 ± 93.43 mg. These values were also taken before downstream processing and do not therefore take into account the losses that would be seen during puri- fication, buffer exchange and final concentration steps.
Another parameter that was investigated was the duration of runs. To fully maximize yield without time constraints, the best indicator that production has ended is when cell density stops increasing. In these runs, this would typ- ically occur around 120 hours. However, in the more successful runs with the PcaLOOL9-producing clones, harvesting was done at points where cell den- sity is likely to have continued to increase further so the plateau in cell growth wasn’t reached yet in these productions. Therefore, yield could have possibly been increased even further by continuing the runs for 1-2 days, although at a slower rate. In this thesis I also proposed an alternative point for harvesting which is the point at which specific productivity plateaus, which could allow for shortening of the fermentation by several days. The loss in yield is quite substantial however, so in reality the optimal harvest point is likely to be somewhere between these two points. Therefore, the point at which specific productivity plateaus can be considered the earliest point at which harvesting can be done and the point at which cell density or volumetric productivity plateaus, the latest point it can be done.
Although this work struggled with various comparability issues across the runs, such as the downstream processing method used, amount of methanol fed, and the dissolved oxygen levels, this is the first time the production of PcaLOOLs was successfully scaled up to bioreactor scale. While the difficul- ties to keep enough variables consistent meant that many fermentation pa- rameters could not be truly validated and optimized, this work suggests many guiding insights into how yields can be maximized further as well as some intuition regarding how to control the fermentation itself.
46
References
[1] J. M. Cregg et al, "Recombinant protein expression in Pichia pastoris,"
Mol. Biotechnol., vol. 16, (1), pp. 23-52, 2000. . DOI: 10.1385/MB:16:1:23.
[2] M. Monschein et al, "Loosenin-like proteins from Phanerochaete car- nosa impact both cellulose and chitin fiber networks (Manuscript)," un- published, private communication, Unpublished.
[3] Anonymous "Pichia Fermentation Process Guidelines," 2002.
[4] D. G. Noseda et al, "Production in stirred-tank bioreactor of recombi- nant bovine chymosin B by a high-level expression transformant clone of Pichia pastoris," Protein Expr. Purif., vol. 123, pp. 112-121, 2016. . DOI:
10.1016/j.pep.2016.03.008.
[5] Y. Wang et al, "Process development of recombinant Aspergillus flavus urate oxidase production in Pichia pastoris intracellularly and its characteri- zation as a potential biosimilar," Process Biochemistry (1991), vol. 102, pp.
376-385, 2021. . DOI: 10.1016/j.procbio.2021.01.026.
[6] d'Anjou and Marc Charles, "Production of Recombinant Antifreeze Proteins in the Methylotrophic Yeast Pichia Pastoris." , Queen's University, 1999.
[7] E. Palmqvist and B. Hahn-Hägerdal, "Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition," Bioresour. Tech- nol., vol. 74, (1), pp. 25-33, 2000. . DOI: 10.1016/S0960-8524(99)00161-3.
[8] M. Poletto, H. L. Ornaghi and A. J. Zattera, "Native Cellulose: Struc- ture, Characterization and Thermal Properties," Materials; Materials (Ba- sel), vol. 7, (9), pp. 6105-6119, 2014. . DOI: 10.3390/ma7096105.
[9] H. V. Scheller and P. Ulvskov, "Hemicelluloses," Annual Review of Plant Biology; Annu Rev Plant Biol, vol. 61, (1), pp. 263-289, 2010. . DOI:
10.1146/annurev-arplant-042809-112315.
[10] S. M. Cragg et al, "Lignocellulose degradation mechanisms across the Tree of Life," Curr. Opin. Chem. Biol., vol. 29, pp. 108-119, 2015. . DOI:
10.1016/j.cbpa.2015.10.018.
[11] D. J. Cosgrove, "Enzymes and other agents that enhance cell wall ex- tensibility," Annu. Rev. Plant Physiol. Plant Mol. Biol., vol. 50, (1), pp. 391- 417, 1999.
47
[12] X. Liu, Y. Ma and M. Zhang, "Research advances in expansins and expansion-like proteins involved in lignocellulose degradation," Biotechnol.
Lett., vol. 37, (8), pp. 1541-1551, 2015. . DOI: 10.1007/s10529-015-1842-0.
[13] D. J. Cosgrove, "Catalysts of plant cell wall loosening," F1000 Re- search; F1000Res, vol. 5, pp. 119, 2016. . DOI: 10.12688/f1000re- search.7180.1.
[14] K. Keegstra et al, "The Structure of Plant Cell Walls: III. A Model of the Walls of Suspension-cultured Sycamore Cells Based on the Interconnec- tions of the Macromolecular Components," Plant Physiol, vol. 51, (1), pp.
188-197, 1973. . DOI: 10.1104/pp.51.1.188.
[15] K. Keegstra et al, "The Structure of Plant Cell Walls: III. A Model of the Walls of Suspension-cultured Sycamore Cells Based on the Interconnec- tions of the Macromolecular Components," Plant Physiol., vol. 51, (1), pp.
188-197, 1973. Available: https://pubmed.ncbi.nlm.nih.gov/16658282 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC367377/. DOI:
10.1104/pp.51.1.188.
[16] Y. B. PARK and D. J. COSGROVE, "A Revised Architecture of Primary Cell Walls Based on Biomechanical Changes Induced by Substrate-Specific Endoglucanases," Plant Physiol., vol. 158, (4), pp. 1933-1943, 2012. . DOI:
10.1104/pp.111.192880.
[17] S. D. Mansfield, C. Mooney and J. N. Saddler, "Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis," Biotechnol. Prog., vol. 15, (5), pp. 804-816, 1999. . DOI: 10.1021/bp9900864.
[18] J. C. Ogier et al, "Production d'éthanol a partir de biomasse lignocel- lulosique Ethanol Production from Lignocellulosic Biomass," Oil & Gas Sci- ence and Technology, vol. 54, (1), pp. 67-94, 2006. . DOI:
10.2516/ogst:1999004.
[19] M. J. Taherzadeh and K. Karimi, "Enzymatic-based hydrolysis pro- cesses for ethanol from lignocellulosic materials: A review," Bioresources, vol. 2, (4), pp. 707, 2007.
[20] W. Tu and J. P. Hallett, "Recent advances in the pretreatment of lig- nocellulosic biomass," Current Opinion in Green and Sustainable Chemis- try, vol. 20, pp. 11-17, 2019. . DOI: 10.1016/j.cogsc.2019.07.004.
[21] V. Arantes and J. N. Saddler, "Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis," Biotechnology for Bio- fuels; Biotechnol Biofuels, vol. 3, (1), pp. 4, 2010. . DOI: 10.1186/1754-6834- 3-4.
48
[22] D. J. Cosgrove, "Expansive growth of plant cell walls," Plant Physiol.
Biochem., vol. 38, (1), pp. 109-124, 2000. . DOI: 10.1016/S0981- 9428(00)00164-9.
[23] H. Suzuki et al, "Sequence diversity and gene expression analyses of expansin-related proteins in the white-rot basidiomycete, Phanerochaete carnosa," Fungal Genet. Biol., vol. 72, pp. 115-123, 2014. . DOI:
10.1016/j.fgb.2014.05.008.
[24] A. Tabuchi, L. Li and D. J. Cosgrove, "Matrix solubilization and cell wall weakening by β‐expansin (group‐1 allergen) from maize pollen," Plant J., vol. 68, (3), pp. 546-559, 2011. . DOI: 10.1111/j.1365-313X.2011.04705.x.
[25] D. J. (. Cosgrove, P. Bedinger and D. M. Durachko, "Group I allergens of grass pollen as cell wall-loosening agents," Proc. Natl. Acad. Sci. U. S. A., vol. 94, (12), pp. 6559-6564, 1997. . DOI: 10.1073/pnas.94.12.6559.
[26] D. J. Cosgrove, "Microbial Expansins," Annu. Rev. Microbiol., vol. 71, (1), pp. 479-497, 2017. . DOI: 10.1146/annurev-micro-090816-093315.
[27] N. Georgelis, N. Nikolaidis and D. J. Cosgrove, "Biochemical analysis of expansin-like proteins from microbes," Carbohydr. Polym., vol. 100, (1), pp. 17-23, 2014. . DOI: 10.1016/j.carbpol.2013.04.094.
[28] H. Kende et al, "Nomenclature for members of the expansin super- family of genes and proteins," Plant Mol. Biol., vol. 55, (3), pp. 311-314, 2004.
. DOI: 10.1007/s11103-004-0158-6.
[29] R. Quiroz-Castañeda E. et al, "Loosenin, a novel protein with cellu- lose-disrupting activity from Bjerkandera adusta," Microbial Cell Factories;
Microb Cell Fact, vol. 10, (1), pp. 8, 2011. . DOI: 10.1186/1475-2859-10-8.
[30] A. de Oliveira L. et al, "The Structure of the Elicitor Cerato-platanin (CP), the First Member of the CP Fungal Protein Family, Reveals a Double ψβ-Barrel Fold and Carbohydrate Binding," J. Biol. Chem., vol. 286, (20), pp.
17560-17568, 2011. . DOI: 10.1074/jbc.M111.223644.
[31] M. Andberg, M. Penttilä and M. Saloheimo, "Swollenin from Tricho- derma reesei exhibits hydrolytic activity against cellulosic substrates with features of both endoglucanases and cellobiohydrolases," Bioresour. Tech- nol., vol. 181, pp. 105-113, 2015. . DOI: 10.1016/j.biortech.2015.01.024.
[32] M. Saloheimo et al, "Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cel- lulosic materials," Eur. J. Biochem., vol. 269, (17), pp. 4202-4211, 2002. . DOI: 10.1046/j.1432-1033.2002.03095.x.
49
[33] K. Gourlay et al, "Swollenin aids in the amorphogenesis step during the enzymatic hydrolysis of pretreated biomass," 2013. . DOI:
10.1016/j.biortech.2013.05.053.
[34] A. Campos-Oliver et al, "Cloning and expression of a hypothetical
loosenin from
Neurospora crassa," Rev Latinoam Biotecnol Ambient Y Algal, vol. 4, (1), pp.
1-7, 2014.
[35] J. M. Cregg et al, "Functional characterization of the two alcohol oxi- dase genes from the yeast Pichia pastoris," Mol. Cell. Biol., vol. 9, (3), pp.
1316-1323, 1989. . DOI: 10.1128/mcb.9.3.1316-1323.1989.
[36] F. W. Krainer et al, "Recombinant protein expression in Pichia pas- toris strains with an engineered methanol utilization pathway," Microbial Cell Factories; Microb Cell Fact, vol. 11, (1), pp. 22, 2012. . DOI:
10.1186/1475-2859-11-22.
[37] S. Macauley‐Patrick et al, "Heterologous protein production using the Pichia pastoris expression system," Yeast, vol. 22, (4), pp. 249-270, 2005. . DOI: 10.1002/yea.1208.
[38] W. Liu et al, "Scaling-up Fermentation of Pichia pastoris to demon- stration-scale using new methanol-feeding strategy and increased air pres- sure instead of pure oxygen supplement," Scientific Reports; Sci Rep, vol. 6, (1), pp. 18439, 2016. . DOI: 10.1038/srep18439.
[39] D. G. Noseda et al, "Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter," Protein Expr. Purif., vol. 92, (2), pp. 235-244, 2013. . DOI:
10.1016/j.pep.2013.08.018.
[40] X. Zhang et al, "Enhanced bioproduction of chitin in engineered Pichia pastoris," Food Bioscience, vol. 47, pp. 101606, 2022. . DOI:
10.1016/j.fbio.2022.101606.
[41] M. Gao et al, "Efficient endo-β-1,3-glucanase expression in Pichia pastoris for co-culture with Agrobacterium sp. for direct curdlan oligosaccha- ride production," Int. J. Biol. Macromol., vol. 182, pp. 1611-1617, 2021. . DOI:
10.1016/j.ijbiomac.2021.05.142.
[42] M. Gao et al, "One-step production of functional branched oligoglu- cosides with coupled fermentation of Pichia pastoris GS115 and Sclerotium rolfsii WSH-G01," Bioresour. Technol., vol. 335, pp. 125286, 2021. . DOI:
10.1016/j.biortech.2021.125286.
[43] D. Wang et al, "Green Process: Improved Semi-Continuous Fermen- tation of Pichia pastoris Based on the Principle of Vitality Cell Separation,"
50
Frontiers in Bioengineering and Biotechnology; Front Bioeng Biotechnol, vol. 9, pp. 777774, 2021. . DOI: 10.3389/fbioe.2021.777774.
[44] S. Singh et al, "Large-scale functional expression of WT and trun- cated human adenosine A2A receptor in Pichia pastoris bioreactor cultures,"
Microbial Cell Factories; Microb Cell Fact, vol. 7, (1), pp. 28, 2008. . DOI:
10.1186/1475-2859-7-28.
[45] M. Jahic et al, "Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures," Microbial Cell Facto- ries; Microb Cell Fact, vol. 2, (1), pp. 6, 2003. . DOI: 10.1186/1475-2859-2- 6.
[46] M. Jahic et al, "Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes," J. Biotechnol., vol. 102, (1), pp. 45- 53, 2003. . DOI: 10.1016/S0168-1656(03)00003-8.
[47] T. Tissopi et al, "Surface display of novel transglycosylating α-gluco- sidase from Aspergillus neoniger on Pichia pastoris for synthesis of iso- maltooligosaccharides," Biochem. Eng. J., vol. 181, pp. 108400, 2022. . DOI:
10.1016/j.bej.2022.108400.
[48] N. Maity et al, "High level production of stable human serum albumin in Pichia pastoris and characterization of the recombinant product," Biopro- cess Biosyst Eng, vol. 45, (2), pp. 409-424, 2022. . DOI: 10.1007/s00449- 021-02670-z.
[49] M. Deng et al, "Efficient Bioproduction of Human Milk Alpha-Lactal- bumin in Komagataella phaffii," J. Agric. Food Chem., vol. 70, (8), pp. 2664- 2672, 2022. . DOI: 10.1021/acs.jafc.1c07908.
[50] Z. E. Duman et al, "High-level heterologous expression of active Chaetomium thermophilum FDH in Pichia pastoris," Enzyme Microb. Tech- nol., vol. 137, pp. 109552, 2020. . DOI: 10.1016/j.enzmictec.2020.109552.
[51] Z. Yang and Z. Zhang, "Codon-optimized expression and characteri- zation of a pH stable fungal xylanase in Pichia pastoris," Process Biochemis- try (1991), vol. 53, pp. 80-87, 2017. . DOI: 10.1016/j.procbio.2016.11.022.
[52] M. Cayetano-Cruz et al, "High level expression of a recombinant xy- lanase by Pichia pastoris cultured in a bioreactor with methanol as the sole carbon source: Purification and biochemical characterization of the enzyme,"
Biochem. Eng. J., vol. 112, pp. 161-169, 2016. . DOI:
10.1016/j.bej.2016.04.014.
[53] Ö Ata et al, "Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to
51
fine-tune bioreactor performance," Bioprocess Biosyst Eng, vol. 38, (5), pp.
889-903, 2014. . DOI: 10.1007/s00449-014-1333-z.
[54] W. J. Holmes et al, "Developing a scalable model of recombinant pro- tein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime," Microbial Cell Factories; Microb Cell Fact, vol. 8, (1), pp. 35, 2009. . DOI: 10.1186/1475-2859-8-35.
[55] Adivitiya et al, "Nitrogen supplementation ameliorates product qual- ity and quantity during high cell density bioreactor studies of Pichia pastoris:
A case study with proteolysis prone streptokinase," Int. J. Biol. Macromol., vol. 180, pp. 760-770, 2021. . DOI: 10.1016/j.ijbiomac.2021.03.021.
[56] A. Caballero-Pérez et al, "Buried Kex2 Sites in Glargine Precursor Ag- gregates Prevent Its Intracellular Processing in Pichia pastoris Muts Strains and the Effect of Methanol-Feeding Strategy and Induction Temperature on Glargine Precursor Production Parameters," Appl. Biochem. Biotechnol., vol.
193, (9), pp. 2806-2829, 2021. . DOI: 10.1007/s12010-021-03567-z.
[57] M. Gao et al, "Expression of a thermostable β-1,3-glucanase from Trichoderma harzianum in Pichia pastoris and use in oligoglucosides hydrol- ysis," Process Biochemistry (1991), vol. 107, pp. 74-82, 2021. . DOI:
10.1016/j.procbio.2021.05.010.
[58] D. Abdulrachman et al, "Heterologous expression of Aspergillus acu- leatus endo-polygalacturonase in Pichia pastoris by high cell density fermen- tation and its application in textile scouring," BMC Biotechnol., vol. 17, (1), pp. 15, 2017. . DOI: 10.1186/s12896-017-0334-9.
[59] J. Hausjell et al, "Recombinant production of a hard‐to‐express mem- brane‐bound cytochrome P450 in different yeasts—Comparison of physiol- ogy and productivity," Yeast, vol. 37, (2), pp. 217-226, 2020. . DOI:
10.1002/yea.3441.
[60] S. Calderon-Salais et al, "Constitutive expression of the active frag- ment of human vasostatin Vs30 in Pichia pastoris SMD1168H," Protein Expr.
Purif., vol. 144, pp. 40-45, 2018. . DOI: 10.1016/j.pep.2017.12.002.
[61] Yuliawati, Aminah and A. M. Fuad, "Purification of recombinant hu- man granulocyte colony-stimulating factor from Pichia pastoris using two ninta chromatography methods," IOP Conference Series.Earth and Environ- mental Science; IOP Conf.Ser.: Earth Environ.Sci, vol. 439, (1), pp. 12044, 2020. . DOI: 10.1088/1755-1315/439/1/012044.
[62] M. Ahmad et al, "Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production," Appl.
Microbiol. Biotechnol., vol. 98, (12), pp. 5301-5317, 2014. . DOI:
10.1007/s00253-014-5732-5.