Process engineering of biopharmaceutical production in moss bioreactors via model-based description and evaluation of phytohormone impact

Abstract

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 μM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-Kynurenine (100 μM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 hours after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first two hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/ g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention

“…Auxin plays crucial roles in plant development (Santner & Estelle, 2009; Weijers & Wagner, 2016), including Physcomitrella development (Decker et al ., 2006; Fujita et al ., 2008; Coudert et al ., 2017; Menand et al ., 2020; Nemec‐Venza et al ., 2022) and biotechnology (Ruiz‐Molina et al ., 2022). Although PIN proteins are central in auxin transport and action (Adamowski & Friml, 2015; Sauer & Kleine‐Vehn, 2019; Ung et al ., 2022), only two of the three canonical Physcomitrella PIN proteins have been fully characterized; the similar and relatively highly expressed PpPINA and PpPINB.…”

“…Auxin plays crucial roles in plant development (Santner & Estelle, 2009;Weijers & Wagner, 2016), including Physcomitrella development (Decker et al, 2006;Fujita et al, 2008;Coudert et al, 2017;Menand et al, 2020;Nemec-Venza et al, 2022) and biotechnology (Ruiz-Molina et al, 2022).…”

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention

“…Auxin plays crucial roles in plant development (Santner & Estelle, 2009; Weijers & Wagner, 2016), including Physcomitrella development (Decker et al ., 2006; Menand et al ., 2007; Fujita et al ., 2008; Coudert et al ., 2017; Nemec-Venza et al ., 2022) and biotechnology (Ruiz-Molina et al ., 2022). Although PIN proteins are central in auxin transport and action (Adamowski & Friml, 2015; Sauer & Kleine-Vehn, 2019; Ung et al ., 2022), only two of the three canonical Physcomitrella PIN proteins have been fully characterized; the similar and relatively highly expressed PpPINA and PpPINB.…”

“…and biotechnology(Ruiz-Molina et al, 2022). Although PIN proteins are central in auxin transport and action(Adamowski & Friml, 2015;Sauer & Kleine- Vehn, 2019;Ung et al, 2022), only two of the three canonical Physcomitrella PIN proteins have been fully characterized; the similar and relatively highly expressed PpPINA and PpPINB.…”