Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

Abstract

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

Keywords: Ustilago maydis; autoinduction; chemical genetics; cytokinesis; inducible promoter; nanobody; regulated secretion; unconventional secretion.

Figure 1

Current schematic model of lock-type secretion and implications for heterologous protein export in U. maydis. (A) Unconventional secretion of chitinase Cts1 occurs during cytokinesis of yeast cells. Prior to budding, a primary septum is assembled at the mother cell side, followed by a secondary septum at the daughter cell side. The two septa delimit a so-called fragmentation zone (FZ), a small compartment filled with different proteins and membrane vesicles (not shown). Position of septa is indicated by arrows. (B) In the wild-type situation, Cts1 accumulates in the fragmentation zone and participates in cell separation. Recent research identified the potential anchoring factor Jps1 and the septation factors Don1 (not shown) and Don3, which are essential for Cts1 secretion. (C) In the absence of Jps1, Cts1 is excluded from the fragmentation zone and unconventional secretion is abolished. Nevertheless, cell separation occurs normally. (D) In the absence of Don3, the secondary septum is not assembled, and cell separation is hampered, leading to the formation of cell aggregates. Cts1 still accumulates at the mother-daughter cell boundary but its unconventional secretion is abolished.

Figure 2

Transcriptional regulation of unconventional secretion via the potential anchoring factor Jps1. (A) Rationale of regulated Jps1 expression on the genetic level. Unconventional secretion factor Jps1 is controlled by the arabinose inducible promoter P_crg and constitutively produced Gus-Cts1 is used as a read-out for quantification of unconventional secretion. T, transcriptional terminator. (B) Micrographs of yeast-like growing cells in the “on” and “off” stage mediated by glucose and arabinose in the medium, respectively. White arrowheads depict the fragmentation zone between mother-daughter cell boundary, open white arrowheads show additional intracellular accumulations of Jps1-Gfp. DIC, differential interference contrast. Scale bar, 10 µm. (C) Gus activity in culture supernatants of indicated AB33 Gus-Cts1 derivatives. Enzymatic activity was individually normalized to average values of positive controls secreting Gus-Cts1 constitutively, which were grown in glucose and arabinose-containing cultures. Values for the positive control in the two media do not differ significantly (p = 0.2022; Figure S2). Strains containing regulated jps1 or jps1-gfp versions show a slight induction of extracellular Gus activity after growth in arabinose-containing medium. Error bars depict standard deviation. The diagram represents results of three biological replicates. Fold change of induced cultures and p-values of Student’s unpaired t-test are shown. Definition of statistical significance: p-value < 0.05.

Figure 3

Transcriptional regulation of unconventional secretion via kinase Don3. (A) Exemplary strategy for transcriptional don3-gfp regulation of unconventional secretion. Upon supplementation of the medium with glucose, the P_crg promoter is inactive, while the addition of arabinose leads to its activation. Constitutive Gus-Cts1 expression is used as a read-out for quantification of unconventional secretion. (B) Western blot of cell extracts (CE, upper panel) and TCA precipitated culture supernatants (TCA) depicting Don3-Gfp and cytosolic Gfp (cell lysis control). Primary antibodies against Gfp were used to detect the respective proteins (anti-Gfp). In cell extracts, Don3-Gfp protein is only present upon induction with arabinose. Glc, glucose supplementation; ara, arabinose supplementation. (C) Induction of unconventional secretion is reversible upon shift between glucose and arabinose supplementation using strain AB33don3Δ/P_crgdon3/Gus-Cts1. Cultivation of cells in cycles consisting of 16-h growth in CM-glc (supplemented with glucose) and 8 h CM-ara (supplemented with arabinose), allows alternating “on” and “off” states of unconventional secretion. After each cycle, the relative extracellular activity of Gus-Cts1 was determined and cell densities were adjusted for the next cycle. The experiment was conducted over 5 consecutive days. Error bars depict standard deviation. The diagram represents results of four biological replicates. Fold change of induced cultures and p-values of Student’s unpaired t-test are shown. Definition of statistical significance: p-value < 0.05.

Figure 4

Post-translational regulation of unconventional secretion via inactivation of Don3 kinase activity. (A) Strategy for post-translational regulation of unconventional secretion using the mutagenized Don3 version Don3* in concert with a bulky ATP analog (NA-PP1). (B) Micrographs of yeast-like growing cells grown in medium containing arabinose. Cells treated with the bulky ATP analog NA-PP1 are indicated. Arrowheads depict the Gfp signal at the mother-daughter cell boundary. DIC, differential interference contrast. Scale bars, 10 µm. (C) Gus activity in culture supernatants of indicated AB33 P_omaGus-Cts1 derivatives. Enzymatic activity was individually normalized to average values of positive controls secreting Gus-Cts1 constitutively, which were grown in arabinose-containing cultures. Values of positive controls in the two media do not differ significantly (p = 0.7317; Figure S4). Strains containing regulated don3* or don3*-gfp versions show a strong induction of extracellular Gus activity after growth in arabinose medium without NA-PP1. The diagram represents results of four biological replicates. Error bars depict standard deviation. Fold change of induced cultures and p-values of Student’s unpaired t-test are shown. Definition of statistical significance: p-value < 0.05. (D) Western blots of cell extracts (CE, upper panel) and TCA precipitated culture supernatants of AB33don3Δ cultures expressing regulated Don3-Gfp and Don3*-Gfp. Primary antibodies against Gfp were used to detect the respective proteins (anti-Gfp). Both fusion proteins are degraded in the supernatant and only free Gfp can be detected. Free Gfp derived from Don3-Gfp was detected in the presence and absence of NA-PP1. However, no free Gfp derived from Don3*-Gfp was detectable in the presence of NA-PP1, indicating inhibition of unconventional secretion.

Figure 5

Time-resolved comparison between transcriptional and translational Don3 regulation. Cells of the Gus-Cts1 reporter strain containing the mutagenized kinase version Don3* (Figure 4A) were pre-incubated in medium supplemented with arabinose only (arabinose − NA-PP1, white columns), with glucose only (light blue columns) or with arabinose and the kinase inhibitor NA-PP1 (arabinose + NA-PP1, dark blue columns). After a washing step to remove media components, cells were resuspended in medium containing arabinose and Gus activity was determined for 8 h at distinct time points. Enzymatic activity was normalized to average values of induced overnight culture. The diagram represents results of four biological replicates. Error bars depict standard deviation. p-values of Student’s unpaired t-test between previously normalized culture and induced culture are shown. Definition of statistical significance: p-value < 0.05.

Figure 6

Establishing an autoinduction process based on transcriptional regulation. (A,B) Reporter strain AB33don3Δ/P_crggfp was cultivated in buffered CM medium supplemented with glucose (glc) and arabinose (ara) in different amounts and ratios as indicated in the diagram. Since in contrast to the experiments before the cultures were incubated for a prolonged time reaching high optical densities, the medium was buffered with 100 mM MES to prevent a drastic pH drop [15]. The two parameters fungal biomass (A) and Gfp fluorescence (B) were recorded online in a BioLector device. Gains: 20 (scattered light); 80 (Gfp).

Figure 7

Evaluation of the autoinduction process for unconventional secretion of an anti-Gfp nanobody. Strain AB33don3Δ/P_crgdon3/NB-Cts1 was inoculated in CM medium supplemented with 1% glucose, 1% arabinose, and buffered with 0.1 M MES. The culture was split into 5 individual flasks for harvest of supernatant proteins, cell extracts and parallel online growth monitoring in BioLector and offline monitoring via photometer. Supernatant was collected at defined time points and unconventionally secreted NB-Cts1 was IMAC purified. Cell extracts were prepared in parallel. For purified supernatant and cell extracts ELISA were performed using purified Gfp as antigen. (A) Schematic representation of the genetic setup for transcriptional don3-gfp regulation of unconventional secretion in autoinduction medium by activating transcription through arabinose after the consumption of glucose (diauxic switch indicated by clock symbol). NB-Cts1 is constitutively produced but trapped in the cell prior to Don3 synthesis. (B) Online monitoring of the cultivation using the BioLector device. Primary ordinate axis shows biomass via backscatter light (gain 20), secondary ordinate axis shows pH, red, and dissolved oxygen tension (DOT), blue. Time points of sampling of parallel grown shake flask cultures are indicated by arrowheads. (C) Enzyme-linked immunosorbent assay (ELISA) using NB-Cts1 purified from culture supernatants at indicated time points. 1×, 5× and 10× concentrated purified supernatants, (D) ELISA using cell extracts harvested at defined protein amounts containing 15 ng, 30 ng, and 125 ng total protein.