Recombinant protein production in P. pastoris GS115/LacB and P. pastoris GS115m/LacB
In a previous study, growth rate and maximal biomass were lower for P. pastoris GS115m/LacB grown in YPR than for P. pastoris GS115/LacB. However, the amount of recombinant protein, β-galactosidase encoded by LacB, in the supernatants of P. pastoris GS115m/LacB cultures was higher than that of P. pastoris GS115/LacB (Yan et al. 2018). This indicated that P. pastoris GS115m/LacB produced the target protein more efficiently. To verify this result, the production of recombinant protein in the two strains grown in YPR medium was investigated based on protein production vs wet cell weight (WCW). The results revealed that Pichia cells efficiently produced target proteins from 12 to 36 h, and the recombinant protein production at 72 h was more than twofold higher in P. pastoris GS115m/LacB cell than in P. pastoris GS115/LacB (Fig. 1). The underlying mechanisms for the improved production of recombinant protein, which could provide some important information for strain engineering, were further investigated.
Global transcriptional profiles of P. pastoris GS115/LacB and P. pastoris GS115m/LacB
Pichia pastoris GS115/LacB and P. pastoris GS115m/LacB were cultured in YPR medium until an OD600 of ~ 2 or ~ 6, respectively. The cells from two biological replicates of P. pastoris GS115/LacB and P. pastoris GS115m/LacB were collected, and total RNA from these cells was prepared for transcriptome analysis using RNA-seq. The RNA-seq data were deposited in the CNGBdb (https://db.cngb.org) with Accession Number CNP0000622 and CNP0000710. The overall expression levels in the two biological replicates of each group were highly similar to each other (R2 beyond 0.98) (Additional file 2: Fig. S1). At an OD600 of ~ 2, a total of 749 DEGs including 281 down-regulated and 468 up-regulated genes were identified among 5,041 genes, including an exogenous gene, LacB. The large number of DEGs indicated that the decrease in rhamnose metabolic flux exerted wide-ranging effects on the transcriptomes of Pichia cells and thereby led to various changes in physiological profiles.
To further examine gene expression profiles, real-time PCR was performed to investigate the relative expression of four up-regulated DEGs (PAS_chr4_0550, PAS_chr1-1_0356, PAS_chr3_0798, and PAS_chr4_0146), four down-regulated DEGs (PAS_chr3_0095, PAS_chr3_0403, PAS_chr4_0799, and PAS_chr3_0257), one non-DEG (PAS_chr3_0229), and two genes of interest (LRA4 and LacB). The trends in expression of the target genes were consistent between real-time PCR and RNA-seq despite the presence of minor differences in the expression levels of certain genes between the two methods (Fig. 2).
Improved expression of LRA3 in P. pastoris GS115m/LacB
Highly transcribed genes usually play crucial roles in organism survival, and their expression changes under different conditions. According to the FPKM value of each gene, the 25 most highly expressed genes in P. pastoris GS115/LacB and P. pastoris GS115m/LacB were identified at an OD600 of ~ 2 (Additional file 3: Table S2) and an OD600 of ~ 6 (Additional file 4: Table S3), respectively. Theoretically, these genes are expected to play important roles in the survival of Pichia cells using rhamnose as the sole carbon source.
Notably, LRA3 was also one of the 25 most highly expressed genes in both strains grown to OD600 ~ 2 and ~ 6, which suggested that LRA3 expression was intensively induced in the presence of rhamnose. At OD600 of ~ 2 and ~ 6, the LRA3 transcription level was ranked 21st and 14th in P. pastoris GS115/LacB, and while it was ranked 2nd and 6th in P. pastoris GS115m/LacB, respectively. These results showed that LRA3 expression level was significantly elevated in P. pastoris GS115m/LacB with rhamnose induction. In addition, it was surprising that the LRA3 transcription level was even higher than that of GAPDH in P. pastoris GS115m/LacB.
Transcription profiles of the genes related to rhamnose metabolism during incubation
Rhamnose was the main carbon source for cell survival when Pichia cells were grown in YPR medium, and the rhamnose utilization rate was therefore a key factor affecting energy production, biomass biogenesis, and physiological profiles in Pichia cells. Down-regulating the expression of key rate-determining step enzymes such as LRA4 would decrease rhamnose utilization efficiency, resulting in insufficient supply of energy and carbon matrices for primary and secondary metabolism, growth, and propagation. To adapt to these conditions, the strain should up-regulate the expression of rhamnose-utilization genes to accelerate rhamnose metabolism. This expectation was borne out by transcriptome analysis.
The genes involved in rhamnose metabolism included five genes such as four enzyme-coding genes (LRA1–4) and a regulator-coding gene (RhaR). As expected, the expression levels of all the genes except for LRA4 in P. pastoris GS115m/LacB were differentially up-regulated more than twofold compared with P. pastoris GS115/LacB at an OD600 of ~ 2 (Fig. 3). Simultaneously, LRA4 maintained its low expression as it was under the control of the weak promoter PLRA2 (Fig. 3). These results indicated that the low production rate of the key rhamnose metabolism-related enzyme LRA4 led to inadequate production of energy and carbon matrices required for normal growth, and P. pastoris GS115m/LacB therefore enhanced the expression of rhamnose metabolism-related genes to increase rhamnose utilization to provide more energy and biomass for cell growth.
To further investigate the expression profiles of these genes during incubation, the genes were examined when the strains were grown to a high cell density (OD600 ~ 6). Relative expression of all genes, except for LRA4, decreased compared with that at an OD600 of ~ 2 (Fig. 3). When grown to OD600 ~ 12, the expression levels of these genes in P. pastoris GS115m/LacB were almost equal to those of P. pastoris GS115/LacB (with the exception of LRA4), which was reported previously (Yan et al. 2018). This could be explained that the decreasing concentration of residual rhamnose in the medium made a declining induction to the related gene expression. Overall, the relative expression of genes other than LRA4 was dynamic; it was closely associated with the concentration of residual rhamnose in the medium and decreased with the consumption of rhamnose.
LacB expression profiles during incubation
Recombinant protein production, an important index for an expression system, was closely and positively dependent on the transcription activity of its promoter. In P. pastoris GS115m/LacB, LacB expression was controlled under PLRA3, and thereby the production of the recombinant protein, β-galactosidase encoded by LacB, was largely subject to the transcriptional activity of PLRA3. As mentioned above, LRA3 was one of the most highly transcribed genes, and LRA3 expression was greatly enhanced in P. pastoris GS115m/LacB (Additional file 3: Table S2). Under the control of the same promoter, PLRA3, the trend in LacB expression was consistent with that of LRA3 (Fig. 4). The production of recombinant protein in P. pastoris GS115m/LacB improved with the increase of PLRA3 transcription activity.
Declined cell viability in P. pastoris GS115m/LacB grown on rhamnose
Generally, rhamnose metabolism was down-regulated due to the decreased expression of the rate-limiting enzyme LRA4. Low rhamnose metabolism was accompanied by low energy supply and reduced sources of carbon-based biomass components. This resulted in decreased growth rate and declined cell biomass in P. pastoris GS115m/LacB grown in YPR medium, which was confirmed by the results of our previous study (Yan et al. 2018). Additionally, it was reported that cell viability was also affected by carbon starvation such as glucose shortage (Oda et al. 2015).
To confirm whether cell viability of P. pastoris GS115m/LacB altered, cell growth assay was performed. Differences in the number and size of cell colonies indicated the various profiles of cell viability and generation time of the tested strains, respectively. Small colonies as well as decreased number of cell colonies were observed in P. pastoris GS115m/LacB compared with P. pastoris GS115/LacB when rhamnose as the carbon source (YPR and MR) (Fig. 5), which indicated prolonged propagation and declined viability in P. pastoris GS115m/LacB cells. This led to a lower biomass of P. pastoris GS115m/LacB grown in YPR and MR, which was described in another study (Yan et al. 2018).
Increased autophagy level in P. pastoris GS115m/LacB during growth on rhamnose
Autophagy is a principal catabolic pathway for degrading cellular components including organelles and dysfunctional proteins. Some nonessential cellular components can be degraded via autophagy to synthesize critical components (Devenish and Prescott 2015; Olsvik et al. 2019). Autophagy occurs at low levels under normal conditions (Huang et al. 2015) and increases under adverse conditions such as nutrient deficiency, hypoxia, and oxidative stress (Onodera and Ohsumi 2005; Scherz-Shouval and Elazar 2011; Shpilka et al. 2015; Weidberg et al. 2011). Similarly, autophagy might be induced by the insufficient carbon metabolism due to the decreased rhamnose utilization.
To investigate that autophagy could be triggered by the mild carbon starvation, the autophagy in P. pastoris GS115/LacB and P. pastoris GS115m/LacB cells was monitored using autophagosomes staining. Intensive autophagy signals were detected in P. pastoris GS115m/LacB cells compared with P. pastoris GS115/LacB cells. Obviously, the carbon starvation arose from the decrease of rhamnose metabolism indeed caused autophagy in P. pastoris GS115m/LacB (Fig. 6). We assumed that P. pastoris GS115m/LacB cells recycled non-essential components to synthesize essential components for cell survival and reduce cell apoptosis via autophagy under carbon starvation.
Undetectable effect on cell apoptosis due to decreased LRA4 expression
Autophagy is interconnected with apoptosis because both of them might be triggered by same signals. Autophagy happened in P. pastoris GS115m/LacB, and apoptosis was thereby concerned. In order to understand whether apoptosis underwent in P. pastoris GS115m/LacB, apoptosis profiles in cells of P. pastoris GS115m/LacB and P. pastoris GS115/LacB was analyzed by flow cytometry using the Annexin V-FITC/PI apoptosis detection kit. Relatively low apoptosis level occurred in both kinds of cells, and no differences were observed in them (Fig. 7). It indicated that apoptosis did not obviously occur in cells of P. pastoris GS115m/LacB and P. pastoris GS115/LacB although the intensive autophagy occurred in P. pastoris GS115m/LacB. The results showed that the decreased rhamnose metabolism only led to a mild carbon source starvation, which was different from the carbon source starvation due to depletion of carbon source, and brought to a slight alteration of physiological state of P. pastoris GS115m/LacB instead of death such as apoptosis.
Low level of reactive oxygen species (ROS) in P. pastoris GS115m/LacB
ROS, which at a high level can induce apoptosis (Sullivan and Chandel 2014), was elevated in production when cells survived nutrient starvation such as inadequate supply of glucose (Wang et al. 2018b). Apoptosis did not occur in P. pastoris GS115m/LacB, indicating a low level of ROS in P. pastoris GS115m/LacB cells. The low levels of ROS in both kinds of cells were further confirmed by flow cytometry after DHR123 staining (Fig. 8).