Enzyme production yields and process productivity
The growth and enzyme production characteristics of the created recombinant strains were evaluated using bioreactor cultivations. GE side-activity of three commercial enzyme cocktails, namely Celluclast® 1.5 L (Novozymes, 27.47 U.L–1), DepolTM 740 L A (Biocatalysts, 10.70 U.L–1) and Depol TM 740 L B (Biocatalysts, 7.72 U.L–1) were tested to serve as a comparative baseline. The bioreactor cultivation for expression of GE returned a final volumetric activity yield of 238.17 U.L–1, and a final recombinant protein titre of 2778.01 mg.L–1 (Fig. 1a; Table 1), well in excess of previous attempts involving methanol induction of GE [7, 24]. Notably, the volumetric activity was well above the side activities of the commercial enzyme cocktails tested here (7.72—27.47 U.L–1). Bioreactor cultivation for CDH production returned a final mean volumetric activity of 329.49 U.L–1 and a final mean recombinant protein concentration of 1489.30 mg.L–1 (Figure 1b; Table 1). Although the protein concentration of secreted CDH reported here (1489.30 mg.L–1) is an improvement on previous attempts at methanol-induced expression, the volumetric activity and specific activity is lower than previously reported [21, 25–27]. Lowered specific activity has been observed in recombinant CDH production by P. pastoris due to a sub-stoichiometric occupation of catalytic sites within the FAD cofactor, as well as hyper-glycosylation [28]. Further, hyper-glycosylation may have affected the specific activity of both target enzymes, since the reported protein sizes (CDH: 127 kDa; GE: 78 kDa) are larger than the expected, in silico protein sizes (ExPASy: CDH: 88.46 kDa; GE: 81.29 kDa) [4, 29].
The general trends observed for the fermentations are as follows (Fig. 1a-b). During the batch phase, an initial lag phase of less than 6 hours was observed, corresponding to minimal volumetric activity, followed by an exponential growth phase, where linear increases in volumetric activity were observed until the end of the batch phase (between 20–22 hours). During the glycerol fed-batch stage, there was a brief exponential growth phase of approximately 6 hours, followed by an extended growth phase. During this continuous growth phase, the agitation of the bioreactor remained at the maximum (1000 rpm), and oxygen sparging was required throughout to maintain sufficient availability of dissolved oxygen (30 %) in the culture.
The maximum specific growth rate was determined during the batch phase under identical growth conditions (Table 1). GE- and CDH-expressing P. pastoris strains had similar μmax values of 0.15 h–1 and 0.16 h–1 respectively, within expected values reported for recombinant P. pastoris strains using glycerol (0.15—0.20 h–1) [30, 31]. Similarly, the biomass yields obtained (120.94, 136.47 gDCW.L–1; Table 1) were comparable to previous bioreactor cultivations of recombinant P. pastoris [18, 32].
A decrease in specific growth rate, modelled as a power regression (GE: 20979x–3.63;R2 = 0.9871; CDH: 885.76x–2.852; R2 = 0.9882), was observed during the fed-batch process (Fig. S1), during which the levels of glycerol remained below detectable HPLC levels. This is attributed to the fact that the amount of biomass in the fermentation vessel increased with time (Fig. S1), while the amount of glycerol fed into the bioreactor remained constant, effectively decreasing the ratio of available carbon source per gram of biomass. This growth rate decline has been suggested to be the concluding stage of an ideal growth rate time course for a number of secreted protein fermentations in P. pastoris [30, 33, 34].
Specific productivity describes the efficiency of the process by quantifying the amount of recombinant enzyme secreted per gram of biomass per hour. Maximum values of specific productivity (qp.max) of protein secretion were reached during the batch phase (1.22, 0.76 mg recombinant protein.biomass–1.h–1 for GE and CDH, respectively; Table 1). During the fed-batch stage, specific productivity remained constant at a lowered value close to the mean specific productivity value (qp.mean) (0.52 and 0.43 mg recombinant protein.biomass–1.h–1 for GE and CDH, respectively), resulting in GE and CDH accumulation throughout the fed-batch stage (Fig 1a-b). This has been observed in previous constant fed-batch cultivations of recombinant PGAP-P. pastoris [33, 35]. The productivity could be increased by manipulating the growth rate such that an extended growth phase at μmax is maintained before the end-phase decline in growth rate [31, 33, 34, 36]. This is achieved by an increasing (linear or exponential) glycerol feeding rate during the fed-batch stage. The optimal duration of the phase at which μmax is maintained differs between recombinant strains and should be optimised on a case-by-case basis.
Concentration of crude enzyme extracts by tangential flow ultrafiltration (TFU)
The heterologous proteins were secreted at moderate (CDH: 50.89 %) to high (GE: 93.61 %) percentages of the total extracellular protein, as determined by densitometry analyses (Fig. 1c). CDH and GE were, therefore, the major proteins present in the fermentation broth. While native, extracellular P. pastoris proteins were present in the fermentation broth (Fig. 1c), these were devoid of any contaminating enzymatic activity (as determined with the use of a negative control strain), which allows for simple and economical downstream processing, contributing to the affordability and accessibility of the process.
The harvested supernatant was concentrated using TFU (Table 2). The microfiltration process returned satisfactory volumetric activity yields (GE: 86.84 %, CDH: 86.54 %; Table 2), and the loss in activity units can be explained by the inherent loss of sample volume in the filtration apparatus itself. The total protein concentration in the enzyme extracts was increased 3.69-, 2.84-fold for GE and CDH, respectively. In terms of volumetric activity, GE and CDH were concentrated 4.95-, and 5.20-fold, respectively. The ultrafiltration process returned volumetric activity yields that were acceptable (49.57 and 50.00 %), but lower than expected [26, 37–39] despite acceptable integrity of membrane (permeate with minimal volumetric activity 0.00—2.95 UTotal.L–1). It is therefore hypothesised that the recombinant protein is being retained or degraded in the filtration apparatus itself, as proteins can adsorb to membranes, often causing permanent fouling [40] and the formation of a protein monolayer on the surface of the membrane [41]. Despite these shortcomings, the final concentrated enzyme products could be used directly on lignocellulose according to enzyme dosages established in previous studies [4, 42].