Physiological optimization of secreted protein production by Aspergillus niger

Physiological optimization of secreted protein production by Aspergillus niger Donald A. MacKenzie, Laurent C.G. Gendron, David J. Jeenes and David B. Archer Department of Genetics and Microbiology, Agricultural and Food Research Council, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich, UK

Physiological factors affecting hen eggwhite lysozyme and native glucoamylase production by Aspergillus niger have been examined in batch culture. Expression of the genes encoding both proteins was controlled by theglucoamylase promoter. In standard expression medium (ACMS/N/P), secreted lysozyme yields were found to be maximal at 20-25°C (8-10 mg l - 1) and markedly reduced at 30-37 ~ C (3-5 mg l - 1). Production of lysozyme exhibited similar induction or repression profiles to that of endogenous glucoamylase such that secreted lysozyme yields could be ordered with respect to growth on the following carbon sources: soluble starch > maltose > glucose > > xylose. Significantly higher yields of up to 30-60 mg l - 1 were obtained in a richer medium containing soya milk, although in contrast to growth in ACMS/N/P, the highest levels of secreted lysozyme were achieved at 37°C. This improvement is attributed partly to an increase in culture biomass concentration and to a reduction in medium acidification. Growth in this medium produced a markedly different pellet morphology.

Keywords: Heterologous expression;Aspergillus niger; lysozyme; glucoamylase; physiological factors

Introduction Filamentous fungi are attractive hosts for the production of foreign proteins because of their high secretory capacity. ~,2 In only a few cases, however, have yields of heterologous protein matched the gram per liter levels obtained for certain homologous enzymes. 3-5 Yield improvements have been achieved by changing the secretion signals attached to the target protein, by altering the growth medium, and by mutating the host to produce a strain with a higher secretory capacity. 4 A better understanding of the secretory pathway and the physiological factors affecting protein secretion should also help to increase product yield. Although the physiology of filamentous fungal growth has been studied extensively for the formation of homologous products such as organic acids 6 and enzymes, 7,8 there are fewer reports detailing which parameters are critical for heterologous protein secretion in these organisms. 9,1° Aspergillus niger has been used successfully to secrete hen eggwhite lysozyme ( H E W L ) at levels of about 10 mg 1 - 1 . 11.12 , 1In these studies, H E W L secretion was improved

Address reprint requests to Dr. MacKenzie at the Department of Genetics and Microbiology, Agricultural and Food Research Council, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK Received 10 February 1993; revised 14 September 1993

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by optimizing the levels of ammonium chloride and sodium phosphate buffer in the medium and the size of spore inoculum used. In this paper, we have investigated the effects of growth temperature and carbon source on H E W L production and report an improvement in yield when a complex medium containing soya milk is used.

Materials and methods Strains a n d culture conditions A. niger lysC25 and Lll are two independent transformants of strain AB4.113 containing the full-length HEWL cDNA under control of the A. niger var. awamori glucoamylase promoter. 11 Standard lysozyme production medium was ACMS/N/P, containing 1% (w/v) soluble starch (Difco) and 50 mM sodium phosphate buffer.ll Where indicated, alternative carbon sources were added to the appropriate concentration from autoclaved 10% (w/v) stocks of D-glucose (BDH Analar), maltose (Sigma), or D-xylose (Sigma). SCM and soya milk medium (SMM) were essentially those described by Ward et al. 14 except that arginine, streptomycin, and antifoam were omitted. In addition, two forms of SCM were prepared, one containing 50 g 1- 1 maltose and the other 150 g 1- 1. White Wave Soya Milk (Unisoy Milk n by-products Ltd., Stockport, UK) was used in SMM (70 ml 1-1). Fungal spore inocula were prepared in sterile 0.05% (v/v) Tween 20 from potato dextrose agar (PDA; Difco) slopes and added to media to a final concentration of 3 x 105 conidia ml - 1. Cultures (100 ml) in 250-ml conical flasks were incubated at 25°C unless otherwise stated and shaken at 150 rev min - 1

© 1994Butterworth-Heinemann

Lysozyme production by A. niger: D. A. MacKenzie et al.

Analytical methods Lysozyme activity in culture supernatants was determined by measuring the rate of lysis of a Micrococcus !ysodeikticus(Sigma) 0.4 mg m1-1 cell suspension. 11 Glucoamylase (GA/Vl) activity was assayed by incubating culture supernatant (5-10 ixl)with 1 ml 1% (w/v) glucose-free starch (Sigma) for 10 rain at 25°C in 50 mM sodium acetate buffer, pH 4.5, and measuring the amount of glucose released by incubating 0.5 ml of the reaction mixture with 1 ml of glucose oxidase reagent, containing 300 Ixg ml - 1 glucose oxidase, 30 Ixg m1-1 horseradish peroxidase, and 100 ~g m1-1 O-dianisidine-dihydrochloride (all Sigma) in 0.3 M Tris, 0.32 M sodium phosphate, 40% (v/v) glycerol buffer, pH 7.0, for 5 min at room temperature. The absorbance at 525 nm was measured and compared with that for a series of glucose standards. Appropriate controls were assayed to determine the concentration of glucose in the culture medium at each sampling time, and this value was subtracted to give the net amount of glucose released by GAM. Glucoamylase activitywas expressed in mg I - 1 of culture medium, where 1 mg GAM is equivalent to 3.37 mg glucose released from starch per minute under the assay conditions used. Cell dry weight was determined as described previously. 12

Photographic analysis Samples of culture (0.5 ml) were collected at the times indicated, and the mycelium was washed three times in 20 ml of either distilled water or glycerol, or suspended directly in fixative [9% (w/v) acidic formaldehyde in 50% (v/v) ethanol]. 15 Fungal pellets were then photographed with back lighting using a Wild Heerbrugg stereo microscope and Photoautomat MPS45 exposure meter.

Results and discussion

Effect of growth temperature on secreted H E W L production When A. niger transformant L l l was grown in ACMS/N/P at various temperatures, secreted lysozyme levels were highest at 20-25°C (Figure la). The time course of H E W L production was very similar to that for another A. niger transformant, lysC25.11 A characteristic peak was observed, followed by a drop in activity likely to be caused by neutral proteases released on autolysis. 16 Lysozyme activity in the medium could be detected earlier for cultures incubated at 30-37°C as a consequence of faster growth at these temperatures. This was reflected by a more rapid acidification of the medium (Figure lb). However, peak H E W L levels at the higher temperatures reached only 30-50% of the maximum values obtained at 20-25°C, suggesting that protease activity, perhaps from more rapid autolysis, was a greater problem. Indeed, production of various recombinant proteins, including several secreted ones, appears to be favored at temperatures lower than those optimal for growth in bacteria, 17 yeast, 18 and filamentous fungi. 19 This may be due to an enhancement of protein folding and/or passage through the secretory pathway at lower temperatures in addition to the postulated reduction in protease activity. In all subsequent experiments, cultures were incubated at 25°C unless otherwise stated.

Effect of various carbon sources on secreted H E W L production Since expression of the H E W L eDNA was under the control of the glucoamylase promoter in transformants L l l and lysC25, it was necessary to check whether H E W L produc-

12

10 8

2

6 4 2 I

e

b

5

==i o

o

1

2

3

4 5 Time (day=)

6

7

8

Figure 1 (a) Secreted lysozyme production and (b) medium pH profile forA. niger L11 grown in ACMS/N/P at 20°C (1~), 25°C ( l ) , 30oc (Q)), and 37°C (0)

tion displayed the same carbon source regulation as that of native glucoamylase. 2° From Table 1 it is seen that when using single carbon sources, soluble starch was the strongest inducer of both lysozyme and glucoamylase production, whereas xylose was a strong repressor. This agrees well with previous reports on GAM production inA. niger.2° Growth on maltose gave lysozyme levels about 50% that obtained with starch, although glucoamylase is normally expressed maximally in starch- or maltose-containing media. 20 In our strain, however, maltose, at least at 1% (w/v), appeared to be a less effective inducer of GAM (Table/).Yields of H E W L and GAM with glucose as carbon source were similar to those from maltose-grown cultures. This is in marked contrast to some strains of A. niger21 and otherAspergillus species 22 where glucoamylase synthesis is subject to catabolite repression by glucose. However, A. niger strains less sensitive to glucose repression do exist, 2° including many selected as high GAM producers for commercial use, 7 and our strain falls into this group. The effect of mixing carbon sources was also examined. As reported previously for glucoamylase production, 2° a combination of starch and glucose was as effective as starch alone in inducing both H E W L and GAM (Table 1). Similarly, the repression caused by xylose could be overcome by the addition of maltose. One major exception, however, was the induction observed for both lysozyme and glucoamylase when xylose was mixed with starch. In previous studies using 15% (w/v) of each carbon source, 2° xylose strongly

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Papers repressed glucoamylase production in the presence of starch when assayed after 4 days' growth at 37°C (when sufficient xylose for repression may still have been present in the culture medium). In the present study, we have evidence that at lower concentrations ofxylose (up to 1%, w/v), induction of both lysozyme and glucoamylase can occur when the w/v ratio of xylose to starch is less than one, and that this is delayed by about 2 days when the ratio is equal to or greater than one (data not shown). This implies that above a certain threshold xylose acts as a repressor in the presence of starch, but that this sugar is preferentially metabolized byA. niger and induction by starch can then proceed. With all sugars tested, when the total carbohydrate concentration in the medium was 2% (w/v) or above, culture mixing became extremely difficult because of the high biomass concentrations attained. Under these conditions, HEWL yields were markedly reduced, except in the case of the 1% (w/v) starch/l% (w/v) glucose combination (Table 1). The use of starch/glucose mixtures to feed productive high-cell-density cultures may therefore be investigated in future bioreactor studies.

60

~

a

20

Effects of growth in soya milk medium on secreted HEWL yieM and culture morphology Soybean products have been added to fungal growth media as sources of organic nitrogen.] In particular, the addition of soya milk has improved secreted bovine chymosin production by A. niger vat. awamori.4,14 Increased yields of 20-30 mg 1- 1 of secreted lysozyme were also obtained when A. niger transformants were grown in SMM at 20-30°C (Figure 2a). Part of this improvement may be attributed to the higher carbon source concentration in SMM compared with ACMS/N/P. This led to higher biomass concentrations of 10-20 g dry wt 1- 1 in SMM compared with 4-5 g dry wt 1-1 in ACMS/N/P. During incubation in SMM at 25°C, growth, as determined by biomass yield (data not shown) or medium acidification (Figure 2b), continued for up to 5-6 weeks. When culture productivity was determined by measuring H E W L levels and cell dry weights, there was very

Table 1 Effect of carbon source on secreted HEWL and glucoamylase production in A. niger lysC25

Carbon source Starch Maltose Glucose Xylose Starch + glucose Starch + xylose Maltose + ×ylose

Concentration (% w/v)

HEWL (mg I-1) a

GAM (mg I-1) a

1.0 1.0 1.0 1.0 0.5 b 1.0 b 0.5 b 1.0 b 0.5 b 1.0 b

8.0 4.5 4.0 0.2 7.2 10.4 5.2 5.0 2.5 4.7

62.9 48.3 40.6 1.5 ND 65.3 ND 51.9 ND 50.8

apeak lysozyme and glucoamylase levels for growth at 25°C in ACM/ N/P plus the appropriate carbon source. Each value isthe average of two determinations b Each sugar ND, Not determined

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0

0

7

14

21

28

35

42

49

56

Time(days) Figure 2 (a) Secreted lysozyme production and (b) medium pH profile forA. niger L l l grown in SMM at 20°C (EZ]), 25°C (11), 30oC (O), and 37°C (Q) and in SMM minus tri-sodium citrate at 25°C (A)

little difference in the peak values for both ACMS/N/P and SMM (3.83 and 3.37 mg HEWL g - ~cell dryweight, respectively). However, biomass concentration cannot be the only factor. As discussed in the previous section, biomass yields increased when ACMS/N/P was supplemented with extra starch, but H E W L levels were reduced. In addition, growth at 25°C in modified SCM (see Materials and methods), another medium containing 15% (w/v) maltose, produced approximately the same amount of fungal biomass but only 50% the yield of secreted H E W L compared with growth in SMM (data not shown). This increased HEWL yield in SMM may therefore be due to the higher buffering capacity of citrate present in this medium, which results in a more gradual reduction in pH (Figure 2b) and an extended period of growth, as determined by increase in cell dry weight. This can be contrasted with the relatively rapid pH drop found with growth in ACMS/N/P (Figure lb) and SCM (data not shown). Although lysozyme is not sensitive to extracellular acid proteases, 16 faster growth and medium acidification may lead to more rapid autolysis, releasing intracellular proteases which can then degrade HEWL. The slow pH drop seen in SMM may prevent the onset of autolysis and lead to the observed increased yield of lysozyme. This is supported by the finding that in SMM lacking citrate, the pH drop was much more rapid and lysozyme yield was reduced (Figure 2a and b). Variation in lysozyrne levels was also observed

Lysozyme production by A. niger: D. A. MacKenzie et al. when cultures were grown in S M M over a range of temperatures (Figure 2a). In contrast to growth in ACMS/N/P, 37°C was found to give the best yields of about 60 mg 1 - 1 Interestingly, growth at this t e m p e r a t u r e also produced the smallest p H drop (Figure 2b), further evidence that m e d i u m acidification is a critical factor in final product yield. Culture morphology of A. niger transformants was markedly different when grown in SMM. A p a r t from the very early stages of growth, A. niger L11 grew in ACMS/N/P as compact pellets 1.5-3.0 m m in diameter (Figure 3a). Their appearance was unchanged by washing with distilled water or glycerol, or by fixation. W h e n grown in SMM, A. niger L l l pellets, although about the same size as those from A C M S / N / P cultures, were much less compact structures, and their a p p e a r a n c e d e p e n d e d on the method of washing. Pellets washed with glycerol (Figure 3b) retained a compact center but displayed a surrounding extended fringe of hyphae similar to those described by Cox and Thomas. 15 Those washed with water a p p e a r e d even more " o p e n " in nature (Figure 3c). However, when SMM-grown pellets were fixed, these structures completely collapsed in on themselves, suggesting that they were much more fragile than those grown in ACMS/N/P (data not shown). In older cultures, from about 10 days onwards, the pellets became much smaller and more compact and were associated with a more dispersed collection of hyphal fragments (Figure 3d). This morphology did not change over the following 6 weeks' incubation. T h e bulk of protein secretion from filamentous fungi is believed to occur at the tips of growing hyphae, 23 and factors that increase the degree of hyphal branching and therefore the n u m b e r of active tips may improve product yield. It was not possible to measure the degree of hyphal branching in these cultures, but the apparent " o p e n n e s s " and increased fragility of SMM-grown pellets may have contributed to the increased yield of secreted lysozyme.

Conclusions F r o m this study of the physiology of lysozyme and glucoamylase production by A. niger, several parameters important in maximizing product yield have been identified. Although the majority of these factors have their effect at the level of general cell growth, some, such as the choice of carbon source, are m o r e specific to the glucoamylase prom o t e r used for gene expression. A combination of optimizing growth temperature and m e d i u m composition had the greatest effect on lysozyme levels. Increased biomass yield and the greater buffering capacity of S M M c o m p a r e d to ACMS/N/P m e d i u m were major factors in this improvement, but other questions have yet to be addressed. For example, the effects of controlling parameters such as pH, dissolved oxygen, agitation, and shear stress can be examined more readily using bioreactor technology. The data presented in this paper should help set the ground rules for improving heterologous protein production in these organisms.

Acknowledgements T h e authors wish to thank Julia Bracewell for technical assistance and Dr. Mary Parker and Jane Scarll for help in photographing fungal pellets.

References 1 2 3 4

5 6 7 8 9

10 11 12 Figure 3 Pellet morphology of A. niger L l l grown at 25°C in (a) ACMS/N/P for 3 days, (b),(c) SMM for 3 days, and (d) SMM for 11 days. Pellets were washed with water in (a), (c), and (d) and with glycerol in (b). Magnification is x 8 in (a), (b), and (c) and x 16 in (d)

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