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Short Communication

Consolidated bioprocessing for sodium gluconate production from cellulose us-

Xiaolong Han, Guodong Liu, Yunjun Pan, Wenxia Song, Yinbo Qu

PII: S0960-8524(17)32161-2
DOI: https://doi.org/10.1016/j.biortech.2017.12.028
Reference: BITE 19283

To appear in: Bioresource Technology

Received Date: 31 October 2017

Please cite this article as: Han, X., Liu, G., Pan, Y., Song, W., Qu, Y., Consolidated bioprocessing for sodium
gluconate production from cellulose using Penicillium oxalicum, Bioresource Technology (2017), doi: https://
doi.org/10.1016/j.biortech.2017.12.028

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Consolidated bioprocessing for sodium gluconate production from cellulose using

Penicillium oxalicum

Xiaolong Hana,b, Guodong Liua, Yunjun Pana,c, Wenxia Songb, Yinbo Qua,c*

a
State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100,

China

b
School of Life Science, Qufu Normal University, Qufu, 273165, China

c
National Glycoengineering Research Center, Shandong University, Jinan, 250100,

China
* Corresponding author: Yinbo Qu

Tel. +86-531-88365954 E-mail: quyinbo@sdu.edu.cn

Abstract

The feasibility of consolidated bioprocessing for sodium gluconate production

from cellulose was studied. A recombinant strain named z19 was constructed from

Penicillium oxalicum wild-type strain 114-2 for simultaneous expression of glucose

oxidase and catalase from Aspergillus niger. While keeping a cellulolytic ability

similar with that of 114-2, z19 secreted certain amounts of glucose oxidase and

catalase. Fed-batch and two-stage temperature control strategy (0-120 h, 30C;

120-192 h, 45C) was utilized for sodium gluconate production from cellulose (filter

paper power), with 13.54 g/L of sodium gluconate obtained at the end of the

fermentation. The results provide an alternative route for producing sodium gluconate

from cellulose in a one-pot reaction.

1
Keywords

Penicillium oxalicum; Cellulose; Consolidated bioprocessing; Sodium gluconate

1 Introduction

Chemicals produced from lignocellulosic materials have become instrumental in

solving the current problems of energy and resource shortage and of environmental

protection (Menon & Rao, 2012). Like the traditional fermentation industry, which

mainly uses grains or tubers as raw materials, the biorefinery of lignocellulosic

materials involves the production of cellulase and hemicellulase, pretreatment and

saccharification of biomass and conversion of fermentable sugars into valuable

chemicals by microorganisms. Consolidated bioprocessing (CBP) is gaining

considerable attention because it facilitates the simultaneous integration of enzyme

production, saccharification and fermentation by one microorganism, thereby

simplifying the biological process (Lynd et al., 2005).

Sodium gluconate has a wide range of applications, such as a surface cleaning

agent for steel and glass, and as a high-efficiency retarder and superplasticizer in the

concrete industry (Ramachandran et al., 2006). Aspergillus niger is mainly applied to

sodium gluconate production through submerged fermentation with starch sugar as raw

material, because its glucose oxidase (GOD) can exclusively and efficiently oxidize

glucose into gluconic acid and H2O2 (Ramachandran et al., 2006). Moreover, H2O2 can

be quickly decomposed into water and oxygen by the catalase (CAT) in A. niger, which

protects the GOD activity from inhibition. That is, GOD and CAT are the only needed
2
enzymes for sodium gluconate production from glucose. The cost of starch sugar

accounts for a large proportion of the total cost of gluconate sodium production

(Ramachandran et al., 2006; Singh & Kumar, 2007), which is not preferable for this

bulk chemical. Thus, numerous studies have attempted to produce gluconate sodium

based on inexpensive lignocelluloses (Ikeda et al., 2006; Zhang et al., 2016a; Zhang et

al., 2016b).

In our previous work, we successfully produced sodium gluconate from the

hydrolysate of delignified corn cob residue through co-immobilized GOD and CAT

method (Han et al., 2018). To reduce the cost of enzymes and simplify the process, we

constructed a recombinant strain in Penicillium oxalicum that simultaneously

overexpressed GOD and CAT from A. niger, and attempted to produce sodium

gluconate through CBP in a one-pot reaction.

2 Materials and methods

2.1 Strains and culture conditions

The fungal strain P. oxalicum 114-2 (CGMCC 5302) producing a lignocellulolytic

enzyme system with diverse components (Liu et al., 2013) was used as the parental

strain. P. oxalicum 114-2 and all mutants were maintained in 30% (v/v) glycerinum

vials and inoculated on wheat bran extract slant. The culture temperature for P.

oxalicum 114-2 and all mutants was 30C, and green spores were generated after

inoculating 3-5 days.

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2.2 Generation of the GOD and CAT co-overexpression mutant

Genomic DNA of A. niger N593 (a kind gift from Adrian Tsang, Concordia

University, Canada) was used as the template to amplify the DNA sequences encoding

GOD (GenBank accession no. XP_001389862) and CAT (XP_001388621) as well as

their downstream sequences, using primers 1/2 and 3/4, respectively. The constitutive

promoter gpdA from Aspergillus nidulans was amplified using the plasmid pAN7-1 as

the template (Mullaney et al., 1985) using primers 5/6 or 5/7. The hygromycin B

resistance cassette hph was amplified with primers 8/9 from the plasmid pSilent-1

(Nakayashiki et al., 2005). The GOD and CAT overexpression cassettes were obtained

by double-joint PCR (Davidson et al., 2002) in the orders of hph-gpdA(p)-god and

hph-gpdA(p)-cat, with nest primers 10/11 or 10/12, respectively. The two cassettes

were gel-purified and transformed into the protoplast of P. oxalicum 114-2

simultaneously. The transformation was performed as previously described (Li et al.,

2015). All the primers are provided in supplementary data.

2.3 Enzyme assays and determination of sodium gluconate concentration

Filter paper enzyme (FPase), endoglucanase, cellobiohydrolase and β-glucosidase

activities were assayed as previously described (Han et al., 2017). GOD activity was

assayed using the method of Bankar et al. with modification as described previously

(Bankar et al., 2008; Han et al., 2018). CAT activity was assayed by a catalase detection

kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the

manufacturer's instructions.
4
 Sodium gluconate concentration was measured using a spectrophotometric method

(Han et al., 2018). Briefly, 500 μL of sample (diluted with distilled water) was mixed

with an equal volume of 0.1 M cupric sulfate solution, and heated in a boiling bath for

5 min, then cooled to room temperature immediately. The generated complex can be

quantified by measuring the absorbance at 700 nm for calculating the concentration of

sodium gluconate.

2.4 Sodium gluconate production

Spores collected from wheat bran extract slant were inoculated to 50 mL seed

medium in 300 mL shaking flasks. The seeds were cultured at 30°C and 200 rpm for

48 h. The production of sodium gluconate was carried out in 500 mL shaking flasks

filled with 100 mL fermentation medium, and 10 mL pre-cultured seeds were

inoculated. Flasks were shaken at 200 rpm. Fermentation temperature was set to 30C

from 0 to 120 h, and then raised to 45C after 120 h with 20 g/L filter paper powder

being added simultaneously. pH was controlled by feeding Na2CO3 frequently. The

fermentation was finished when gluconate sodium concentration in all the samples

plateaued.

The seed medium contained 10 g/L delignined corn cob residue from xylitol

production, 10 g/L wheat bran, 10 g/L peptone, 10 g/L glucose, 2 g/L (NH4)2SO4, 3 g/L

KH2PO4, and 0.5 g/L MgSO4·7H2O.

The fermentation medium contained 20 g/L delignined corn cob residue, 6 g/L

microcrystalline cellulose, 46.5 g/L wheat bran, 10 g/L soybean cake powder, 2 g/L
5
(NH4)2SO4, 2.79 g/L NaNO3, 1 g/L urea, 3 g/L KH2PO4, and 0.5 g/L MgSO4·7H2O.

The yield of sodium gluconate was calculated by:

(1)

where indicates sodium gluconate concentration (g/L) at the end of

reaction; represents the concentration of the added cellulose (filter

paper powder, g/L); 0.743 is the molar conversion factor from glucan to equivalent

sodium gluconate.

3 Results and discussion

3.1 Construction of a GOD and CAT co-overexpression recombinant strain

Commercial GOD and CAT preparations are usually produced by A. niger, so we

amplified the DNA sequences encoding these two enzymes from A. niger. First, we

tried to separately express GOD and CAT from A. niger in P. oxalicum A11Δ

(modified from a low-background host strain Δ15A, Hu et al., 2015) using the

promoter of amylase gene amy15A. The results of SDS-PAGE, enzyme assays and

bromocresol green-based qualitative assay of gluconic acid production (data not

shown) indicated that GOD and CAT from A. niger could be expressed in P. oxalicum

correctly. Then, we constructed two overexpression cassettes using the A. nidulans

gpdA promoter and the selectable marker gene hph (Fig. 1a). The two overexpression

cassettes, hph-gpdA(p)-god and hph-gpdA(p)-cat were used to transform the protoplasts

of P. oxalicum wild-type strain 114-2 simultaneously, and 37 transformants were

6
obtained after several rounds of transformation. The obtained transformants were

cultured in fermentation medium for 96 h, and detected for FPase, GOD and CAT

activities. The results indicated that most of the 37 transformants contained only one

of the two overexpression cassettes (Fig. 1b). One transformant, z19, showed the

production of both GOD and CAT. At 96 h, the FPase, GOD and CAT activities of z19

achieved 0.295 U/mL, 28.62 U/mL and 638.24 U/mL respectively.

3.2 Consolidated bioprocessing for gluconate sodium production

The feasibility of CBP for gluconate sodium production from cellulose by the

mutant z19 was evaluated. A two-stage temperature control strategy was employed. As

shown in Fig. 2a, the FPase activity of z19 at 120 h in the fermentation broth was 0.33

U/mL, similar to that of parental 114-2 strain. The endoglucanase and

cellobiohydrolase activity profiles of z19 and parent strain 114-2 were also similar,

suggesting that cellulase production was not affected by the expression of GOD and

CAT. The GOD and CAT activities of z19 at 120 h reached 37.21 U/mL and 741.32

U/mL, respectively. Compared with 114-2, a significant improvement in the GOD and

CAT activities was observed in z19. The temperature of the culture was raised to 45C

after 120 h, at which P. oxalicum could not grow, while cellulases, GOD and CAT

activities were retained (data not shown). The filter paper powder added at 120 h was

hydrolyzed by the cellulases secreted during the enzyme production stage. Then, the

obtained glucose in the fermentation broth of z19 was catalyzed into sodium

gluconate by GOD, where the concentration of sodium gluconate reached 13.54 g/L
7
after 72 h of conversion (Fig. 2b). In contrast, sodium gluconate was hard to detect in

the fermentation broth of parental strain 114-2 under the same conditions.

Using lignocellulose as a feedstock for sodium gluconate production can

substantially reduce the food consumption and the raw material cost (Zhang et al.,

2016b). Ikeda et al. obtained 80-100 g/L gluconic acid from waste paper hydrolysate

by A. niger in a turbine blade reactor (Ikeda et al., 2006). Gluconobacter oxydans also

showed excellent gluconic acid producing capacity using the hydrolysate of dry dilute

acid pre-treated and biodetoxified corn stover feedstock, and maximum sodium

gluconate was produced at the titer of 132.46 g/L (Zhang et al., 2016a). However,

commercial cellulases used in saccharification of lignocellulosic biomass are more

expensive than α-amylase and glucoamylase used in starch sugar production if

calculating the cost per mole of glucose produced. CBP eliminates the cost of

commercial cellulase preparation, and associates cellulase production with cellulosic

material saccharification in one pot (Lynd et al., 2005). In this study, “native

cellulolytic strategy” was selected because P. oxalicum secreted a balanced

lignocellulose-degrading enzyme system (Liu et al., 2013). The new enzyme cocktail

secreted by P. oxalicum z19 directly converted cellulose into sodium gluconate,

indicating that the engineering of P. oxalicum towards the CBP for sodium gluconate

production was successful.

However, the productivity of sodium gluconate (0.071 g/L/h at 192 h) is expected

to be further raised by strain improvement and process optimization. One of the main
8
problems that hampered the production of gluconate sodium from cellulose was the

limited capacity of the mutant z19 to secrete cellulases, which discouraged the

hydrolysis of cellulose to fermentable sugars (the yield of sodium gluconate on the

added filter paper power was 50.3%). With the knowledge of the transcriptional

regulatory network for cellulase production in P. oxalicum, genetic manipulation of

the transcription factors in cellulase expression such as ClrB, CreA, XlnR, and AmyR

has been shown to significantly improve the level of cellulase production (Li et al.,

2015), which is expected to be integrated with the co-overexpression of GOD and

CAT for efficient conversion of cellulose to sodium gluconate.

Another problem is that D-glucono-1,5-lactone as the real product of GOD is a

strong inhibitor of β-glucosidases (Peng et al., 2017), which might explain the

reduced β-glucosidase in z19 relative to parental stain (Fig. 2a). Considering that

β-glucosidase is a rate-limiting enzyme in cellulose hydrolysis, saccharification of

cellulose could be inhibited by the simultaneous conversion of glucose to

D-glucono-1,5-lactone, particularly at high concentrations. Supplementation of an

aldonolactonase which can catalyze the hydrolysis of D-glucono-1,5-lactone to

D-gluconic acid improved β-glucosidase activity and glucose yield during

lignocellulose saccharification (Peng et al., 2017). Therefore, overexpression of

aldonolactonase would be a target for the construction of CBP-microorganisms for

sodium gluconate production. In addition, over-expression of β-glucosidases using the

inducible promoter from bgl2 gene improved the β-glucosidase activity of P. oxalicum
9
by 65-folds (Yao et al., 2016), which is also expected to counteract the inhibitory

effect on the activity of β-glucosidase.

Conclusion

Genetically engineered P. oxalicum z19 successfully expressed the GOD and CAT

from A. niger. No significant difference was detected in overall cellulase activities

between z19 and the parental strain 114-2. Fed-batch and two-stage temperature

control strategy was applied to the CBP for sodium gluconate production, and 13.54

g/L of sodium gluconate was obtained in a one-pot reaction. The simplified

manufacture process and the ability to use low-cost cellulosic materials will make the

CBP strategy an economically attractive approach for sodium gluconate production.

Supplementary data

Supplementary data includes Table S1.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation

of China (Grant Nos. 31370086, 31670079 and 31200065) and Shandong Natural

Science Foundation (ZR2017BC088).

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Figure Captions

Fig. 1. Generation of a GOD and CAT co-overexpression recombinant strain from P.

oxalicum wild-type strain 114-2. (a) Construction of the hph-gpdA(p)-god cassette and

the hph-gpdA(p)-cat cassette. (b) Extracellular FPase, GOD and CAT activities of

transformants after 96 h of cultivation in fermentation medium.

Fig. 2. Production of sodium gluconate from cellulose by two-stage temperature

control strategy. (a) Enzyme production during 0-120 h at 30C; (b) Sodium gluconate

production during 120-192 h at 45C with feeding 20 g/L filter paper powder. The

values shown are the mean of three biological replicates and the error bars indicate

standard deviations from the mean values.

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14
15
Highlights

 Penicillium oxalicum z19 co-overexpressed GOD and CAT from Aspergillus

niger.

 Two-stage temperature control strategy was applied for sodium gluconate

production.

 13.54 g/L sodium gluconate was obtained from cellulose in one-pot reaction.

16
Graphical abstract

17