Production of d-Xylonate from Corn Cob Hydrolysate by a

Research Article pubs.acs.org/journal/ascecg

Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Production of D-Xylonate from Corn Cob Hydrolysate by a Metabolically Engineered Escherichia coli Strain Yipeng Zhang,† Shiting Guo,† Yuxian Wang,† Xiao Liang,† Ping Xu,‡ Chao Gao,† and Cuiqing Ma*,† †

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State Key Laboratory of Microbial Technology & Shenzhen Research Institute, Shandong University, No. 72 Binhai Road, Qingdao, 266237, P. R. China ‡ State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China S Supporting Information *

ABSTRACT: Corn cob is the secondary agricultural residue that can be easily hydrolyzed into hydrolysate with abundant D-xylose. Besides D-xylose, D-glucose, and L-arabinose are also utilizable sugars in corn cob hydrolysate. Here, we established a coutilization system through which recombinant Escherichia coli can use D-glucose and L-arabinose supporting its growth and convert D-xylose in corn cob hydrolysate into D-xylonate. First, biosynthetic pathway of D-xylonate was overexpressed and two xylonate dehydratases were knocked out in E. coli W3110 to enhance D-xylonate production from D-xylose. Then, the genes responsible for acetate, ethanol, and lactate synthesis were knocked out to decrease these byproducts production during the growth of the recombinant strain. Three successive strategies were employed to enhance the D-xylonate productionusing lactose as the inducer, eliminating carbon catabolite repression, and inactivating the lactose degradation. Finally, 108.2 g/L D-xylonate was produced with a yield of 1.09 g of D-xylonate/g of D-xylose using D-xylose as the substrate and D-glucose as the carbon source through fed-batch fermentation. When corn cob hydrolysate was used, 91.2 g/L D-xylonate was produced with a specific productivity of 1.52 g/[L·h]. This study is valuable not only for producing D-xylonate but also for providing a coutilization system to obtain other important chemicals from corn cob hydrolysate. KEYWORDS: Biomass, Biotransformation, Coutilization, Escherichia coli, D-Xylonate

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1).17 The highest titers of D-xylonate using strains of Gluconobacter, Pseudomonas, and Enterobacter were 109, 162, and 190 g/L, respectively.20−22 Genetically engineered bacteria and yeast can also be used in 23−28 D-xylonate production. For example, xylonate production has been achieved with the recombinant E. coli.19,26 However, the concentration and productivity of the reported xylonateproducing recombinant E. coli are insufficient for industrial production. Until now, the reported recombinant E. coli can produce D-xylonate at a concentration of 39.2 g/L.26 Extra Dglucose at a concentration of 10 g/L was needed to support the growth of the recombinant strain.26 Corn cob hydrolysate contains D-xylose, D-glucose, and L-arabinose as its major utilizable sugars. E. coli can efficiently metabolize D-xylose, Dglucose, and L-arabinose for the production of various chemicals.29−31 Additionally, E. coli has clear genetic background and multiple genetic engineering tools.32 Various derivatives of D-xylonate, such as 3,4-dihydroxybutyrate and 1,2,4-butantriol, can be easily acquired through introducing specific pathways into D-xylonate producing recombinant E.

INTRODUCTION Corn cob is the secondary agricultural residue in the world;1 more than 70 million metric tons of corn cob was produced by USA and China in 2008.2,3 It is rich in hemicellulose and can easily be hydrolyzed to obtain corn cob hydrolysate with Dxylose, D-glucose, and L-arabinose as its utilizable sugars.4,5 However, corn cob hydrolysate has not been effectively utilized recently. In China, corn cob hydrolysate is mainly used to prepare D-xylose, which is used to produce xylitol through chemical reduction.6−8 The annual production of xylitol is expected to be about 2.4 × 105 tons by 2020.9,10 With the increasing yield of maize,11 the corn cob production has exceeded the demands of the xylitol industry and is becoming an environmental pollutant due to the lack of its effective utilization.12 D-Xylonate is a five-carbon organic acid that can be used as a chelator, dispersant, and precursor for various platform chemicals, such as 3,4-dihydroxybutanal, 3,4-dihydroxybutyrate, 1,2,4-butantriol, and 1,4-butanediol.13−18 Because of its potential for industrial production and versatile usage, Dxylonate has been named one of the top 30 most desirable chemicals by the US Department of Energy.19 Many species of bacteria such as Gluconobacter, Pseudomonas, Enterobacter, and related genera can produce D-xylonate from D-xylose (Table © XXXX American Chemical Society

Received: September 20, 2018 Revised: November 23, 2018 Published: December 18, 2018 A

DOI: 10.1021/acssuschemeng.8b04839 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Comparison of Xylonate Production by Different Microorganisms strain Gluconobacter oxydans Pseudomonas fragi Enterobacter cloacae Saccharomyces cerevisiae S. cerevisiae Kluyveromyces lactis Pichia kudriavzevii E. coli EWX4 E. coli BL21 ΔxylAB/pA-xdhxylCa E. coli XGL4 E. coli XGL4

method and substrate

pH

concentration (g/L)

natural producer batch; D-xylose 5.5 batch; D-xylose 6.5 batch; D-xylose 6.5 engineered strain batch; D-glucose and D-xylose 5.5 batch; D-glucose and D-xylose 5.5 batch; D-glucose and D-xylose 5.5 fed-batch; D-glucose and D-xylose 5.5 batch; D-glucose and D-xylose 7.0 batch; glucose and xylose 7.0 fed-batch; D-glucose and D-xylose 7.0 fed-batch; corn cob hydrolysate 7.0

yield (g/g)

productivity (g/[L·h])

ref

109 162 190

1.1 1.06 1.05

2.5 1.4 1.6

20 21 22

3.8 43 19 171 39.2 27.3 108.2 91.2

0.36 0.8 0.60 1.0 0.98 0.97 1.09 1.05

0.03 0.44 0.16 1.4 1.09 1.8 1.8 1.52

23 24 16 25 26 19 this study this study

a

The configuration of xylonate produced by this strain was not reported and xylonate produced by other strains was D-xylonate.

Figure 1. Metabolic engineering strategies for efficient production of D-xylonate from corn cob hydrolysate using E. coli W3110 as host strain. The xylose dehydrogenase (xylB) and xylonolactonase (xylC) were heterologous expressed in E. coli W3110 to catalyze D-xylose to D-xylonate. Other genes and enzymes: XylE, xylose transporter; XylFGH, xylose ABC transporter; PTS, phosphotransferase system; GalP, galactose permease; AraE, arabinose transporter; AraFGH, arabinose ABC transporter; LacY, lactose permease; EMP, Embden-Meyerhof-Parnas pathway; HMP, Hexose Monophosphate pathway; xylA, xylose isomerase; yjhG and yagF, xylonate dehydratases; glk, glucose kinase; pgi, glucose-6-phosphate isomerase; araA, L-arabinose isomerase; araB, ribulokinase; araD, L-ribulose-5-phosphate 4-epimerase; lacZ, β-galactosidase; ldhA, lactate dehydrogenase; adhE, ethanol dehydrogenase; poxB, pyruvate oxidase; ackA, acetate kinase. Red crosses indicated that the genes were inactivated in this study. Brown and green typefaces indicated that the genes (xylB and xylC) were heterologous and came from Caulobacter crescentus.

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coli.13,14 Thus, we also used E. coli as the host strain and intended to establish a method for the efficient production of D-xylonate from corn cob hydrolysate in this work. Wild type E. coli W3110 cannot synthesize D-xylonate from D-xylose but can utilize D-xylose, D-glucose or L-arabinose as its sole carbon source. In this study, xylose dehydrogenase (xylB) and xylonolactonase (xylC) from Caulobacter crescentus were overexpressed and genes related to D-xylonate catabolism and byproducts production were knocked out. Lactose was used instead of IPTG for the induction of xylBC expression. The ptsG and lacZ were knocked out to increase the induction efficiency of lactose (Figure 1). Finally, high production of Dxylonate from corn cob hydrolysate was achieved through fedbatch fermentation using the recombinant E. coli strain constructed in this work.

MATERIALS AND METHODS

Chemicals and Enzymes. Corn cob hydrolysate was a kind gift from Shandong Futase Co., Ltd. (Heze, Shandong, China). Briefly, corn cob was mixed with 2.0% (v/v) sulfuric acid and pretreated at 120 °C for 2 h. The liquid fraction was separated by filtration, decolored by activated carbon at 65 °C for two times and then deionized by anion-exchange resin and cation-exchange resin. The concentrated hydrolysate contained 118.5 g/L D-xylose, 11.5 g/L Dglucose, 11.8 g/L L-arabinose, 1.4 g/L formate, 0.83 g/L ethanol, 0.34 g/L acetate, and 13.5 ppm furfural. D-Xylose, D-glucose, and Larabinose account for 79.7%, 8.2%, and 8.5% of its total sugar content. D-Xylose (99%) was purchased from Shandong Xitang Biotechnology, Co., Ltd. (Jinan, Shandong, China). D-Xylonate and L-arabinose were purchased from Sigma (Louis, Missouri, USA). Restriction enzymes were purchased from Thermo Fisher (Waltham, Massachusetts, USA). Polymerase chain reaction (PCR) primers were provided by Beijing Qingke Xinye Biotechnology, Co., Ltd. (Qingdao, Shandong, China). T4 DNA ligase and FastPfu DNA polymerase were purchased from Thermo Fisher (Waltham, Massachusetts, USA) B


Research Article

ACS Sustainable Chemistry & Engineering

primers ΔxylA-F1/ΔxylA-R1 were used to directly amplify xylA mutant fragment from xylA::kan mutant strain which was purchased from The Coli Genetic Stock Center.36 The PCR products had approximately 300 bp up and down homologous arms of xylA and kanamycin resistance cassette. The ackA, adhE, poxB, ptsG, and yjhG mutant fragments were also obtained by this method. (2) Primers ΔyagF-F1/ΔyagF-R1, ΔyagF-F2/ΔyagF-R2, and ΔyagF-F3/ΔyagFR3, were used to amplify approximately 300 bp up homologous arm of yagF, kanamycin resistance cassette, and 300 bp down homologous arm of yagF, respectively. Three fragments were recombined together by recombination PCR to form yagF mutant fragment. The ldhA and lacZ mutant fragments were also obtained by this method. Plasmid pKD4 was used as template for the PCR amplification of kanamycin resistance cassette for gene disruption.35 Analysis of Product Inhibition of D-Xylonate. E. coli XG2 was inoculated into 500-ml shake flask containing 100 mL of LB and cultured at 30 °C with shaking at 180 rpm for 1 h. Then cells of E. coli XG2 were collected by centrifugation at 6000 rpm for 10 min, washed twice with 100 mM phosphate buffer (pH 7.4). The influence of the D-xylonate concentration on D-xylose biotransformation was determined in 100 mM phosphate buffer containing whole cells of E. coli XG2 (OD600nm = 20). The reaction was initiated by adding the whole cells, D-xylose (40 g/L), and D-xylonate (0, 20, 40, 60, and 80 g/L), followed by incubation at 37 °C with shaking at 120 rpm. The concentrations of D-xylose and D-xylonate in the reaction mixtures were quantitatively analyzed by high-performance liquid chromatography (HPLC). Batch and Fed-Batch Fermentations. Batch fermentation was conducted in a 1.4-L bioreactor (Infors AG, Bottmingen, Switzerland) with an operating volume of 1 L. Fed-batch fermentation was conducted in a 5-L bioreactor (B. Braun Biotech International GmbH, Germany) with an initial broth volume of 4 L. The fermentation using D-xylose as substrate and D-glucose as carbon source was conducted in M9 fermentation medium containing 10 g/L D-glucose and 40 g/L Dxylose. Alternatively, the corn cob hydrolysate was fed into the fermentation broth to make the D-glucose and L -arabinose concentration at about 4 g/L and D-xylose concentration at about 40 g/L. Batch fermentation and fed-batch fermentation were both performed at 37 °C with an aeration rate of 1 vvm and an agitation speed of 600 rpm. Samples were withdrawn periodically to determine the cell density, concentrations of D-glucose, D-xylose, L-arabinose, Dxylonate, and byproducts. The pH was maintained at 7.0 by automatic addition of 14% NH4OH. Analytical Methods. The culture media were withdrawn periodically and centrifuged at 13000 g for 10 min and the supernatants were used for detection. The concentration of D-glucose was measured enzymatically by using a bioanalyzer (SBA-40D, Shandong Academy of Sciences, China) after appropriate dilution. The concentration of D-xylose and L-arabinose were analyzed by using HPLC (Agilent 1100 series) equipped with Aminex HPX-87P column (300 × 7.8 mm; Bio-Rad, USA) and RID detector as described by Redondo et al.37 The mobile phase was pure water, the flow rate was 0.7 mL/min, and the column temperature was 75 °C. D-Xylonate was detected by HPLC equipped with Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, USA) and DAD detector at a wavelength of 210 nm. Other organic acids were detected by HPLC equipped with Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, USA) and RID detector. The mobile phase was 10 mM H2SO4, the flow rate was 0.4 mL/min, and the column temperature was 55 °C.38

and TransGen Biotech (Beijing, China), respectively. All other chemicals were of analytical grade and commercially available. Bacterial Strains, Plasmids, and Growth Conditions. The strains and plasmids used in this study are listed in Table 2. E. coli

Table 2. Bacterial Strains and Plasmids Used in This Work characteristica

name E. coli DH5α E. coli W3110 E. coli X1 E. coli X2 E. coli X3 E. coli X4 E. coli XG1 E. coli XG2 E. coli XGL1 E. coli XGL2 E. coli XGL3 E. coli XGL4 pETPtac pET28axylBC pETPtacxylBC pTKRED pCP20 pKD4

strain F−, φ80d lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44λ−, thi−1, gyrA96, relA1 F−, λ−, IN(rrnD-rrnE)1, rph−1 E. E. E. E. E. E. E.

coli coli coli coli coli coli coli

W3110ΔxylA X1ΔyjhGΔyagF X1 containing pETPtac-xylBC X2 containing pETPtac-xylBC X2ΔldhAΔackAΔpoxBΔadhE XG1 containing pETPtac-xylBC XG1ΔptsG

reference or source Novagen CGSC4474 this this this this this this this

study study study study study study study

E. coli XGL1 containing pETPtac-xylBC

this study

E. coli XGL1ΔlacZ

this study

E. coli XGL3 containing pETPtac-xylBC

this study

plasmid expression vector in which the promoter PT7 of pET28a was replaced by Ptac; Kanr cloning vector with genes xylB and xylC encoding xylose dehydrogenase and xylonolactonase from C. crescentus; Kanr expression vector with genes xylB and xylC encoding xylose dehydrogenase and xylonolactonase from C. crescentus; Kanr plasmid expressing λRed recombinase genes for gene knock out; Sper plasmid expressing Flp recombinase to remove kanamycin resistance cassette during gene knock out; Cmr template for amplification of the kanamycin resistance cassette; Kanr

34 b

this study lab stock CGSC14177 CGSC7632

a Kanr, kanamycin resistant; Sper, spectinomycin resistant; Cmr, chloramphenicol resistant. bThe vector included two genes xylB and xylC, which were synthesized by Tongyong Biosystem Co., Ltd. (Chuzhou, Anhui, China).

DH5α was used for general cloning purposes and propagated in Luria−Bertani (LB) medium at 37 °C under aeration. The pTKRED and pCP20 were used for the gene knockout in E. coli W3110. The pETPtac with a promoter Ptac was used for overexpression of genes in E. coli W3110. Kanamycin, chloramphenicol, and spectinomycin were added at a concentration of 50, 40, and 50 μg/mL when necessary. M9 minimal medium33 was used for the comparison of the capability of D-xylonate production between engineered strains. The batch and fed-batch fermentation media were M9 minimal medium supplemented with 2 and 5 g/L yeast extract, respectively. DNA Manipulation in E. coli W3110. The primers used in this study are listed in Table S1. The genes xylB and xylC of C. crescentus were synthesized by Tongyong Biosystem Co., Ltd. (Chuzhou, Anhui, China) and cloned into pETPtac plasmid34 with the restriction site of EcoRI and HindIII. Knockout mutants of E. coli W3110 were generated by one-step inactivation method.35 The DNA mutant fragment used in this method had two homologous arms for homologous recombination and an FRT-flanked kanamycin resistance cassette for replacing target gene and screening mutant strain. Two methods were used for obtaining DNA mutant fragments. Briefly, (1)

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RESULTS AND DISCUSSION Construction of D-Xylonate Biosynthesis Pathway in E. coli W3110. E. coli W3110 can use D-xylose or D-xylonate as its sole carbon source, but cannot synthesize D-xylonate from D-xylose (Figure 2). For the high-yield production of Dxylonate, both the D-xylose and D-xylonate catabolic pathways must be blocked, while the D-xylonate production pathway must be enhanced. Thus, xylose isomerase (xylA) related to Dxylose catabolism and two xylonate dehydratases (yjhG and C


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producing D-xylonate from D-xylose. As D-xylonate catabolism is blocked in E. coli X4, this strain could produce a higher concentration and yield of D-xylonate than E. coli X3. Elimination of Byproduct Formation. Inactivation of Dxylose and D-xylonate catabolism pathways abolishes D-xylose assimilation and thus extra carbon sources are required for the growth of recombinant strain.16,26 Considering that LB was unsuitable for the massive production of D-xylonate, M9 medium containing 10 g/L D-glucose was used to support the growth of E. coli X4 in our successive experiments. As shown in Figure 3A, E. coli X4 will produce byproducts such as acetate, lactate, and ethanol that can decrease the utilization efficiency of D-glucose and hinder the D-xylonate separation process. Therefore, the genes ackA, poxB, adhE, and ldhA, which are responsible for acetate, ethanol, and lactate production, were all deleted in E. coli X4 to generate strain E. coli XG2. Compared to E. coli X4, E. coli XG2 produced less acetate, ethanol, and lactate but exhibited a decreased D-glucose consumption (Figure 3A). Batch fermentation using E. coli XG2 was then conducted in M9 medium with 40 g/L D-xylose and 10 g/L D-glucose. As shown in Figure 3B, 47.3 g/L D-xylonate was produced from 44.2 g/L D-xylose with a productivity of 1.18 g/[L·h]. There was no production of lactate and ethanol throughout the fermentation process, and the final concentration of acetate was only 0.85 g/L (Figure 3B). Under the same conditions, E. coli X4 produced 40.6 g/L D-xylonate and accumulated 1.14 g/ L acetate at the end of fermentation. (Figure S1). Analysis of Product Inhibition of D-Xylonate toward Recombinant E. coli. Similar to that in previous works,26 recombinant E. coli XG2 could not produce D-xylonate with a concentration high enough for industrial-scale production. The product inhibition of D-xylonate toward recombinant E. coli XG2 was then analyzed through whole-cell biotransformation under various D-xylonate concentrations. The biotransformation reaction mixtures containing whole cells of E. coli XG2, 40 g/L D-xylose, and D-xylonate at various concentrations (0, 20, 40, 60, and 80 g/L) were incubated at 37 °C with shaking at 120 rpm. Notably, the transformation of D-xylose was not significantly affected by the addition of more exogenous Dxylonate (Figure 4). D-Xylonate could accumulate to 117 g/L with its initial concentration at 80 g/L. Thus, product inhibition was not a decisive factor limiting the production of D-xylonate. E. coli XG2 has the potential to produce high concentrations of D-xylonate. Lactose-Induced Production of D-Xylonate. The expression of xylB and xylC was under the control of the Ptac promoter, which can be induced in the presence of lactose or IPTG. In previous studies involving D-xylonate production in recombinant E. coli19,26 and in our experiment previously, IPTG was used to induce the expression of xylB and xylC.

Figure 2. Growth of different E. coli strains in M9 medium with Dxylose (A) or D-xylonate (B) as the carbon sources. The experiment was conducted in a 300 mL shake flask containing M9 medium with 5 g/L D-xylose or 5 g/L D-xylonate at 37 °C on a rotary shaker at 180 rpm.

yagF), which are genes involved in D-xylonate catabolism, were sequentially deleted from the E. coli W3110 genome. The resulting strain E. coli X1 lost the ability to utilize D-xylose, and E. coli X2 lost the ability to utilize D-xylose and D-xylonate (Figure 2). Then, xylB and xylC were both overexpressed in E. coli X1 and E. coli X2 generating E. coli X3 and E. coli X4, respectively. As expected, E. coli X3 regained the ability to grow on D-xylose because of the expression of D-xylonate biosynthesis pathway and its innate D-xylonate catabolism. E. coli X4 still cannot grow on either D-xylose or D-xylonate despite expressing genes related to the D-xylonate biosynthesis pathway. Production of D-xylonate from D-xylose using these recombinant strains was carried out in LB medium containing 10 g/L D-xylose. As shown in Table 3, E. coli W3110 could assimilate D-xylose for its growth but the biomass of the recombinant strains decreased because of the disruption of Dxylose and D-xylonate assimilation pathway. E. coli X3 and E. coli X4 expressing both xylB and xylC have the capability of

Table 3. Biomass, D-Xylose Consumption, D-Xylonate Production, and Yield of Different E. coli Strainsa strain E. E. E. E. E.

coli coli coli coli coli

W3110 X1 X2 X3 X4

biomass (OD600nm) 5.07 4.61 4.39 3.52 3.11

± ± ± ± ±

0.075 0.131 0.029 0.085 0.100

D-xylose

consumed (g/L)

3.26 ± 0.067 ND ND 6.03 ± 0.29 6.81 ± 0.10

D-xylonate

(g/L)

ND ND ND 6.56 ± 0.62 7.63 ± 0.10

yield (g/g) ND ND ND 1.09 ± 0.10 1.12 ± 0.01

The experiment was conducted in a 300 mL shake flask with LB containing 10 g/L D-xylose at 37 °C on a rotary shaker at 180 rpm. IPTG at a concentration of 1 mM was added at the initial of fermentation. ND means not detected.

a

D


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Figure 3. Effects of byproduct elimination on the production of D-xylonate. (A) D-Glucose consumption and byproduct formation by E. coli X4 and E. coli XG2 when cultivated in M9 medium containing 10 g/L D-xylose and 10 g/L D-glucose. (B) D-Xylonate production by batch fermentation of E. coli XG2 using IPTG as the inducer. The experiment was conducted in M9 medium with 40 g/L D-xylose and 10 g/L D-glucose approximately. IPTG at a concentration of 1 mM was added at the initial of fermentation.

productivity was 1.71 g/[L·h], which was higher than that of E. coli XG2 (1.26 g/[L·h]). Inactivation of Lactose Degradation Pathway. Besides D-xylose, the transport of lactose is also under the control of carbon catabolite repression. Carbon catabolite repression allows E. coli to metabolize D-glucose preferentially prior to lactose. Delayed response to induction with lactose often occurs in systems using lactose as the inducer. The inactivation of ptsG could enhance the transport of lactose and increase the expression of xylB and xylC. The enhancement of the productivity of E. coli XGL2 may be due to the enhanced Dxylose transport and D-xylonate biosynthesis. E. coli XG2 could not metabolize lactose in the presence of D-glucose (Figure 5A), whereas E. coli XGL2 could utilize 5 g/L lactose added in the fermentation system (Figure 5B). To increase the induction efficiency further, the β-galactosidase gene (lacZ) in E. coli XGL2 was knocked out to generate strain E. coli XGL4. As shown in Figure 5C, the inactivation of lacZ could block the consumption of lactose throughout the fermentation process. E. coli XGL4 could produce 44.7 g/L D-xylonate from 40.5 g/L D-xylose with a higher productivity of 1.86 g/[L·h], which may be the result of enhanced induction efficiency of lactose. Fed-Batch Fermentation Using D-Xylose As the Substrate. To decrease the toxicity toward cell growth, reduce the cost of production, and be adapted to massive production, IPTG was replaced by lactose as the inducer to express xylB and xylC. Then, ptsG was inactivated to increase the transport of lactose and D-xylose. Since promoting lactose transport would also increase lactose metabolism, lacZ was inactivated to further enhance the induction efficiency. After all this optimization, the concentration and productivity of Dxylonate increased step by step (Figure 5D). Fed-batch fermentation was then conducted with E. coli XGL4 using D-xylose as the substrate to produce D-xylonate. In previous works that used lactose as the inducer, the concentration used was between 10 mM and 250 mM.39 Since the deletion of lacZ can block lactose degradation, lactose was used at a low concentration of 10 mM to induce Dxylonate biosynthesis. The experiment was conducted in a 5-L bioreactor containing 4 L medium with an initial D-xylose concentration of 40 g/L. D-Glucose at a concentration of 10 g/ L was added to supporting the growth of the recombinant strain according to previous work.26 Solid D-xylose was added to the medium when the D-xylose concentration was lower than 20 g/L. As shown in Figure 6, 108.2 g/L D-xylonate was

Figure 4. Analysis of product inhibition of D-xylonate toward E. coli XG2. The experiment was conducted in 100 mM phosphate buffer containing whole cells of E. coli XG2 (OD600nm = 20), D-xylose (40 g/ L), and D-xylonate (0, 20, 40, 60, 80 g/L) at 37 °C and on a rotary shaker at 120 rpm. After 24 h of biotransformation, the concentration of D-xylonate in the reaction mixtures was quantitatively analyzed by HPLC.

Considering the possible toxicity of IPTG toward cell growth39−43 and the consequent effects of this cytotoxicity on D-xylonate production, lactose was also used to induce the expression of xylB and xylC in E. coli XG2. The effects of IPTG and lactose on cell growth and D-xylonate production were assessed through shake-flask fermentation using the M9 medium. The biomass of the system using lactose as the inducer was higher than that achieved using IPTG. Because Dxylonate production is catalyzed by whole cells of E. coli XG2 in the fermentation system, higher concentrations of Dxylonate was also acquired using lactose as the inducer (Figure S2). Batch fermentation using lactose as the inducer was then conducted in a 1.4-L bioreactor with a broth volume of 1 L. As shown in Figure 5A, 41.7 g/L D-xylonate was obtained from 40.8 g/L D-xylose by E. coli XG2 in 33 h. The average productivity (1.26 g/[L·h]) was slightly higher than that of the system using IPTG (1.18 g/[L·h]). Thus, lactose was used as the inducer for D-xylonate production in subsequent experiments. Knockout of ptsG to Increase D-Xylonate Production. The presence of D-glucose would inhibit the transport of Dxylose and consequently affect D-xylonate production. Therefore, the D-glucose transporter encoding gene ptsG was knocked out in E. coli XG2. The generated strain E. coli XGL2 was then used for D-xylonate production using a mixture of D-glucose and D-xylose. As shown in Figure 5B, 41 g/L Dxylonate was obtained from 38.6 g/L D -xylose. The E


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Figure 5. Optimization of the induction strategy for D-xylonate production by recombinant E. coli. (A) Time-course of batch fermentation by E. coli XG2. (B) Time-course of batch fermentation by E. coli XGL2. (C) Time-course of batch fermentation by E. coli XGL4. (D) Comparison of cell density, D-xylonate production, D-xylonate productivity, and lactose consumption of E. coli XG2, E. coli XGL2, and E. coli XGL4 at 24 h. The E. coli XG2, E. coli XGL2, and E. coli XGL4 were cultured in M9 medium containing 40 g/L D-xylose, 10 g/L D-glucose, and 5 g/L lactose approximately.

obtained from 99.3 g/L D-xylose after 60 h. The productivity of D-xylonate was 1.8 g/[L·h] and the yield of D-xylonate was 1.09 g/g.

Figure 7. D-Xylonate production by fed-batch fermentation of E. coli XGL4 with corn cob hydrolysate. The fed-batch fermentation was conducted in M9 medium containing corn cob hydrolysate (40 g/L Dxylose, 4 g/L D-glucose, and 4 g/L L-arabinose) and 10 mM lactose approximately. Condensed corn cob hydrolysate was added to the medium to a final D-xylose concentration of about 30 g/L when the Dxylose concentration was lower than 10 g/L.

Figure 6. D-Xylonate production from D-xylose by fed-batch fermentation of E. coli XGL4. The fed-batch fermentation was conducted in M9 medium containing 40 g/L D-xylose, 10 g/L Dglucose, and 10 mM lactose approximately. Solid D-xylose was added to the medium to a final D-xylose concentration of about 40 g/L when the D-xylose concentration was lower than 20 g/L.

lactose. The major byproduct was acetate, which accumulated to a low concentration of 0.55 g/L at the end of fermentation. D-Xylose, the second most abundant sugar in nature, can be used as a potential feedstock for generating various chemicals. Efficient recombination systems have been developed for metabolic engineering of E. coli. Many genetically engineered E. coli strains have been used in production of chemicals from D-xylose. However, as shown in Table S2, the theoretical yields and final concentrations of many chemicals produced by recombinant E. coli from D-xylose were relatively low. In this work, the recombinant E. coli strain XGL4 was developed to

Utilization of the Corn Cob Hydrolysate in Fed-Batch Fermentation. Fed-batch fermentation using the corn cob hydrolysate as the carbon source by strain E. coli XGL4 was also carried out. As shown in Figure 7, 91.2 g/L D-xylonate was obtained from 86.7 g/L D-xylose after 60 h. The productivity of D-xylonate was 1.52 g/[L·h], and the yield of D-xylonate was 1.05 g/g. During fermentation, 8.4 g/L D-glucose and 8.83 g/L L-arabinose were consumed, and there was no consumption of F


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potential feedstock for different chemicals, various recombinant strains may be constructed and used in the production of other valuable chemicals based on the coutilization systems introduced in this work.

biotransform D-xylose into D-xylonate, a valuable chemical with high theoretical yield and final concentration. Many biotechnological routes have been developed for the production of D-xylonate from D-xylose (Table 1). Recombinant E. coli strain EWX4 can produce 39.2 g/L D-xylonate from 40 g/L D-xylose, and 10 g/L D-glucose.26 In this study, the limiting factor for the high production of D-xylonate by E. coli was identified. Then, three successive strategies were utilized to enhance the production of D-xylonate (Figure 5D). After systematic metabolic engineering, 108.2 g/L D-xylonate was obtained from 99.3 g/L D-xylose and 10.2 g/L D-glucose by the final recombinant strain E. coli XGL4 (Figure 6). As a fivecarbon organic acid, production of D-xylonate was accompanied by pH decrease of the reaction system. However, many producers of D-xylonate could produce D-xylonate at low pH.16,19,23−25 For example, P. kudriavzevii could produce 171 g/L D-xylonate at a low pH of 5.5.25 Although growth of E. coli is optimal at neutral pH range, a two-step fermentation process which combines growth at neutral pH and D-xylonate production at acidic pH might be developed for higher Dxylonate production by recombinant E. coli. Besides reaction pH, the induction mode may also influence the performance of the xylonate producers. When 0.5 mM IPTG was added for the induction of xylBC in E. coli EWX4, the productivity of Dxylonate is 1.09 g/[L·h].26 In this work, 1 mM IPTG or 10 mM lactose were used as inducers and the productivity of Dxylonate by E. coli XGL4 were 1.18 g/[L·h] or 1.26 g/[L·h], respectively. Further enhancement of the D-xylonate production might be acquired through optimum of the concentration of lactose or using constitutive expression systems. Of the numerous producers of D-xylonate, Gluconobacter oxydans exhibited relatively high D-xylonate productivity and yield.19 Recently, G. oxydans has also been used to produce calcium xylonate from wheat straw prehydrolysate.44 However, this strain required a complex growth medium and converted wheat straw prehydrolysate into a mixture of xylonate and gluconate, which can only be used in a concrete admixture. E. coli XGL4 can produce 91.2 g/L D-xylonate in minimal medium using the corn cob hydrolysate as the substrate. DGlucose and L-arabinose in corn cob hydrolysate were used as the carbon source to support the growth of the recombinant E. coli strain XGL4 with decreased byproduct formation, and only D-xylonate accumulated in the final fermentation solution. Considering its desirable characteristics, E. coli XGL4 may offer a promising alternative for industrial D-xylonate production. Corn cob hydrolysate is an abundant renewable material with D-xylose, D-glucose, and L-arabinose account for 79.7%, 8.2%, and 8.5% of its total sugar content. Therefore, the complete and efficient utilization of this renewable material requires the presence of microorganisms that can effectively transform D-xylose into target products. In this study, we proposed a coutilization strategy for an integrated biorefinery based on corn cob hydrolysate. E. coli strain XGL4 was constructed for the efficient production of D-xylonate from corn cob hydrolysate. D-Glucose and L-arabinose in corn cob hydrolysate were used as the carbon source for the growth of the recombinant strain, while D-xylose in the hydrolysate was transformed into the target product, D-xylonate. D-Xylonate is a precursor of many important chemicals including 3,4dihydroxybutanal, 3,4-dihydroxybutyrate, 1,2,4-butantriol, and 1,4-butanediol. The efficient production of these important chemicals may be realized by successfully expressing the related pathways in E. coli strain XGL4. Since D-xylose is a

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CONCLUSIONS In summary, systematic approaches were used to optimize Dxylonate production through recombinant E. coli. D-Xylonate production with a concentration of 108.2 g/L and a productivity of 1.8 g/[L·h] was realized by the recombinant E. coli strain XGL4 using D-xylose as the substrate and Dglucose as the carbon source. Compared with other previously reported recombinant E. coli strains that have been used for the D-xylonate production, the engineered strain XGL4 has significant advantages such as achieved high product concentration and productivity. Furthermore, the E. coli strain XGL4 could use corn cob hydrolysate as the substrate for Dxylonate production. By using a coutilization strategy for the integrated biorefinery of corn cob hydrolysate, 91.2 g/L Dxylonate with the productivity of 1.52 g/[L·h] was produced by E. coli XGL4. The coutilization strategy presented in this study is not only a promising alternative for D-xylonate production but may also serve as a good example for the production of other important chemicals based on renewable biomass.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b04839.

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Experimental details and supplementary data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-532-58631561. ORCID

Ping Xu: 0000-0002-4418-9680 Chao Gao: 0000-0002-5205-0670 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by National Natural Science Foundation of China (31470164 and 31670041), Special Funds for Public Welfare Research and Capacity Building in Guangdong Province of China (2017A010105020), Young Scholars Program of Shandong University (2015WLJH25), Shandong Provincial Funds for Distinguished Young Scientists (JQ201806), and Open Project Program of the State Key Laboratory of Microbial Metabolism (MMLKF17-11).

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Production of d-Xylonate from Corn Cob Hydrolysate by a

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