Next Article in Journal
Biodepolymerization of Polyamide Fibers Using Yarrowia lipolytica as Whole-Cell Biocatalyst
Previous Article in Journal
Enhancing the Content of Hesperidin and Nobiletin in Citrus Wines through Multi-Strain Fermentation
Previous Article in Special Issue
Optimization of Fermentation Process of Wheat Germ Protein by Aspergillus niger and Analysis of Antioxidant Activity of Peptide
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 10
Issue 5
10.3390/fermentation10050240
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Optimizing Curdlan Synthesis: Engineering Agrobacterium tumefaciens ATCC31749 for Enhanced Production Using Dextrin as a Carbon Source

by
Tingting Yu
1,2,
Yu Wang
1,2,
Wei Wang
1,2,
Yonggang Zhang
1,2,
Yanmin Zhang
1,2,
Hongyu Han
1,2,
Yang Liu
1,2,
Siduo Zhou
1,2 and
Xueqian Dong
1,2,*
1
Faculty of Food Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
2
Shandong Research and Design Institute of Food & Fermentation Industry, Jinan 250013, China
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(5), 240; https://doi.org/10.3390/fermentation10050240
Submission received: 19 March 2024 / Revised: 26 April 2024 / Accepted: 27 April 2024 / Published: 30 April 2024
(This article belongs to the Special Issue Optimization and Analysis of Fermentation Process for Microbial Products)
Browse Figures
Versions Notes

Abstract

:
A key goal in current research on industrial curdlan production is the expansion of carbon sources for fermentation. In this study, a recombinant bacterial strain, sp-AmyAXCC, capable of fermenting and synthesizing curdlan using dextrin as a carbon source, was produced via heterologous expression of IPTG-inducible α-amylase from Xanthomonas campestris NRRL B-1459 in Agrobacterium tumefaciens ATCC31749. External expression of the enzyme was confirmed by western blotting, and the expression levels of exogenous proteins during the fermentation process were monitored. Additionally, the properties of the curdlan product were characterized using attenuated total reflectance-Fourier transform infrared spectroscopy and X-ray diffraction. The recombinant strain produced curdlan at a titer of 30.40 ± 0.14 g/L, gel strength of 703.5 ± 34.2 g/cm2, and a molecular weight of 3.58 × 106 Da, which is 33% greater than the molecular weight of native curdlan (2.69 × 106 Da). In the batch fermentation of sp-AmyAXCC with 12% dextrin as a carbon source, the titer of curdlan was 66.7 g/L with a yield of 0.56 g/g, and a productivity rate of 0.62 g/L/h at 108 h. The results of this study expand the substrate spectrum for Agrobacterium fermentation in curdlan production and provides guidance for further industrialization of curdlan production.

1. Introduction

Curdlan, (C6H10O5)n, is an extracellular, non-branched β-1,3-glucan produced through microbial fermentation under nitrogen-limited conditions. Typically, each curdlan molecule consists of 300–500 glucose residues [1] (Figure 1). Aqueous curdlan solutions can form either low-intensity heat-reversible or high-intensity heat-irreversible gels with different structures and properties at different temperatures [2]. Curdlan was first discovered and reported by Tokuya et al. [3] in 1966 and was approved by the U.S. Food and Drug Administration (FDA) in 1996 for use as a food additive. In recent years, the applications of curdlan have expanded continuously; it is now widely used in food processing, aquaculture, oil recovery, biopharmaceuticals, daily chemicals, and other fields. In particular, curdlan is an irreplaceable, high-quality raw material used in the high-end food manufacturing industry [4,5]. Curdlan and its derivatives also exhibit physiological functions, such as antibacterial [6], antitumor [7], antiviral [8], and immune-enhancing [9] activity. Chemical modifications of curdlan, such as sulfation [10], carboxymethylation [11], cationization [12], amination [13], phosphorylation [14], click chemistry [15], oxidation, and esterification [16], can be employed to prepare water-soluble and biologically active curdlan derivatives applicable in the field of biomedicine.
Microbes capable of curdlan synthesis include Agrobacterium sp. [17], Rhizobium spp. [18], Escherichia coli [19], and Bacillus subtilis [20]. Among these, Agrobacterium spp. have been widely used in the industrial production of curdlan [21]. Numerous studies have been conducted to enhance curdlan production, including screening or constructing high-yield strains [22,23], optimizing fermentation control strategies [24,25], and improving separation and extraction technologies [26]. Carbon sources are crucial factors in the fermentation industry. Currently, the primary carbon sources for industrial curdlan production are sucrose and glucose. However, sucrose is relatively expensive, and high concentrations of glucose severely inhibit bacterial growth. Therefore, one of the current research hotspots in industrial curdlan production is the expansion of the carbon sources for fermentation. An example of this research is the work of Wan et al. [27], who demonstrated the feasibility of Agrobacterium sp. to utilize cassava starch as a substrate for curdlan biosynthesis by supplementing exogenous β-amylase and pullulanase during fermentation. Genetic engineering of curdlan-producing strains has become an efficient means of promoting industrial production. As an example, Shin et al. [28] constructed an Agrobacterium sp. strain with a 2.5-fold enhanced capacity to utilize cellobiose to synthesize curdlan by introducing an energy-efficient cellobiose phosphorolysis pathway under the control of strong promoters. Gao et al. [29] increased curdlan production by 19% or 17% by deleting the EPS production regulator-encoding gene exoR or EPS production repressor protein-encoding gene exoX, respectively, in Agrobacterium sp. Based on these studies, it is feasible to introduce exogenous enzymes related to substrate utilization into Agrobacterium sp. to give the bacteria the ability to use carbon sources other than sucrose and glucose to produce curdlan.
Heterologous expression, which allows for the production of large quantities of specific proteins in different host systems, is a widely used technique that typically involves introducing a target gene (e.g., a DNA sequence encoding a target protein) into a host cell and utilizing the host cell’s biological machinery to transcribe and translate the target gene to produce the desired target protein. Heterologous expression has now become a major production method to increase the protein yield of many industrial enzymes such as α-amylase. In order to achieve efficient expression, expression vectors are usually screened and constructed. This mainly includes the selection of suitable expression vectors and strong promoters, etc. PBBR1 plasmids have antibiotic screening markers, small plasmid size (<5.3 kb), and extended multiple cloning sites (MCS) within the lacZa gene. They also have the advantage of mobility and compatibility. The strength of promoter activity is the key to realizing the genetic engineering of exogenous expression, and different types of promoters have different mechanisms of action, including constitutive, inducible, and so on.
Starch and dextrin are widely available, low-cost carbon sources that have long been used for the production of another microbial polysaccharide, xanthan gum. The strain Xanthomonas campestris can utilize starch to produce xanthan gum because of its highly efficient starch utilization system, which includes amylase, the gene for which has been cloned and expressed in E. coli. [30]. The most common curdlan-producing strain, Agrobacterium sp. ATCC31749, naturally lacks the relevant enzymes that can utilize starch and dextrin. In this study, a recombinant strain, sp-AmyAXCC, was constructed by introducing the α-amylase from X. campestris NRRL B-1459 into Agrobacterium sp. ATCC31749, under the control of Ptac. The IPTG-induced expression of the His-tagged α-amylase in sp-AmyAXCC was confirmed by western blot. The engineered strain was confirmed to synthesize curdlan using dextrin as a carbon source. The α-amylase activities, as well as other parameters, were monitored in a batch fermentation of curdlan using sp-AmyAXCC as the producer and dextrin as the carbon source. In addition, several properties of the curdlan produced from dextrin were characterized. Taken together, the results of this study expand the substrate spectrum for curdlan fermentation by Agrobacterium sp. and promote the industrial production of curdlan.

2. Materials and Methods

2.1. Chemicals

Peptone, dextrin, beef paste, yeast extract, (NH4)2HPO4, KH2PO4, MgSO4, CaCO3, glycerin, HEPES, and NaCl were purchased from Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Corn syrup was purchased from Angel Yeast Co. Ltd. (Yichang, China). IPTG, anhydrotetracycline (AHT), xylose, rhamnose, and arabinose were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Strains, Plasmids, and Culture Conditions

The strains and plasmids used in this study are listed in Table 1. The oligonucleotides used in this study are listed in Table 2.
Luria-Bertani (LB) medium was used to cultivate E. coli strains and used as the seed medium for the Agrobacterium sp. The medium used for curdlan fermentation contained (L−1) 50 g dextrin, 4 g (NH4)2HPO4, 2 g KH2PO4, 2 g MgSO4, 2 g CaCO3, and 1 g corn syrup, and the initial pH was adjusted to 7.0. All media were sterilized at 121 °C for 20 min. For antibiotic selective media, kanamycin was added at 50 μg/mL.

2.3. Promoter Screening

Green fluorescent protein (GFP) was used as an indicator for promoter screening. Plasmids pBBR-Ptac/Ptet/Pxyl/PRha/PBAD-GFP, in which GFP expression was controlled by the corresponding promoter, were transformed into Agrobacterium sp. ATCC31749. The transformants were inoculated to 5 mL LB medium with kanamycin and cultured at 30 °C to OD600 = 0.6–0.8. The corresponding inducers (IPTG for Ptac, AHT for Ptet, xylose for Pxyl, rhamnose for PRha, and arabinose for PBAD) were added to the medium, and the cultures were induced overnight. Green fluorescence was observed under blue-light irradiation.

2.4. Plasmid Construction and Transformation

Genomic DNA from X. campestris NRRL B-1459 was extracted using a TIANamp Bacteria DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). Amino acids MRASTDQETHPMHATSRPCPRTFWQRAHQLLLIALTLLLTTASAQAD of X. campestris α-amylase (WP_029217145.1) was predicted to be the signal peptide. To avoid incompatibility of the signal peptide of X. campestris α-amylase with the heterologous host, it was substituted with a signal peptide from Agrobacterium sp., and the signal peptide of the BMP family ABC transporter substrate-binding protein (WP_262515555.1) was applied. The α-amylase encoding gene was amplified using genomic DNA of X. campestris NRRL B-1459 as a template and amy-F1/amy-R1 as primers. The PCR product was used as the template for another round of PCR with amy-F2/amy-R2 as primers to introduce a signal peptide encoding sequence and a 6 × His-tag encoding sequence at its 5′ end and 3′ end, respectively. Homologous arms for T5 exonuclease-dependent DNA assembly were also included in amy-F2/amy-R2. The pBBR-Ptac-GFP plasmid was digested using EcoR I and Nde I. The second round PCR product and double enzyme digestion product were then used for the construction of recombinant plasmid pBBR-Ptac-α-amylase following the One Step SeamlessMix (Ruibo Biotech Co., Ltd., Beijing, China).
The pBBR-Ptac-α-amylase plasmid was transformed into Agrobacterium sp. by electroporation according to the instructional manual of MicroPulser Electroporator (Bio-Rad, Hercules, CA, USA). Transformants were screened for kanamycin resistance and identified by colony PCR. The correct clone was named sp-AmyAXCC and was stored.

2.5. Expression and Activity Assay of Recombinant α-Amylase

The sp-AmyAXCC was inoculated into LB medium with 50 μg/mL kanamycin and cultivated at 30 °C. When the OD600 nm reached 0.6~0.8, 0.5 mM (final concentration) isopropyl β-D-thiogalactopyranoside (IPTG) was added. After 12 h of induction, the culture was centrifuged, and the supernatant was collected to determine the protein concentration and α-amylase activity. Total protein in the supernatant was extracted via ammonium sulfide precipitation, and the precipitate was resuspended in Tris buffer (50 mM Tris, 200 mM NaCl, pH 8.0). Western blotting with His-tag antibodies was used to confirm the extracellular expression of α-amylase.
A BCA protein assay kit (Tiangen) was used to measure protein concentrations, with bovine serum albumin as the standard. Dextrin is hydrolyzed to glucose by α-amylase, which can be reduced by dinitrosalicylic acid (DNS), and α-amylase activity was analyzed using the DNS assay [31]. To determine the amylase activity, the crude enzyme sample (2 mL) was added to the soluble starch solution (1% w/v, 0.5 mL) in phosphate-citrate buffer (50 mM, pH 6.0), DNS was added, and the mixture was incubated at 65 °C for 30 min followed by a boiling water bath for 5 min and cooling in ice water. One amylase unit was taken as the amount of enzyme required to release 1 μmol of glucose per minute under the given assay conditions.

2.6. Batch Fermentation in Stirred Bioreactors

Batch fermentation was conducted in a 10-L stirred bioreactor (Bailun Biological Technology, Shanghai, China) with 7 L of the initial medium. The fermentation was initiated after inoculation of seed cultures (10% v/v) at an OD600nm of 14.95 and carried out at 30 °C. The initial pH was adjusted to 7.0 and was not controlled during the fermentation process. The aeration rate was 1.0 vvm, and the agitation speed was 300 rpm. IPTG was added to a final concentration of 0.5 mM after 5 h of inoculation. Samples were collected periodically to determine the biomass, α-amylase activity, curdlan yield, and gel strength. Biomass and curdlan yields were determined using the gravimetric method [32]. The sample was centrifuged and 1 mol/L NaOH was added to dissolve the curdlan. After the mixture was centrifuged at 8000× g for 10 min, the supernatant was collected, the curdlan was precipitated through the addition of 1 mol/L HCl, the pH was adjusted to 7.0, and 95% (v/v) ethanol was added to the supernatant; the mixture was again centrifuged to remove the supernatant, and the precipitate was collected. The precipitate was washed thrice with distilled water, dehydrated with three volumes of ethanol, and freeze-dried. The test was repeated three times for each sample.

2.7. Thermal Gelation Property

Lyophilized curdlan powder was homogenized at 8000 rpm for 10 min, dissolved in deionized water to a final concentration of 20 g/L, and the curdlan solution (10 mL) was transferred into a glass tube (18 mm × 180 mm). The tube was incubated in a water bath (95 °C) for 10 min and was cooled to ensure the gel formation. A 10 mm long gel section was cut from the bottom of the tube for gel strength measurement using a texture analyzer (Brookfield CT3, Brookfield, WI, USA). The parameters of the compression-mode test were as follows: probe model, TA/35; test speed, 250 mm/min. The gel strength (g) was defined as the maximum force required for gel cracking.

2.8. Molecular Weight Determination of Curdlan

The molecular weight (Mw) of curdlan was measured using an Agilent 1260 Infinity II GPC/SEC (Agilent, Waldbronn, Germany) with double-detector light scattering (LS) and refractive index (RI) [23]. Polymethyl methacrylate (PMMA) was used as a calibration standard to establish this method. The freeze-dried curdlan was completely dissolved in dimethyl sulfoxide (DMSO) with stirring for approximately 4 h at 80 °C. A total of 20 μL of the curdlan DMSO solution (5.0 mg/mL) was injected into the column (PL Polaegel-M 7.5 × 50 mm and PL Polaegel-M 7.5 × 300 mm) by using DMSO as the mobile phase at a flow rate of 1.0 mL/min at 45 °C [33]. The average Mw was calculated using the Agilent 1260 Infinity II GPC/SEC software based on the LS and RI data.

2.9. Fourier-Transform Infrared Spectroscopy and X-ray Diffractometry

The attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra were obtained using a Fourier transform spectrophotometer (Tensor II; Bruker, Karlsruhe, Germany). Curdlan samples were analyzed as described by Wang et al. [34]. In brief, the prepared curdlan solutions were freeze-dried and crushed into a powder, and the samples were assayed using the potassium bromide (KBr) disc method with an average of 64 scans at a resolution of 8 cm−1 in the range of 4000–500 cm−1. The XRD pattern of curdlan gum was measured by an XRD diffractometer (Smartlab, Tokyo, Japan). Cu-Kα radiation (λ = 0.154 nm) is produced at 40 kV and 30 mA. XRD analysis was performed at room temperature, at 2θ range of 5° to 80°, a step size of 0.02°, and a scanning speed of 20°/min [35].

3. Results and Discussion

3.1. Promoter Screening

Promoters play important roles in metabolic processes. Using inducible promoters to regulate the expression of recombinant enzymes allows them to be expressed only when needed, thus avoiding the extra metabolic burden on cells as much as possible. To obtain an ideal promoter to regulate the expression of α-amylase, five inducer promoters, PBAD, PRha, Ptet, Ptac, and Pxyl, were tested in ATCC31749 in this study. Plasmid PBBR-Ptac/Ptet/Pxyl/PRha/PBAD-GFP, derived from pBBR1MCS-2, was transferred into ATCC31749, and GFP fluorescence was assayed to evaluate the efficacy and stringency of the promoters. As shown in Figure 2A, only cells harboring GFP regulated by the IPTG-inducible promoter Ptac showed a strong difference in fluorescence with or without an inducer, indicating that Ptac has both high efficacy and stringency in ATCC31749. As can be seen in Figure 2B,C, the strains in which GFP expression was controlled by PBAD or Ptet did not exhibit significant green fluorescence in the presence of the respective inducers (l-arabinose or AHT). This result suggests the low efficiency of these promoters in ATCC31749. In contrast, the strains in which GFP expression was controlled by Pxyl or PRha showed substantial green fluorescence before and after induction with the corresponding inducers (xylose or l-rhamnose) (Figure 2D,E). This indicates high efficiency but poor stringency between the two promoters. Poorly regulated promoters may respond to various conditions or signals, resulting in non-specific gene expression. This can lead to the activation of exogenous genes under undesired conditions, causing unnecessary metabolic processes or protein synthesis. Such nonspecific activation may affect cell growth and metabolic efficiency. Based on these results, Ptac was selected to control recombinase expression.

3.2. Construction of sp-AmyAXCC and Identification of α-Amylase Expression

Agrobacterium sp. are widely used curdlan producers. The strain ATCC31749 is naturally deficient in starch and dextrin utilization. An efficient amylase is required to produce curdlan from dextrin. X. campestris is a well-known producer of xanthan gum that efficiently utilizes starch as a substrate. Abe et al. [36] identified an α-amylase that hydrolyzes α-cyclodextrin (α-CD) into maltose and glucose at a high rate in X. campestris. Therefore, the X. campestris strain NRRL B-1459, which was preserved in our laboratory and exhibits remarkable extracellular amylase activity was chosen to be the supplier of α-amylase. To facilitate the validation of protein expression, a 6 × His-tag was added to the C’-terminus of α-amylase. As shown in Figure 3A, a specific region of the amylase gene was successfully amplified from X. campestris NRRL B-1459 genomic DNA. Plasmid pBBR-Ptac-α-amylase was constructed by substituting the GFP encoding gene in pBBR-Ptac-GFP with the PCR product (Figure 3B) and was then transformed into Agrobacterium sp. ATCC31749 to obtain strain sp-AmyAXCC (Figure 3C).
To validate the extracellular expression of X. campestris α-amylase in sp-AmyAXCC, the constructed strain was cultivated and induced with IPTG for 12 h. Western blot analysis using a His-tag specific antibody on the sample of sp-AmyAXCC revealed a single band with an Mw ranging from 40 to 53 kDa, which is absent for wild-type ATCC31749 and corresponds to the target protein (Figure 4). This further confirms the expression of α-amylase in Agrobacterium sp. ATCC31749. After 12 h of induction, the amylase activity in the sp-AmyAXCC supernatant was 584.5 U/mL, and the specific enzyme activity was estimated to be 5467 U/mg. These results indicate that the sp-AmyAXCC strain constructed in this study can successfully express active α-amylase extracellularly under IPTG induction.

3.3. Curdlan Productions by sp-AmyAXCC

Gel strength is a crucial indicator of the gelatin properties of curdlans. Typically, strain ATCC31749 cannot produce curdlan when using starch or dextrin as the substrate, which is consistent with the findings of Wan et al. [27]. As shown in Figure 5, when the recombinant strain is cultured with 5% glucose and dextrin as substrates, curdlan yields are 29.38 ± 2.1 g/L and 30.40 ± 1.4 g/L, with gel strengths of 657.8 ± 21.6 and 703.5 ± 34.2 g/cm2, respectively. Moreover, the molecular weight of the product from the recombinant strain (3.58 × 106 Da) is higher than that of curdlan (2.69 × 106 Da), representing a 33% increase in molecular weight. When the recombinant strain was cultured with 12% dextrin as the substrate, the curdlan yield was 56.6 ± 1.6 g/L, with a gel strength of 871.2 ± 27.9 g/cm2. In contrast, almost no curdlan was produced when 12% glucose was used as the substrate, possibly due to the higher concentration of glucose, triggering the glucose effect and proving detrimental to bacterial growth. When glucose is used as a carbon source, it is typically added separately [37]. Additionally, a high concentration of added glucose may result in the coloration of the synthesized curdlan, necessitating additional steps for decolorization, thereby increasing the complexity of subsequent product processing. Thus, the construction of this recombinant strain is important for the production and processing of curdlan.

3.4. Batch Fermentation of sp-AmyAXCC with Dextrin as Carbon Source

To validate the ability of sp-AmyAXCC to utilize dextrin to produce curdlan, batch fermentation was carried out in a 10 L stirred fermenter (Figure 6). As a carbon source, dextrin was added to the fermentation broth at an initial concentration of 120 g/L. However, it was difficult to quantify dextrin during fermentation; thus, the concentration was not recorded. pH is a key factor influencing curdlan synthesis [37]. The optimal initial pH for cell growth is 7.0; whereas the optimal pH for curdlan production is 7.0 or 5.5–6.2. The initial pH of the fermentation broth was set at 7.0 to facilitate bacterial growth. The pH decreased from 7.0 to 5.8 and remained stable throughout the fermentation process, making it suitable for curdlan synthesis. IPTG was added into the culture after 5 h of inoculation to a final concentration of 0.5 mM to induce the expression of α-amylase. The α-amylase activity increased continuously after induction and reached the maximum value of 1954.9 U/mL at 72 h of fermentation. Curdlan was successfully synthesized from dextrin using exogenous α-amylase. Although the α-amylase activity decreased gradually after 84 h to a final value of 1678.2 U/mL, the titer of curdlan continued to increase and even reached a rather high growth rate during 84–108 h. This may be because the products of dextrin degradation, maltose, and glucose, which can be naturally utilized by Agrobacterium sp., accumulate in the fermentation broth and can be rapidly utilized to produce curdlan. At 108 h, the titer of curdlan was 66.7 g/L, with a yield of 0.56 g/g and a productivity of 0.62 g/L/h. Furthermore, the gel strength of the curdlan products was assayed during fermentation. This value increased continuously and reached 972.5 g/cm2 after 108 h. Through the specific fermentation results in Table 3, we found that the yield performance of this strain is very impressive in the case of dextrin as substrate. Compared to other substrates, dextrin as a substrate also has the potential to achieve high yields of available curdlan.

3.5. Characterization of Curdlan Product

The characteristics of the product were compared with curdlan, including ATR-FTIR spectrum, X-ray diffraction, gel strength, and molecular weight. As shown in Figure 7, the FTIR spectrum of the samples exhibited characteristic peaks common to curdlan (3323, 2950, 1366, 1156, 887 cm−1), similar to the results of previous studies on curdlan [24]. A broad peak at 3323 cm−1 indicates O-H stretching due to hydrogen bonding, and the symmetric -CH2- stretching vibration is observed at 2950 cm−1. A characteristic peak representing C=O stretching vibration was observed at 1156 cm−1, and a broad peak at 887 cm−1 indicates the linear β-1,3-glucan structure [42]. The X-ray diffraction results showed characteristic diffraction peaks at 19.8° and 29.8°, which closely aligned with those of curdlan in the recombinant strain product. From the above experimental results, it can be proved that the fermentation product of the recombinant strain is consistent with the structure of the available curdlan. When the recombinant strain was cultured with 12% dextrin as substrate, the gel strength of the product was 871.2 ± 27.9 g/cm2, which increased by 26.2% (690.1 ± 30.7 g/cm2) compared with that of the curdlan gel. And the molecular weight of the product of the recombinant strain (3.58 × 106 Da) was higher than that of curdlan (2.69 × 106 Da) with a 33% increase in molecular weight.

4. Conclusions

In this study, a recombinant strain, sp-AmyAXCC, with an IPTG-induced heterogenous α-amylase, was successfully constructed based on strain A. tumefaciens ATCC31749. Protein expression of the recombinant strain was analyzed by western blotting. The target protein exhibited an amylase activity of 584.5 U/mL and a specific activity of 5467 U/mg. Compared to the wild-type strain, the recombinant strain demonstrated the ability to utilize dextrin as a fermentation substrate to synthesize curdlan. In the batch fermentation of sp-AmyAXCC with 12% dextrin as a carbon source, the titer of curdlan was 66.7 g/L, with a yield of 0.56 g/g and a productivity of 0.62 g/L/h at 108 h. The gel strength of curdlan reached 972.5 g/cm2. Structural characteristics of the curdlan produced by the recombinant strain were similar to those produced by the wild-type strain. This study expands the substrate range of curdlan production to dextrin, providing new scopes for reducing curdlan production costs and enhancing curdlan yield.

Author Contributions

Conceptualization, X.D.; data curation, T.Y. and Y.W.; formal analysis, T.Y., W.W. and H.H.; funding acquisition, X.D.; investigation, T.Y. and Y.W.; methodology, T.Y. and W.W.; project administration, S.Z.; resources, Y.Z. (Yonggang Zhang) and Y.Z. (Yanmin Zhang); supervision, Y.Z. (Yonggang Zhang) and X.D.; validation, Y.Z. (Yanmin Zhang); writing—original draft, T.Y. and Y.W.; writing—review and editing, Y.L. and X.D. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2021YFC2103200) and Major innovation projects in science, education, and industry (2022JBZ01-08).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author, [X.D.], upon reasonable request.

Acknowledgments

We sincerely thank Tianyi Jiang (Shandong Jianzhu University) for providing crucial guidance in conceptualizing experimental strategies, managing, and coordinating the entire experimental process, as well as offering professional advice on the revision and review of the initial draft of the paper. We express our heartfelt gratitude to Jiang for his assistance, as his contributions have played a crucial role in refining this paper.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Hundschell, C.S.; Wagemans, A.M. Rheology of common uncharged exopolysaccharides for food applications. Curr. Opin. Food Sci. 2019, 27, 1–7. [Google Scholar] [CrossRef]
  2. Gieroba, B.; Sroka-Bartnicka, A.; Kazimierczak, P.; Kalisz, G.; Pieta, I.S.; Nowakowski, R.; Pisarek, M.; Przekora, A. Effect of gelation temperature on the molecular structure and physicochemical properties of the curdlan matrix: Spectroscopic and microscopic analyses. Int. J. Mol. Sci. 2020, 21, 6154. [Google Scholar] [CrossRef] [PubMed]
  3. Harada, T. Succinoglucan 10C3: A new acidic polysaccharide of Alcaligenes faecalis var. myxogenes. Arch. Biochem. Biophys. 1965, 112, 65–69. [Google Scholar] [CrossRef] [PubMed]
  4. Verma, D.K.; Niamah, A.K.; Patel, A.R.; Thakur, M.; Sandhu, K.S.; Chávez-González, M.L.; Shah, N.; Aguilar, C.N. Chemistry and microbial sources of curdlan with potential application and safety regulations as prebiotic in food and health. Food Res. Int. 2020, 133, 109136. [Google Scholar] [CrossRef]
  5. Chen, Y.; Wang, F. Review on the preparation, biological activities and applications of curdlan and its derivatives. Eur. Polym. J. 2020, 141, 110096. [Google Scholar] [CrossRef]
  6. Zhu, F.; Du, B.; Xu, B. A critical review on production and industrial applications of beta-glucans. Food Hydrocoll. 2016, 52, 275–288. [Google Scholar] [CrossRef]
  7. Bao, M.; Ehexige, E.; Xu, J.; Ganbold, T.; Han, S.; Baigude, H. Oxidized curdlan activates dendritic cells and enhances antitumor immunity. Carbohydr. Polym. 2021, 264, 117988. [Google Scholar] [CrossRef] [PubMed]
  8. Wang, D.; Kim, D.H.; Yoon, J.-J.; Kim, K.H. Production of high-value β-1, 3-glucooligosaccharides by microwave-assisted hydrothermal hydrolysis of curdlan. Process Biochem. 2017, 52, 233–237. [Google Scholar] [CrossRef]
  9. Yang, X.; Zheng, M.; Hao, S.; Shi, H.; Lin, D.; Chen, X.; Becvarovski, A.; Pan, W.; Zhang, P.; Hu, M. Curdlan prevents the cognitive deficits induced by a high-fat diet in mice via the gut-brain axis. Front. Neurosci. 2020, 14, 384. [Google Scholar] [CrossRef]
  10. Vessella, G.; Esposito, F.; Traboni, S.; Di Meo, C.; Iadonisi, A.; Schiraldi, C.; Bedini, E. Exploiting diol reactivity for the access to unprecedented low molecular weight curdlan sulfate polysaccharides. Carbohydr. Polym. 2021, 269, 118324. [Google Scholar] [CrossRef]
  11. Sato, F.; Nakamura, Y.; Katsuki, A.; Khadka, S.; Ahmad, I.; Omura, S.; Martinez, N.E.; Tsunoda, I. Curdlan, a microbial β-glucan, has contrasting effects on autoimmune and viral models of multiple sclerosis. Front. Cell. Infect. Microbiol. 2022, 12, 16. [Google Scholar] [CrossRef] [PubMed]
  12. Cai, Z.; Zhang, H. Recent progress on curdlan provided by functionalization strategies. Food Hydrocoll. 2017, 68, 128–135. [Google Scholar] [CrossRef]
  13. Nurzynska, A.; Klimek, K.; Swierzycka, I.; Palka, K.; Ginalska, G. Porous curdlan-based hydrogels modified with copper ions as potential dressings for prevention and management of bacterial wound infection—An in vitro assessment. Polymers 2020, 12, 1893. [Google Scholar] [CrossRef] [PubMed]
  14. Suflet, D.M.; Popescu, I.; Prisacaru, A.I.; Pelin, I.M. Synthesis and characterization of curdlan–phosphorylated curdlan based hydrogels for drug release. Int. J. Polym. Mater. 2021, 70, 870–879. [Google Scholar] [CrossRef]
  15. Han, J.; Wang, X.; Liu, L.; Li, D.; Suyaola, S.; Wang, T.; Baigude, H. “Click” chemistry mediated construction of cationic curdlan nanocarriers for efficient gene delivery. Carbohydr. Polym. 2017, 163, 191–198. [Google Scholar] [CrossRef] [PubMed]
  16. Yan, J.-K.; Pei, J.-J.; Ma, H.-L.; Wang, Z.-B. Effects of ultrasound on molecular properties, structure, chain conformation and degradation kinetics of carboxylic curdlan. Carbohydr. Polym. 2015, 121, 64–70. [Google Scholar] [CrossRef] [PubMed]
  17. Wan, J.; Wang, Y.; Jiang, D.; Gao, H.; Yang, G.; Yang, X. Effects of carbon sources on production and properties of curdlan using Agrobaterium sp. DH-2. Prep. Biochem. Biotech. 2020, 50, 857–864. [Google Scholar] [CrossRef] [PubMed]
  18. Ben Salah, R.; Jaouadi, B.; Bouaziz, A.; Chaari, K.; Blecker, C.; Derrouane, C.; Attia, H.; Besbes, S. Fermentation of date palm juice by curdlan gum production from Rhizobium radiobacter ATCC 6466™: Purification, rheological and physico-chemical characterization. LWT-Food Sci. Technol. 2011, 44, 1026–1034. [Google Scholar] [CrossRef]
  19. Mohsin, A.; Sun, J.; Khan, I.M.; Hang, H.; Tariq, M.; Tian, X.; Ahmed, W.; Niazi, S.; Zhuang, Y.; Chu, J. Sustainable biosynthesis of curdlan from orange waste by using Alcaligenes faecalis: A systematically modeled approach. Carbohydr. Polym. 2019, 205, 626–635. [Google Scholar] [CrossRef]
  20. Prakash, S.; Rajeswari, K.; Divya, P.; Ferlin, M.; Rajeshwari, C.T.; Vanavil, B. Optimization and production of curdlan gum using Bacillus cereus PR3 isolated from rhizosphere of leguminous plant. Prep. Biochem. Biotech. 2018, 48, 408–418. [Google Scholar] [CrossRef]
  21. Yuan, M.; Fu, G.; Sun, Y.; Zhang, D. Biosynthesis and applications of curdlan. Carbohydr. Polym. 2021, 273, 118597. [Google Scholar] [CrossRef] [PubMed]
  22. Yu, X.; Zhang, C.; Yang, L.; Zhao, L.; Lin, C.; Liu, Z.; Mao, Z. CrdR function in a curdlan-producing Agrobacterium sp. ATCC31749 strain. BMC Microbiol. 2015, 15, 25. [Google Scholar] [CrossRef] [PubMed]
  23. Gao, H.; Xie, F.; Zhang, W.; Tian, J.; Zou, C.; Jia, C.; Jin, M.; Huang, J.; Chang, Z.; Yang, X. Characterization and improvement of curdlan produced by a high-yield mutant of Agrobacterium sp. ATCC 31749 based on whole-genome analysis. Carbohydr. Polym. 2020, 245, 116486. [Google Scholar] [CrossRef]
  24. Liang, Y.; Zhu, L.; Ding, H.; Gao, M.; Zheng, Z.; Wu, J.; Zhan, X. Enhanced production of curdlan by coupled fermentation system of Agrobacterium sp. ATCC 31749 and Trichoderma harzianum GIM 3.442. Carbohydr. Polym. 2017, 157, 1687–1694. [Google Scholar] [CrossRef]
  25. Liang, Y.; Zhu, L.; Gao, M.; Zheng, Z.; Wu, J.; Zhan, X. Influence of Tween-80 on the production and structure of water-insoluble curdlan from Agrobacterium sp. Int. J. Biol. Macromol. 2018, 106, 611–619. [Google Scholar] [CrossRef] [PubMed]
  26. Yang, M.; Zhu, Y.; Li, Y.; Bao, J.; Fan, X.; Qu, Y.; Wang, Y.; Hu, Z.; Li, Q. Production and optimization of curdlan produced by Pseudomonas sp. QL212. Int. J. Biol. Macromol. 2016, 89, 25–34. [Google Scholar] [CrossRef] [PubMed]
  27. Wan, J.; Shao, Z.; Jiang, D.; Gao, H.; Yang, X. Curdlan production from cassava starch hydrolysates by Agrobacterium sp. DH-2. Bioproc. Biosyst. Eng. 2022, 45, 969–979. [Google Scholar] [CrossRef] [PubMed]
  28. Shin, H.-D.; Liu, L.; Kim, M.-K.; Park, Y.-I.; Chen, R. Metabolic engineering of Agrobacterium sp. ATCC31749 for curdlan production from cellobiose. J. Ind. Microbiol. Biot. 2016, 43, 1323–1331. [Google Scholar] [CrossRef]
  29. Gao, M.; Liu, Z.; Zhao, Z.; Wang, Z.; Hu, X.; Jiang, Y.; Yan, J.; Li, Z.; Zheng, Z.; Zhan, X. Exopolysaccharide synthesis repressor genes (exoR and exoX) related to curdlan biosynthesis by Agrobacterium sp. Int. J. Biol. Macromol. 2022, 205, 193–202. [Google Scholar] [CrossRef]
  30. Kerdsup, P.; Tantratian, S.; Sanguandeekul, R.; Imjongjirak, C. Xanthan production by mutant strain of Xanthomonas campestris TISTR 840 in raw cassava starch medium. Food Bioprocess Tech. 2011, 4, 1459–1462. [Google Scholar] [CrossRef]
  31. Xiao, Z.; Storms, R.; Tsang, A. A quantitative starch: Iodine method for measuring alpha-amylase and glucoamylase activities. Anal. Biochem. 2006, 351, 146–148. [Google Scholar] [CrossRef] [PubMed]
  32. Gao, H.; Zhang, W.; Zhang, J.; Huang, Y.; Zhang, J.; Tian, J.; Niu, Y.; Zou, C.; Jia, C.; Chang, Z. Methionine biosynthesis pathway genes affect curdlan biosynthesis of Agrobacterium sp. CGMCC 11546 via energy regeneration. Int. J. Biol. Macromol. 2021, 185, 821–831. [Google Scholar] [CrossRef]
  33. Szwengiel, A.; Wiesner, M. Effect of metal ions on levan synthesis efficiency and its parameters by levansucrase from Bacillus subtilis. Int. J. Biol. Macromol. 2019, 128, 237–243. [Google Scholar] [CrossRef] [PubMed]
  34. Yu, L.; Wu, J.; Liu, J.; Zhan, X.; Zheng, Z.; Lin, C.C. Enhanced curdlan production in Agrobacterium sp. ATCC 31749 by addition of low-polyphosphates. Biotechnol. Bioprocess Eng. 2011, 16, 34–41. [Google Scholar] [CrossRef]
  35. Sèbe, G.; Ham-Pichavant, F.; Pecastaings, G. Dispersibility and emulsion-stabilizing effect of cellulose nanowhiskers esterified by vinyl acetate and vinyl cinnamate. Biomacromolecules 2013, 14, 2937–2944. [Google Scholar] [CrossRef]
  36. Abe, J.-i.; Onitsuka, N.; Nakano, T.; Shibata, Y.; Hizukuri, S.; Entani, E. Purification and characterization of periplasmic alpha-amylase from Xanthomonas campestris K-11151. J. Bacteriol. 1994, 176, 3584–3588. [Google Scholar] [CrossRef] [PubMed]
  37. Yang, H.; Li, W.; Chen, S.; Guo, X.; Huang, F.; Zhu, P. Optimization and modeling of curdlan production under multi-physiological-parameters process control by Agrobacterium radiobacter mutant A-15 at high initial glucose. Biotechnol. Bioprocess Eng. 2021, 26, 1012–1022. [Google Scholar] [CrossRef]
  38. Liu, Z.; Xu, Y.; Wang, Z.; Zhu, L.; Li, Z.; Jiang, Y.; Zhan, X.; Gao, M. Promoting substrates uptake and curdlan synthesis of Agrobacterium sp. by attenuating the exopolysaccharide encapsulation. Carbohydr. Polym. 2023, 315, 120941. [Google Scholar] [CrossRef]
  39. Anane, R.F.; Sun, H.; Zhao, L.; Wang, L.; Lin, C.; Mao, Z. Improved curdlan production with discarded bottom parts of Asparagus spear. Microb. Cell Factories 2017, 16, 1–8. [Google Scholar] [CrossRef] [PubMed]
  40. West, T.P. Effect of nitrogen source concentration on curdlan production by Agrobacterium sp. ATCC 31749 grown on prairie cordgrass hydrolysates. Prep. Biochem. Biotech. 2016, 46, 85–90. [Google Scholar] [CrossRef]
  41. Wu, S.; Lu, M.; Fang, Y.; Wu, L.; Xu, Y.; Wang, S. Production of curdlan grown on cassava starch waste hydrolysates. J. Polym. Environ. 2018, 26, 33–38. [Google Scholar] [CrossRef]
  42. Binder, D.; Frohwitter, J.; Mahr, R.; Bier, C.; Grünberger, A.; Loeschcke, A.; Peters-Wendisch, P.; Kohlheyer, D.; Pietruszka, J.; Frunzke, J. Light-controlled cell factories: Employing photocaged isopropyl-β-d-thiogalactopyranoside for light-mediated optimization of lac promoter-based gene expression and (+)-valencene biosynthesis in Corynebacterium glutamicum. Appl. Environ. Microb. 2016, 82, 6141–6149. [Google Scholar] [CrossRef] [PubMed]
Fermentation 10 00240 g001
Figure 1. Molecular structure of curdlan.
Figure 1. Molecular structure of curdlan.
Fermentation 10 00240 g001
Fermentation 10 00240 g002
Figure 2. Fluorescence assays of ATCC31749-derived strains in which GFP expressions were regulated by different promoters. (A) GFP expression of ATCC 31749 with pBBR-Ptac-GFP. (B) GFP expression of ATCC 31749 with pBBR-PBAD-GFP. (C) GFP expression of ATCC 31749 with pBBR-Ptet-GFP. (D) GFP expression of ATCC 31749 with pBBR-PRha-GFP. (E) GFP expression of ATCC 31749 with pBBR-Pxyl-GFP.
Figure 2. Fluorescence assays of ATCC31749-derived strains in which GFP expressions were regulated by different promoters. (A) GFP expression of ATCC 31749 with pBBR-Ptac-GFP. (B) GFP expression of ATCC 31749 with pBBR-PBAD-GFP. (C) GFP expression of ATCC 31749 with pBBR-Ptet-GFP. (D) GFP expression of ATCC 31749 with pBBR-PRha-GFP. (E) GFP expression of ATCC 31749 with pBBR-Pxyl-GFP.
Fermentation 10 00240 g002
Fermentation 10 00240 g003
Figure 3. Construction of strain sp-AmyAXCC by introducing α-amylase from X. campestris NRRL B-1459 into Agrobacterium sp. ATCC31749. (A) PCR amplification of α-amylase encoding gene. Lane M: Trans2K Plus II DNA Marker; lane 1: positive control; lane 2: negative control; lane 3: PCR product with X. campestris NRRL B-1459 genomic DNA as the template. (B) Double enzyme digestion of plasmid pBBR-Ptac-α-amylase. Lane M: Trans2K Plus II DNA Marker; lane 1 and lane 2: pBBR-Ptac-α-amylase digested by EcoR I and Nde I. (C) Colony PCR validation of the transformants. Lane M: Trans5K DNA Marker; lane 1: positive control; lane 2: negative control; lane 3–6: PCR products of different colonies.
Figure 3. Construction of strain sp-AmyAXCC by introducing α-amylase from X. campestris NRRL B-1459 into Agrobacterium sp. ATCC31749. (A) PCR amplification of α-amylase encoding gene. Lane M: Trans2K Plus II DNA Marker; lane 1: positive control; lane 2: negative control; lane 3: PCR product with X. campestris NRRL B-1459 genomic DNA as the template. (B) Double enzyme digestion of plasmid pBBR-Ptac-α-amylase. Lane M: Trans2K Plus II DNA Marker; lane 1 and lane 2: pBBR-Ptac-α-amylase digested by EcoR I and Nde I. (C) Colony PCR validation of the transformants. Lane M: Trans5K DNA Marker; lane 1: positive control; lane 2: negative control; lane 3–6: PCR products of different colonies.
Fermentation 10 00240 g003
Fermentation 10 00240 g004
Figure 4. Validation of the extracellular expression of α-amylase by sp-AmyAXCC. Western Blot of the total proteins of ATCC31749 and sp-AmyAXCC culture supernatants. Lane M: Mw Marker; lane 1: ATCC31749 culture supernatant; lane 2: sp-AmyAXCC culture supernatant.
Figure 4. Validation of the extracellular expression of α-amylase by sp-AmyAXCC. Western Blot of the total proteins of ATCC31749 and sp-AmyAXCC culture supernatants. Lane M: Mw Marker; lane 1: ATCC31749 culture supernatant; lane 2: sp-AmyAXCC culture supernatant.
Fermentation 10 00240 g004
Fermentation 10 00240 g005
Figure 5. Fermentation characteristics of recombinant strain for curdlan production under glucose and dextrin substrate conditions.
Figure 5. Fermentation characteristics of recombinant strain for curdlan production under glucose and dextrin substrate conditions.
Fermentation 10 00240 g005
Fermentation 10 00240 g006
Figure 6. Profile of curdlan fermentation with sp-AmyAXCC using dextrin as carbon source.
Figure 6. Profile of curdlan fermentation with sp-AmyAXCC using dextrin as carbon source.
Fermentation 10 00240 g006
Fermentation 10 00240 g007
Figure 7. Comparison of the characteristics of curdlan produced by the recombinant strain and ATCC31749. (A) ATR-FTIR spectra of curdlan produced by the recombinant strain and ATCC31749; (B) XRD spectra of curdlan produced by the recombinant strain and ATCC31749.
Figure 7. Comparison of the characteristics of curdlan produced by the recombinant strain and ATCC31749. (A) ATR-FTIR spectra of curdlan produced by the recombinant strain and ATCC31749; (B) XRD spectra of curdlan produced by the recombinant strain and ATCC31749.
Fermentation 10 00240 g007
Table 1. Strains and plasmids.
Table 1. Strains and plasmids.
Strain or PlasmidRelevant CharacteristicsSource or Reference
Strains
Agrobacterium sp. ATCC31749Wild-type, capable of produce curdlanLab’s collection
X. campestris NRRL B-1459Wild-type, capable of produce xanthan gumLab’s collection
sp-AmyAXCCAgrobacterium sp. ATCC31749 harboring the plasmid pBBR-Ptac-α-amylaseThis study
E. coli DH5αSupE44, DlacU169(φ80lacZDM15) hsdR17, recA1, endA1, gyrA96, thi-1, relA1 λ-, used for gene cloneLab’s collection
Plasmid
pBBR-Ptac-GFP-fireflyExpression plasmid, with Ptac, Kan RLab’s collection
pBBR-Ptet-GFP-fireflyExpression plasmid, with Ptet, Kan RLab’s collection
pBBR-Pxyl-GFP-fireflyExpression plasmid, with Pxyl, Kan RLab’s collection
pBBR-PRha-GFP-fireflyExpression plasmid, with PRha, Kan RLab’s collection
pBBR-PBAD-GFP-fireflyExpression plasmid, with PBAD, Kan RLab’s collection
pBBR-Ptac-α-amylaseExpression plasmid, Kan R This study
Table 2. Oligonucleotides used in this study.
Table 2. Oligonucleotides used in this study.
PrimerSequence (5′-3′)Use
amy-F2CAATTTCACACAGGAGGAATTCATGAAAAAACTGGTCATCAdding signal peptide at the N’-terminus of α-amylase
amy-F1CAATAAACCGGTAAGCCATATGTCAGTGGTGGTGGTGGTGGTGAAmplification of α-amylase encoding gene (forward)
amy-R1CCTCCGCTGAAGCCGCCAGCGTCCGTATCGAGGAAmplification of α-amylase encoding gene (reverse)
amy-R2AGCCATATGGTGGTGGTGGTGGTGGTGTCATGGATGCGTCTCCTTAdding 6 × His-tag at the C’-terminus of α-amylase
Table 3. Curdlan production rates of Agrobacterium strain: comparison of substrates.
Table 3. Curdlan production rates of Agrobacterium strain: comparison of substrates.
StrainsSubstrateSubstrate(g/L)Titer (g/L)Yield (g Curdlan/g Substrate)Productivity (g/L/h)Reference
sp-AmyAXCCDextrin120.066.70.560.62This study
ATCC31749Sucrose90.038.60.430.40[23]
ATCC31749Sucrose90.048.00.790.33[23]
ATCC31749Maltose90.036.50.410.38[23]
ATCC31749Mannitol90.042.40.470.44[23]
ATCC31749Glucose80.021.90.440.30[24]
ATCC31749Wheat bran80.047.90.60.33[24]
ATCC31749Glucose50.014.40.290.20[38]
ATCC31749Cellobiose50.011.20.30.15[28]
ATCC31749 with pHCE-CBPCellobiose50.025.90.550.36[28]
ATCC31749Asparagus spear juice50.040.20.560.24[39]
ATCC31749Prairie cordgrass10.09.90.010.07[40]
ATCC31749Cassava starch waste20.021.20.400.22[41]
ATCC31749Glucose40.072.00.610.75[19]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yu, T.; Wang, Y.; Wang, W.; Zhang, Y.; Zhang, Y.; Han, H.; Liu, Y.; Zhou, S.; Dong, X. Optimizing Curdlan Synthesis: Engineering Agrobacterium tumefaciens ATCC31749 for Enhanced Production Using Dextrin as a Carbon Source. Fermentation 2024, 10, 240. https://doi.org/10.3390/fermentation10050240

AMA Style

Yu T, Wang Y, Wang W, Zhang Y, Zhang Y, Han H, Liu Y, Zhou S, Dong X. Optimizing Curdlan Synthesis: Engineering Agrobacterium tumefaciens ATCC31749 for Enhanced Production Using Dextrin as a Carbon Source. Fermentation. 2024; 10(5):240. https://doi.org/10.3390/fermentation10050240

Chicago/Turabian Style

Yu, Tingting, Yu Wang, Wei Wang, Yonggang Zhang, Yanmin Zhang, Hongyu Han, Yang Liu, Siduo Zhou, and Xueqian Dong. 2024. "Optimizing Curdlan Synthesis: Engineering Agrobacterium tumefaciens ATCC31749 for Enhanced Production Using Dextrin as a Carbon Source" Fermentation 10, no. 5: 240. https://doi.org/10.3390/fermentation10050240

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop