Patent Application
20240158818
This application claims priority to and the benefit of Korean Patent Application No. 10-2022-0102333 filed in the Korean Intellectual Property Office on Aug. 16, 2022, the entire content of which is incorporated herein by reference.
The sequence listing that is contained in the file named “Sequence_Listing_M347.0002US1.xml” which is 3 kilobytes as measured in Microsoft Windows operating system and was created on Jan. 10, 2024, and is filed electronically herewith and incorporated by reference.
The present disclosure relates to a recombinant E. coli strain that produces xylitol from xylose, a method for preparing the same, and a use thereof.
Xylitol is in high demand worldwide, primarily because of its widespread use in the food and pharmaceutical industries. In addition, xylitol may be utilized as a building block molecule for the production of high value-added chemicals.
Currently, the industrial production of such xylitol is made through chemical synthesis involving the catalytic hydrogenation of xylose. However, when xylitol is produced by chemical reaction, there are issues such as yield reduction due to side reaction products, high energy consumption, and high temperature and high pressure conditions. Accordingly, the production of biotechnological xylitol has emerged as a promising low-cost, sustainable alternative to chemical synthesis.
Xylitol may be created naturally by microorganisms (for example, molds, yeasts and bacteria). Natural xylitol producers mainly include yeast species such as Pichia stipitis, Hansenula polymorpha TCC 34438, Kluyveromyces marxianus IMB5, Meyerozyma guilliermondii, Debaryomyces hansenii, Candida guilliermondii, and Kluyveromyces marxianus (CCA 510). In yeast and other xylitol-producing microorganisms, xylose is catalyzed to xylitol via NADH or NADPH-dependent xylose reductase (XR). Xylitol is an intermediate extruded out of cells during xylose metabolism through the oxidoreductase pathway. Extrusion of xylitol is due to a cofactor imbalance between NADPH-dependent XR and NAD+-dependent xylitol dehydrogenase (XLH). The latter catalyzes the reaction of xylitol to xylulose, which is phosphorylated to xylulose-5-phosphate and eventually assimilated into central carbon metabolism (CCM).
Consumption of xylitol by natural producers makes it difficult to produce xylitol in high yield. Accordingly, further engineering of natural producers is necessary to improve xylitol accumulation and avoid xylitol assimilation into CCM. However, some yeast species, such as P. stipites and M. guilliermondii, are considered unsafe, limiting their use in the food industry. These natural producers are difficult to genetically engineer due to limited genetic information and customized genetic engineering tools.
The use of E. coli as a host for xylitol production has the advantages of simple culture conditions and rapid recombinant protein expression. However, xylitol production in E. coli requires insertion of heterologous XR, and not all heterologous XRs, especially yeast-derived XRs, show optimal expression in bacterial strains. Compatible and having optimal expression are native or endogenous genes often found in host strains. However, no endogenous enzyme catalyzed by xylitol has been known to date.
An aspect of the present disclosure is directed to providing a recombinant E. coli strain (transformed E. coli strain) capable of producing xylitol from xylose by expression of an endogenous enzyme.
Another aspect of the present disclosure is directed to providing a method for producing the recombinant E. coli strain.
Another aspect of the present disclosure is directed to providing a method for producing xylitol from xylose using the recombinant E. coli strain.
As a result of intensive research efforts to overcome the issues of the related art, the present inventors have found that plasmid-based expression of the endogenous YahK gene encoding NADPH-dependent aldehyde reductase (ALR) in E. coli enables xylitol production. The present disclosure is the first to identify an endogenous enzyme called YahK as a catalyst for xylitol production. In addition, in contrast to the existing knowledge that the xylose isomerase pathway (XIP) is the only xylose metabolic pathway in wild-type E. coli, the present disclosure further increased productivity of xylitol by using a strain blocking the pathway.
According to one aspect of the present disclosure, there is provided an expression vector including a gene encoding a YahK enzyme.
As used herein, the term “expression vector” refers to a genetic construct including essential regulatory elements such as a promoter so that a target gene may be expressed in a suitable host cell. According to an embodiment of the present disclosure, the expression vector may be a plasmid.
According to an embodiment of the present disclosure, the gene may have a nucleotide sequence of SEQ ID NO: 1, but considering the degeneracy of the genetic code, the gene may be a gene having 80% of homology, preferably 85% of homology, more preferably 90% of homology, and most preferably 95% of homology with the nucleotide sequence represented by SEQ ID NO: 1.
According to an aspect of the present disclosure, there is provided a transformant transformed with an expression vector including a gene encoding the YahK enzyme.
According to an embodiment of the present disclosure, the transformant may have an ability to produce xylitol from xylose.
According to an embodiment of the present disclosure, the transformant may have a blocked xylose isomerase pathway, and xylitol productivity from xylose may be further increased by using a transformant in which the xylose isomerase pathway is blocked as described above.
According to an embodiment of the present disclosure, the transformant may be Escherichia coli, for example, E. coli W3310. In addition, the transformant may be a recombinant E. coli strain in which the YahK enzyme is overexpressed and the xylose isomerase pathway is blocked. According to an embodiment of the present disclosure, the transformant may be E. coli W3310 ΔxylAB pTRCHIS2A-yahK of strain accession number KCTC15024BP.
According to another aspect of the present disclosure, there is provided a method for producing xylitol, wherein the method includes: culturing a transformant transformed with the expression vector including the gene encoding the YahK enzyme in a substrate containing xylose (stage 1); and obtaining the xylitol from the culture cultured in stage 1 (stage 2).
The transformant may refer to what has been described in relation to the transformant above, and may preferably be Escherichia coli, for example, E. coli W3310. Preferably, the transformant may be a transformant overexpressing the YahK enzyme and blocking the xylose isomerase pathway, more preferably E. coli W3310 ΔxylAB pTRCHIS2A-yahK of strain accession number KCTC15024BP.
As a method for culturing the transformant of an embodiment of the present disclosure, a conventional method used for culturing a host may be used. As the culture method, any method used for culturing ordinary microorganisms, such as batch, fluidized batch, continuous culture, reactor, etc., may be used. As a medium for culturing a transformant obtained by using a bacterium such as E. coli as a host, a complete medium or a synthetic medium such as LB medium and NB medium may be exemplified.
Carbon sources are necessary for the growth of microorganisms, and include, for example, saccharides such as glucose, fructose, sucrose, maltose, galactose, and starch; lower alcohols such as ethanol, propanol, and butanol; polyhydric alcohols such as glycerol; organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid, and gluconic acid; and fatty acids such as propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid. According to an embodiment of the present disclosure, the substrate in stage 1 may further contain glycerol. According to an embodiment of the present disclosure, the glycerol may be contained in an amount of 0.2 to 0.8% (v/v).
According to an aspect of the present disclosure, there is provided a composition for producing xylitol including the aforementioned transformant. The composition of an embodiment of the present disclosure may include, for example, a polypeptide, broth, and cell lysate suitable for culturing the transformant to produce xylitol.
According to an embodiment of the present disclosure, a recombinant E. coli strain (E. coli W3110ΔxylAB pTRCHIS2A-yahK) overexpressing the YahK enzyme and blocking the xylose isomerase mechanism was developed and succeeded in biosynthesizing xylose into xylitol. According to an embodiment of the present disclosure, xylitol may be produced from xylose in a one-stage biosynthetic reaction. In addition, since there is no production of by-products and the reaction occurs at room temperature, issues of the related art due to chemical reactions, such as yield reduction due to side reaction products and high temperature conditions, may be solved.
Hereinafter, the present disclosure will be described in more detail through examples. Objects, features and advantages of the present disclosure will be easily understood through the following examples. The present disclosure is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced herein are provided to sufficiently convey the spirit of the present disclosure to those skilled in the art to which the present disclosure pertains. Accordingly, the present disclosure should not be limited by the following examples.
[Construction of Strains and Plasmids]
All strains and recombinant plasmids used in this experiment are listed in Table 1 below. Previously constructed strains were reused and recombinant plasmids were transformed using a one-step TSS (transformation and storage solution) protocol [Chung C T, Niemela S L, Miller R H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA. 1989 April; 86(7):2172-5. doi: 10.1073/pnas.86.7.2172.].
E. coli W3110 ΔxylAB; pTrcHis2A_yahK
Purification of YahK
pTrcHis2A having a yahK gene including a 6×-histidine tag at the C-terminus was transformed into E. coli W3110 (DE3) (see
Shake-Flask Fermentation
The modified M9 (MM9) medium was used, including 1×M9 medium with 1 g/L peptone, 0.5 g/L yeast extract and 1.0 mM MgSO4. For shake flask fermentation, a 300 mL Erlenmeyer flask containing 100 mL of MM9 with appropriate antibiotics and specific sugar substrate(s) was used. Each flask was inoculated with 1 mL of the overnight culture and incubated for 72-96 hours at 37° C. with agitation at 180 rpm. On the other hand, a similar method was applied to scale-up shake flask fermentation except that a 500 mL baffled Erlenmeyer flask containing 200 mL of MM9 with appropriate antibiotics and sugar substrate inoculated with 2 mL of the overnight culture was used. 1 mL samples were collected at designated time intervals to monitor growth and metabolite concentrations as described in previous studies (Cabulong et al., 2017, 2018).
Checking of Xylitol
Xylitol accumulation was identified using GC-MSD and HPLC analysis. GC-MSD analysis was performed using the method described in Valdehuesa et al. (2014) with minor modifications. Briefly, 500 μL of clarified fermentation broth was acidified by adding 80% (v/v) of sulfuric acid. The acidified solution was lyophilized and dissolved in 250 μL of pyridine. The pyridine mixture was then derivatized by adding 250 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and incubated at 70° C. for 30 minutes. The prepared samples were then analyzed using an HP 6890 GC/HP5973 Mass Selective Detector GC-MSD. A 1.0 μL sample was injected at a 50:1 split ratio with a helium carrier gas at a flow rate of 2.0 mL/min. Then, the GC-MS chromatograms of the fermentation samples were compared with an existing in-house standard database and a database of commercially available xylitol. Meanwhile, HPLC analysis of the fermentation broth was compared with standard xylitol using the method described above.
Bioreactor Experiment
Bioreactor studies were performed in a 5 L bioreactor with 2 L of MM9 supplemented with specific antibiotic(s) and sugar substrate(s). 2 mL of starter cultures collected from colonies on LB-Agar medium and cultured in 5 mL LB medium were transferred to flasks with 100 mL LB medium. The flask was incubated overnight at 37° C. with stirring at 180 rpm. Cells were harvested by centrifugation and resuspended in 100 mL of MM9 prior to inoculation into the bioreactor. Fermentation parameters were controlled at 37° C., pH 7, 250 rpm agitation and airflow of 0.5 vvm. The pH was adjusted using 1N NaOH and 4N H2SO4 solution.
Whole-Cell Biocatalyst Experiment
Cells were reanimated in an LB-Agar medium and incubated for 12 hours at 37° C. Colonies from LB-agar were inoculated into 15 ml of conical tubes containing 5 ml of an LB medium and cultured for 12 hours at 37° C. with stirring at 180 rpm. 2 mL of the starter culture was then transferred to a 500 mL baffled Erlenmeyer flask containing 200 mL of an LB medium and appropriate antibiotics. Cultures were incubated according to the conditions mentioned above. IPTG was added when the optical density of the culture reached 0.3-0.4 at 600 nm for induction. After 12 hours, cells were harvested by centrifugation at 4° C. and washed twice with 50 mM of Tris-HCl buffer (pH 7.0). Cells were then resuspended in an MM9 medium and specific amounts of cells were inoculated into 500 mL of baffled Erlenmeyer flasks with 200 mL of an MM9 medium containing specific sugar substrate(s) and antibiotics to adjust the OD at 600 nm. After 24 hours of incubation, a 1 ml sample was collected for analysis.
Optimization of ethylene glycol (EG) production in E. coli strain W3110 via the Dahms pathway requires overexpression of an efficient aldehyde reductase (ALR) gene that catalyzes the conversion of glycoaldehyde to ethylene glycol. Accordingly, candidate ALR genes yahK, yjgB and yqhD were screened to optimize EG production in E. coli. Each gene amplified from the genome of E. coli strain W3110 was cloned into the pTRCHIS2A vector downstream of the trc promoter. Unexpectedly, 1.89 g/L and 0.84 g/L of xylitol were detected from 4 g/L D-xylose in E. coli strains overexpressing yahK and yqhD, respectively, after 72 hours of fermentation, but only minimal amounts of EG were produced (
Subsequently, xylitol produced in XOL1 and EG3 was further checked through GC-MSD and HPLC analysis. Briefly, the peak at 16.938 minutes detected through HPLC analysis of the fermentation specimen and the fragmentation pattern observed through GC-MSD analysis were the same peaks as commercially available xylitol and the fragmentation pattern of xylitol, identifying from the database that the product was xylitol (see
The xylose reductase activity of YahK was measured by monitoring the oxidation of NADPH at 340 nM with minor modifications to the procedure of Sche and Horecker (1996). A total volume of 1.0 mL was prepared containing 100 mM of D-xylose, 1.15 mM of NADPH, 50 mM of Tris-HCl (pH 7.5), and enough enzyme to create an absorbance change between 0.02 and 0.1 ΔE per minute at 37° C. Specific activity is expressed in units per milligram of protein (U/mg protein), where unit (U) is defined as micromol NAD(P)H reduced or oxidized per minute. The optimal pH for YahK was determined at various pH conditions using the following buffers. 50 mM of citrate (pH 3.0-6.0), 50 mM of Tris-Cl (pH 7.0-8.0), and 50 mM of glycine-NaOH (pH 9.0-10.0) at 37° C. The influence of temperature was measured in 50 mM of Tris-Cl (pH 7.5) at various temperatures (20, 30, 40, 50 and 60° C.) using the same mixture. In addition, substrate specificity of YahK for other sugars was performed using the conditions mentioned. Kinetic parameters of xylose reductase relative to D-xylose were determined using 1-100 mM of xylose under the conditions described above.
Substrate Specificity of YahK
The activity of YahK was tested in the presence of several different sugars. The results are graphically shown in
Activity Depending on Temperature and pH
YahK activity was tested according to temperature or pH change, and the results are shown in B and C of
Removal of the natural xylose pathway in E. coli to alter the xylose metabolic pathway destabilizes access to the pentose phosphate pathway, thus impeding carbon flow towards the formation of precursors important for cofactors, cell metabolism, cell maintenance, redox homeostasis and regulation of oxidative stress, resulting in lower yields of target products. YahK of D-xylose enhanced xylitol production through a synergistic action with the xylose isomerase pathway and the Dahms pathway.
The XOL1 strain showed rapid consumption of xylose within 6 hours, then slowed down, while xylitol production started after 24 hours (
The XOL2 strain in which XIP was intact and XR was overexpressed showed an increase in xylitol production and a slow and constant decrease in xylose over time (
The XOL2 strain produced 4 g/L of xylitol, showing higher productivity than the XOL1 strain, and as expected, rapid biomass accumulation peaking at 24 hours was observed (
Overall, the low xylitol titer in the engineered strains are due to the presence of a xylose pathway that competes with xylose as the sole carbon source. It is also noteworthy that even when additional carbon sources are available, it is still impossible to obtain high concentrations of xylitol due to the presence of other xylose pathways.
To increase xylitol titers from xylose, the natural xylose isomerase pathway was blocked to allow xylose to be metabolized only for xylitol production. The strain was constructed by transforming pTRCHIS2A-yahK into an ECI strain to create the XOL3 strain. Since xylose is only used for xylitol production, additional substrates are required to support the production of cofactors or precursors required for growth and cell maintenance of the engineered strain. The supply of NADPH, an important cofactor for YahK to catalyze the conversion of D-xylose to D-xylitol, also comes from cosubstrates. Glycerol and glucose were compared as potential cosubstrates for xylitol production.
Briefly, 0.4% v/v of glycerol or 2.5 g/L of glucose was added to an MM9 medium containing 10 g/L of D-xylose. As shown in
The XOL2 and XOL3 strains underwent scale-up and fed-batch fermentation in 5-L fermentors, respectively. In this experiment, IPTG was not added to rule out whether the periodic addition of glycerol and the presence of the xylose isomerase pathway in E. coli strains might increase xylitol production.
In the case of the XOL2 strain (in other words, a strain having a xylose isomerase pathway, E. coli W3310 pTRCHIS2A-yahK), it was produced from 22.04 g/L of xylitol within 48 hours from 50 g/L of D-xylose, reaching 44% of the theoretical yield with a productivity of 0.61 g/L/h (
The periodic supply of 0.5% (v/v) of glycerol every 48 hours also tested in the XOL3 strain (in other words, a strain deficient in the xylose isomerase pathway, E. coli W3310 ΔxylAB pTRCHIS2A-yahK). After 84 hours, 20.77 g/L of xylitol was produced from 25 g/L of xylose, leaving a residual 4 g/L of xylose (
The very high xylitol titers and yields created after fed-batch fermentation may be attributed to the deletion of xylB in the XOL2 strain, which prevents accumulation of the toxic intermediate xylitol-5-phosphate. XylB of E. coli is known to phosphorylate xylitol to xylitol phosphate, inhibiting the growth of E. coli on D-xylose and reducing xylitol production (Akinterinwa and Cirino, 2009).
As may be understood in the production of xylitol from D-xylose, yahK expression is possible even in the absence of an inducer. However, since yahK expression is located downstream of the IPTG-inducible trc promoter, the addition of IPTG may substantially increase xylitol production in the XOL3 strain. However, too much protein may cause toxicity and metabolic constraints on the host, so it is necessary to determine the appropriate amount of IPTG to increase the YahK concentration at an appropriate level.
E. coli W3310 ΔxylAB pTRCHIS2A-yahK was cultured in M9 minimal medium with 30 g/L of xylose and 0.6% v/v of glycerol. Among the tested IPTG concentrations, those without IPTG (
Xylitol production through whole cell biocatalyst (WBC) has many advantages over cell-free biosynthesis and general fermentation processes. Unlike cell-free biosynthesis, enzyme purification is not required and the addition of exogenous cofactors is not required. This is because cells have unique machinery for cofactor production and recycling, making the process economically feasible (
Both glucose and glycerol were tested as cosubstrates for xylitol production (
In the next experiment, it has been tested whether reducing the cosubstrate at various IPTG concentrations using 1% of glycerol (v/v) would give the same or better results. To this end, the IPTG concentration was varied between 0 mM and 1 mM (
Although the above has been described with reference to preferred embodiments of the present disclosure, those skilled in the art will understand that various modifications and changes may be made without departing from the spirit and scope of the present disclosure as described in the claims below.
Depository Institution: Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Biotechnology and Bioscience
Accession No.: KCTC15024BP
Deposit Date: 20220701
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0102333 | Aug 2022 | KR | national |
