Part:BBa_K4941009

PsXR

Xylose reductase-encoding gene derived from Pichia stipites.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 601
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 601
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 601
  Illegal BglII site found at 89
  Illegal BglII site found at 210
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 601
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 601
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design

        Through literature reading, we selected xylose reductase from Kluyveromyces sp. IIPE453 (KsXR, BBa_K4941010, GenBank: KJ563917.1), xylose reductase from Pichia stipites (PsXR, BBa_K4941009, GenBank: X59465.1), and xylose reductase from Debaryomyces nepalensis NCYC 3413 (DnXR, BBa_K4941008, GenBank: KT239024.1).

Build

        In order to verify the catalytic performance of three different sources of xylose reductase in E. coli BL21, we need to construct three xylose reductase expression plasmids, including pET28a-KsXR-kana (BBa_K4941034), pET28a-PsXR-kana (BBa_K4941033), pET28a-DnXR-kana (BBa_K4941035). Here we take the process of constructing pET28a-PsXR as an example: i) Firstly, the DNA fragment of PsXR (GenBank: X59465.1) was amplified by PCR with primers PsXR_F and PsXR_R; ii) Next, the DNA fragment of the vector plasmid pET28a (BBa_K4941040) was amplified by PCR to obtain linearized pET28a; iii) Then, the linearized pET28a and PCR-amplified PsXR fragment were assembled by Gibson to obtain pET28a-PsXR; iv) The Gibson-assembled reaction mixture was transformed into Escherichia coli DH5α; and v) Positive transformants were sequenced by Sangon Biotech (Shanghai, China). In order to be able to screen transformants using the resistance screening markers, we replaced the kan resistance marker in the pET28a-PsXR plasmid using Gibson assembly (replaced with the Cm resistance marker).

Fig.1: a. Fragments of xylose reductase-encoding genes from three different sources; b. Gibson assembly;c. Positive transformants of pet28a- PsXR, pET28a-DnXR, and pET28a-PsXR; d. Sequence comparison results of constructed plasmids.

Result

        The correctly sequenced xylose reductase expression plasmids (pet28a-KsXR-kana (BBa_K4941034), pet28a-PsXR-kana (BBa_K4941033), pet28a-DnXR-kana (BBa_K4941035)) and T7 RNAP expression plasmids (BBa_K4941065) were transferred into E. coli BL21. Here is an example of transformation of T7RNAP plasmid (BBa_K4941065) and pet28a-PsXR- kana plasmid. i) Firstly, in order to be able to screen the transformants by resistance screening markers, we replaced the kan resistance marker in the pet28a-PsXR- kana plasmid by using Gibson's assembly method (replacing it with the Cm resistance marker) to obtain the strain pet28a-PsXR (BBa_K49410100) (using CM as the screening marker); ii) Next, the T7 RNAP expression plasmid PlacUV5 (BBa_K4941065) was transformed into BL21 and coated on LB plates containing kan resistance using the chemical transformation method; iii) The grown positive transformants were selected to be accessed into Kan-resistant 5 mL LB tubes for 3-4 h to make the receptor cells, and plasmid pet28a-PsXR was transformed into the above transformants using chemical transformation method and coated on LB plates containing Kan resistance and Cm resistance on LB plates; iv) Positive transformants were selected for colony PCR test confirmation and prepared for subsequent fermentation tests.

        The transformants were selected to be transferred into LB+Kana+CM liquid medium and cultured in shaker at 37℃ 220rpm for 12h. The seed solution was transferred into 30ml TB medium and cultured at 37℃ 220rpm for 3h, and then the expression of xylose reductase was induced by adding the inducer 30ul IPTG and then cultured at 28℃ 160rpm for 16h. Subsequently, xylose was added to 5g/L, and the reaction was performed at 30℃ 220rpm for 36h, and samples were taken every 12h for HPLC detection.

        The results showed that BL21- pet28a-PsXR catalyzed the production of 4.6 g/L xylitol from the substrate 5 g/L xylose after 36h. The xylose reductase derived from Pichia stipites was demonstrated to have the ability to catalyze xylose to produce xylitol in the E. coli BL21 protein expression system, and xylitol production by fermentation in Escherichia coli is realized.

fig.2 : a. Xylitol yields of xylitol-expressing strains expressing three xylose reductase enzymes; b. Standard curve of xylitol in HPLC assay.

Design

        With our newly constructed T7RNAP expression library based on the lacUV5 promoter mutant, we optimized the expression using the pET expression system against xylose reductase (PsXR, GenBank: X59465.1) of Pichia stipitis origin. This was done to find the most suitable expression intensity in order to further improve the conversion to xylose and increase xylitol production.

Build

        In order to verify the effect of three catalytic properties of the pET expression system on PsXR by modulating the intensity of T7RNAP expression, and thus affecting the catalytic properties of PsXR, we simultaneously transformed T7RNAP expression plasmids and pet28a-PsXR containing different lacUV5 promoters into E. coli BL21 hosts. Here is an example of the transformation process for the T7RNAP plasmid driven by PlacUV5MB7 (BBa_K4941056) and the pET28a-PsXR plasmid:

        i) Firstly, plasmid PlacUV5MB7-T7RNAP-T7t (BBa_K4941083) was transformed into BL21 using the chemical transformation method and plated onto LB plates containing kanamycin resistance;

        ii) Next, the positively grown transformants were selected and transferred into Kanamycin-resistant 5 mL LB tubes for 3-4 hours to allow the receptor cells to grow. The pET28a-PsXR plasmid was then transformed into the above transformants using the chemical transformation method and plated onto LB plates containing both Kanamycin and Chloramphenicol resistances;

        iii) Positive transformants were selected for colony PCR test confirmation and prepared for subsequent fermentation testing.

Result

        Three transformants containing different lacUV5 promoters were selected and transfected into 96-well culture plates containing LB liquid medium. The plates were then cultured in a shaker at 37 °C and 220 rpm for 12 hours. The seed solution was further transferred into 48-well culture plates containing 2 ml of fermentation medium and cultured at 37 °C and 220 rpm for 3 hours. Afterwards, 30 μl of IPTG was added as an inducer, and the culture was continued at 28 °C and 160 rpm for 16 hours to allow for the expression of xylose reductase.For xylose reductase expression, the plates were incubated at 16°C and 160 rpm for 16 hours. Subsequently, xylose was added to a final concentration of 8 g/L, and the reaction was carried out at 30°C and 220 rpm for 36 hours. HPLC detection was performed to analyze the reaction.

        The results showed that MB7-PsXR successfully catalyzed the production of 6.8 g/L of xylitol from the substrate of 8 g/L xylose after 36 hours. This outcome demonstrates the feasibility of the strategy employed, which involved optimizing the pET expression system through the construction of a promoter library for T7RNAP expression.

Fig.3: Xylitol production under different promoter variants.