Part:BBa_K4941098

pet28a-KsXR-CM

The pet28a plasmid for KsXR expression with a CM resistance gene can be used for colony screening. lac I expresses a inhibitor and thus inhibits the target gene expression, which can be lifted by the addition of IPTG and induces KsXR gene expression.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 686
  Illegal XhoI site found at 879
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1228
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 257
  Illegal BsaI site found at 516
  

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.