Microbe for producing ethanol by utilizing light energy
A technology for ethanol and alcohol dehydrogenase, which is applied in the directions of microorganisms, microorganism-based methods, biofuels, etc., and can solve problems such as no ethanol production
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Embodiment 1
[0085] Embodiment 1 has the acquisition of the carrier pXT117 of antibiotic marker, promotor and Mobilis pdc gene
[0086] The commercially available pET28b (Novagen, USA) was used as the vector for construction.
[0087] The rbc promoter (ie, Prbc) nucleic acid sequence was synthesized according to the reported sequence information of Synechocystis (such as BA000022.2, X65960.1 of Genebank), and its sequence is shown in SEQ ID No.5.
[0088] The nucleic acid sequence of the pdc gene is synthesized according to the reported nucleic acid sequence of the pdc gene of Molecules (such as X59558.1 of Genebank), and its sequence is shown in SEQ ID No.2. In order to clone into pET28b and later construction needs, Spe I site and NotI site were added at the 3' end of the pdc gene sequence during synthesis.
[0089] In addition, an omega interposon with resistance genes against spectinomycin and streptomycin, namely the omega fragment (see Prentki, P., and H.M. Krisch; 1984; Invitro ins...
Embodiment 2
[0092] Example 2 Carrying out point mutation of 290Tyr of PDC protein
[0093] By point mutation technology, TAC on the pdc gene encoding PDC is mutated into ACC, thereby replacing tyrosine Tyr at position 290 of PDC with threonine Thr.
[0094] Use the pXT117 plasmid obtained in step 1 as a template to carry out overlap PCR (overlap PCR), and the primers used include:
[0095] Primer No. 1: 5'-ATGAGTTATACTGTCGGTACC-3';
[0096] Primer No. 2: 3'-TTCGGACAATTGTTCGAGGAGATC-5';
[0097] Primer No. 3: 3’-AGAAGTTGCTG TGG AGGTGGTGACCAA-5';
[0098] Primer No. 4: 5'-TCTTCAACGAC ACC TCCACCACTGGTT-3'.
[0099] The underlined part is the position used to generate the point mutation from TAC (encoding Tyr) to ACC (encoding Thr).
[0100] First use primers No. 1 and 3, and primers No. 2 and 4 to amplify the upper fragment and the lower fragment by PCR respectively. Then, the upper fragment and the lower fragment are used as templates at the same time, and the full-length fragments...
Embodiment 3
[0112] Example 3 Acquisition of shuttle plasmids for transforming cyanobacteria with antibiotic markers, promoters and the Molecularis pdc gene with 290Tyr point mutation
[0113] Digest pXT117-T290 with SphI and NotI, fill in the sticky interface, and recover the Ω-Prbc-pdc fragment.
[0114]The shuttle vector pKW1188SL used to transfect cyanobacteria was cut with EcoRI, and the plasmid was transformed into Escherichia coli DH5α (Escherichia coli, Escherichia coli). No. 3, Courtyard No. 1, the preservation date is October 14, 2011, and the preservation number is CGMCC No.5349. For its construction and structure, see Xiaoming Tan, LunYaoetc., 2011, Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria, Metabolic Engineering 13, 169-176), fill in with T4 DNAPolymerase, and recover a fragment of about 5.4kb.
[0115] The obtained fragment of Ω-Prbc-pdc was ligated with the fragment of pKW1188SL digested with EcoRI, and the shuttl...
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