Novel malonyl-CoA biosensor based on type III polyketide synthase, and use thereof
A technology of polyketide synthase and malonyl, which is applied in the direction of acyltransferase, biochemical equipment and methods, measurement/testing of microorganisms, etc., and can solve the problems that autofluorescent microorganisms cannot be used
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Embodiment 1
[0304] Example 1. Construction of malonyl-CoA biosensor based on type III polyketide synthase
[0305] 1.1. Performance testing of various type III polyketide synthases
[0306] Restriction enzymes used in this and the following examples were purchased from New England Labs (USA) or Enzynomics (Korea), and PCR polymerase was purchased from Biofact (Korea). other individual tags. In addition, the foreign genes introduced in this Example and the following Examples are summarized in Table 1 below together with the accession numbers and their sequences in databases containing information thereon. Also, unless otherwise stated, the E. coli used for cloning was the DH5α strain, which was grown in LB medium (per liter: 10 g tryptone, 5 g yeast extract, and 10 g NaCl) or on LB agar plates (1 / 5 %, v / v). Additionally, add appropriate concentrations of antibiotics (50 μg / mL kanamycin, 100 μg / mL ampicillin and / or 100 μg / mL spectinomycin) if needed.
[0307] In the present invention,...
Embodiment 2
[0363] Example 2. High Throughput Screening of Strains with Increased Malonyl-CoA Production Ability by Using the RppA Biosensor
[0364] 2.1. By introducing a synthetic control sRNA library of Escherichia coli genome scale to have increased malonyl- High-throughput screening of strains capable of producing CoA
[0365] After constructing the RppA malonyl-CoA biosensor, confirming its successful operation, and demonstrating the applicability and scalability of the biosensor, the synthetic control sRNA technology developed by the present inventors was introduced to screen for increased Malonyl-CoA producing strains (KR 10-1575587, US 9388417, EP13735942.8, CN 201380012767.X, KR10-1690780, KR 10-1750855, US 15317939, CN201480081132.X; Na13 et al. (201480081132.X; ), Nat Biotechnol 31:170-174; Yoo SM, Na D, Lee SY (2013), Nat Protoc 8:1694-1707). In addition, to include all major genes in E. coli, a previously constructed E. coli genome-scale synthetic control sRNA library (...
Embodiment 3
[0371] Example 3. Increased yield of useful product using knockdown gene targets screened by RppA biosensor
[0372] Unless otherwise specified, the flask culture conditions used in this example and the following examples are as follows. The colonies were inoculated into test tubes containing 10 mL of LB medium, and cultured in an incubator at 37 °C and 200 rpm. 1 mL of cell culture was inoculated into a baffled flask containing 50 mL of modified R / 2 medium. When the strain that has been grown at 37 °C and 200 rpm reaches an OD of 0.8 600 When the value was reached, the strain was treated with 0.5 mM IPTG to induce production, and it was cultured at 30° C. and 200 rpm for 48 hours. Add glycerol or glucose as carbon source.
[0373] 3.1. Increased 6-methylsalicylic acid production using selected knockdown gene targets
[0374] In the next step, the knockdown gene targets selected in the above examples and capable of increasing malonyl-CoA production are used to produce us...
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