Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same
一种乳酸酯、共聚物的技术，应用在生物化学设备和方法、植物学设备和方法、使用载体引入外来遗传物质等方向，能够解决不可用、PLA共聚物难以合成等问题
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Embodiment 1
[0037] Embodiment 1: the preparation of recombinant escherichia coli
[0038] The recombinant Escherichia coli W transformed with pPs619C1400CPPCT532ReAB plasmid was prepared. The Escherichia coli strain W (ATCC9637) according to this example can metabolize sucrose as a carbon source.
[0039] The pPs619C1400CPPCT532ReAB plasmid prepared for the present invention was constructed to express four main enzymes. These major enzymes are essential for the biosynthesis of poly(3-hydroxybutyrate-co-lactate), a Biodegradable polymer. These main enzymes can be phaC1 Ps6-19 400 (which is a polymerase from Pseudomonas sp.6-19 (KCTC 11027BP)), propionyl-CoA transferase (CP-PCT) from Clostridium propionicum (which is a polymerase that converts CoA from acetyl-CoA A transfers to lactate to be converted to lactyl-CoA), and ketothiolase (phaA) from Ralstonia eutropha RE ) and acetoacetyl-CoA reductase (phaB RE ) (which is an enzyme for the synthesis of 3-hydroxybutyl-CoA from sucrose). ...
Embodiment 1-1
[0041] Example 1-1: Preparation of substrate-specific mutants of PHA synthase by Pseudomonas sp.6-19
[0042] Among various PHA synthases, type II PHA synthases are known as medium-chain-length PHA (MCL-PHA) synthases for polymerizing substrates with relatively many carbon atoms, and it is expected that MCL-PHA synthases in It is very useful in the production of PLA copolymers. Although the phaC1 synthase from Pseudomonas sp.61-3 is a type II synthase, it differs from the phaC1 obtained according to the present invention Ps6-19 With a high degree of homology, it has been reported that phaC1 synthase has a relatively wide range of substrate specificity (Matsusaki et al., J.Bacteriol., 180:6459, 1998), and it has been reported that it is suitable for the production of short chain length PHA (SCL -PHA) mutants (Takase et al., Biomacromolecules, 5:480, 2004). Based on the above studies, as disclosed in Korean Patent Application No. 10-2006-0116234, the present inventors prepared...
Embodiment 1-2
[0120] Example 1-2: Preparation and screening of a mutant library of propionyl-CoA transferase from Clostridium propionicum
[0121] As known, when CP-PCT is overexpressed in E. coli, it causes severe metabolic disturbance and shows toxicity. Usually, in the isopropyl-βD-thio-galactoside (IPTG)-inducible protein expression system using tac promoter or T7 promoter (this system is widely used to express recombinant proteins), all the All recombinant E. coli died. Thus, PLA and PLA copolymers were successfully synthesized using a constitutive expression system that weakly but continuously expresses genes as the microorganism grows. In order to introduce random mutations in the cp-pct gene, pPs619C1300-CPPCT disclosed in Korean Patent Application No. 10-2006-0116234 was used as a template, adding Mn 2+ , using the primers of SEQ ID NO: 39 and 40, error-prone PCR was performed under the condition of varying dNTP concentration.
[0122] SEQ ID NO: 39: 5'-cgc cgg cag gcc tgc agg-3...
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