78 research outputs found
Precise flux redistribution to glyoxylate shunt for efficient production of value-added chemicals
DoctorMetabolic engineering is redesigning the cellular metabolism for various purposes and has made a significant contribution to the development of biotechnology. For chemical production, several strategies involving overexpression of the product synthetic pathway, removal of the competing pathway, and expression optimization of a metabolic key node have been a powerful solutions. Apart from the technical issues, the most important prerequisite for efficient engineering is an in-depth understanding of cellular metabolism.
In this study, the characteristics of glyoxylate shunt were analyzed and exploited for chemical production in accordance with the principle of metabolic engineering. The glyoxylate shunt is a branch of the TCA cycle, and functionally it can convert the TCA intermediates with relatively simple enzymatic steps rather than the TCA cycle. In addition, an anaplerotic reaction is involved that allows a cell to grow with small carbon compounds. Despite such characteristics, it cannot be actively utilized in metabolic engineering and its utilization strategy was also limited. In this regard, the novel strategy to precisely regulate the glyoxylate shunt was designed and applied to the production of various chemicals.
The followings are summarized contents in this study.
First, the efficient conversion of acetate into valuable chemicals, itaconate and L-tyrosine, were described. Acetate is the promising carbon source in that it can be obtained much abundantly and cheaply from various sources, however, its growth inhibition issues have limited its applications for chemical production. In this study, such issues have been solved through the metabolic engineering-based strategies including screening of acetate-tolerant Escherichia coli and optimization of glyoxylate shunt pathway. The efficient production of itaconate, which can be produced from TCA intermediate was demonstrated. Furthermore, the strategies were effectively applied to the production of L-tyrosine from a glycolytic node, showing that the novel strategy designed in this study can be widely applied to the production of various chemicals from acetate.
Second, the novel strategy was applied to the production of 5-aminolevulinic acid (ALA) from glucose. The glyoxylate shunt was employed for TCA conversion instead of the native TCA cycle for precise flux redistribution, and it allowed significantly enhanced ALA production with robust cell growth. This achievement demonstrates that the strategy is not limited to the use of small carbon compounds such as acetate but can be used more broadly, showing enough potential of the strategy.λ°μ΄μ€ 리νμ΄λ리λ λ€μν λ°μ΄μ€λ§€μ€λ‘λΆν° μ μ©ν νν©λ¬Όμ΄λ μλμ§λ₯Ό λ§λλ 곡μ μ΄λ€. μμ μμμ κ³ κ°μ΄λ κ°κ²© λ³λκ³Ό κ°μ λ¬Έμ λ‘ λ°μ΄μ€ 리νμ΄λ리λ μ§μμ μΌλ‘ κ΄μ¬ λ°μ μμΌλ©°, μμΌλ‘ λ³΄λ€ λΉμ€ μλ μν μ μ°¨μ§νκ² λ κ²μ΄λ€. λ°μ΄μ€ 리νμ΄λ리λ λκ² λ―Έμλ¬Όμ λ°ν¨λ₯Ό μ΄μ©νλλ°, μμ°μ μ‘΄μ¬νλ λ―Έμλ¬Όμ λ¬Όμ§ λμ¬λ μμ λ€μ μμ₯κ³Ό μ¬μμ°μ μ΄μ μ΄ λ§μΆ°μ Έ μμ΄ λλΆλΆμ κ²½μ° λ―Έμλ¬Όμ λ¬Όμ§ λμ¬λ₯Ό μνλ λͺ©μ μ λ§κ²λ μ¬μ€κ³νλ μΌμ΄ νμμ μ΄λ€. ν΄λΉ μν μ μννλ νλ¬Έ λΆμΌκ° λμ¬ κ³΅νμ΄λ©°, μ΄λ¬ν μ΄μ λ‘ λ°μ΄μ€ 리νμ΄λ리 μ°μ
μ λμ¬ κ³΅νμ λ°μ κ³Ό ν¨κ» κ·Όκ°μ λ€μ Έμλ€.
λμ¬ κ³΅νμμ νΉμ νν©λ¬Ό μμ°μ μν΄μλ νν©λ¬Ό μμ° νλ‘λ₯Ό κ³Όλ°νμν€κ±°λ λΆμ°λ¬Ό μμ° νλ‘λ₯Ό ν¬ν¨ν λΆνμν λμ¬ νλ‘λ₯Ό μ κ±°μν€λ λ°©λ²μ ν΅ν΄ μνλ λμ¬ νλ¦μ μ¦νμν€λ μ λ΅μ΄ μ¬μ©λ μ μλ€. λΆνμν λμ¬ νλ‘κ° μΈν¬μ μ±μ₯κ³Ό κ°μ νμμ μΈ λμ¬ νλ‘μ μ°κ΄λμ΄ μμ κ²½μ°μλ μ μ μμ λ¨μ λ°νμ΄λ μ κ±°κ° μλ ν΄λΉ μ μ μλ€μ λ°ν μ΅μ νλ₯Ό ν΅ν΄ μνλ λμ¬ νλ¦μ λ³΄λ€ ν₯μμν¬ μ μλ€. ꡬ체μ μΌλ‘ μ§λμΉκ² λ§μ λμ¬ νλ¦μ΄ νν©λ¬Ό μμ°μΌλ‘ μ§νλ κ²½μ°μλ μΈν¬λΉ μμ°μ±μ μ΄λ μ λ ν₯μλ μ μμ§λ§ μΈν¬ μ±μ₯μ΄ μ¬νκ² μ ν΄λλ€λ μ μμ μ 체 μμ°μ±μ΄ λκ² μ μ§λ μ μκΈ°μ ν΄λΉ λ
Έλ μ μ μλ€μ μ κ΅νκ² μ‘°μ νκ³ μ΅μ ννλ μΌμ΄ νΉνλ μ€μνκ² μ¬κ²¨μ§λ€. λμ¬ κ³΅νμμ μ΄λ¬ν μ λ΅λ€μ΄ ν¨κ³Όμ μΌλ‘ μ μ©λκΈ° μν΄μ 무μ보λ€λ μ€μν κ²μ λ¨Όμ λμ¬ νλ‘λ₯Ό κΉκ² μ΄ν΄νλ κ²μ΄λ€.
λ³Έ μ°κ΅¬μμλ μ΄λ¬ν λμ¬ κ³΅νμ κΈ°λ³Έ μ리μ μ
κ°νμ¬, λ¨Όμ κΈλ¦¬μ₯μ€μ° νλ‘μ νΉμ±μ μ΄ν΄νκ³ λ€μν νν©λ¬Ό μμ°μ μ μ©νκ³ μ νλ€. κΈλ¦¬μ₯μ€μ° νλ‘λ TCA νλ‘μ κ³κ°μ§ νλ‘λ‘ λμ₯κ· , μλ¬Ό, κ³°ν‘μ΄μ μ‘΄μ¬νλ€. μ΄λ μ΄μμνΈλ μ΄νΈ 리μμ (isocitrate lyase)μ λ§μ° μ νμμ (malate synthase) λ ν¨μλ‘ κ΅¬μ±λλλ°, μ΄μμνΈλ μ΄νΈ 리μμ λ μ΄μμνΈλ μ΄νΈλ₯Ό μμ μ°(succinate)κ³Ό κΈλ¦¬μ₯μ€μ°(glyoxylate)λ‘ μ νμν€λ©°, λ§μ° μ νμμ λ κΈλ¦¬μ₯μ€μ°μ μμΈνΈ-μ‘°ν¨μ(acetyl-CoA)μ μ€ν©νμ¬ μ΅μ’
μ μΌλ‘ λ§μ°μ λ§λ λ€. μ΄λ₯Ό ν΅ν΄ μ΅μ’
μ μΌλ‘ μ΄μμνΈλ μ΄νΈκ° μμ μ°κ³Ό λ§μ°μΌλ‘ μ νλκ² λλ κ²μ΄λ©°, μ΄ κ²½μ° TCA νλ‘μλ λ€λ₯΄κ² νμ μ μ€μ΄ λ°μλμ§ μλλ€. λλΆμ΄, μμΈνΈ-μ‘°ν¨μμ μ€ν© λ°μμ ν¬ν¨νκ³ μκΈ° λλ¬Έμ λ체 보좩 λ°μ(anaplerotic reaction)μ μννλ€λ νΉμ±μ κ°μ§λ€. νΉν, ν΄λΉ λ°μμ μμΈνΈμ°κ³Ό κ°μ μμ μ κΈ° νν©λ¬Όμ λμ¬νλ λ° μμ΄μλ μ μΌν 보좩 λ°μμ μννκ² λμ΄ κ΅μ₯ν μ€μν μν μ μννλ€.
μ΄λ¬ν νΉμ±μλ λΆκ΅¬νκ³ , κΈλ¦¬μ₯μ€μ° νλ‘μ νμ©μ κΈλ¦¬μ₯μ€μ°μ΄ μλ €μ§ μμλ
λμ κ΅μ₯ν μ νμ μΌλ‘ μ΄λ£¨μ΄μ Έ μλ€. κΈλ¦¬μ₯μ€μ° νλ‘ μ μ μλ€μ μ μ¬ μ΅μ μΈμμΈ iclR μ μ μμ μ κ±°λ₯Ό ν΅ν΄ μμ½κ² κΈλ¦¬μ₯μ€μ° νλ‘λ₯Ό μ¦νμν¬ μ μλ€κ³ μλ €μ‘κ³ , μ΄λ₯Ό νμ©ν λ¨μ νλ‘ μ¦νλ§μ΄ νν©λ¬Ό μμ°μ μ μ©λκ³€ νλλ°, κ·Έ μμ μμ μ°μ΄λ ν¨λ§λ₯΄μ°(fumaric acid)κ³Ό κ°μ TCA νλ‘μ μ§μ μ μΈ μ°κ΄μ κ°λ νν©λ¬Όλ€μ κ΅νλμλ€. μ΄λ μ¬μ€ ν΄λΉ μ λ΅μ ν΅ν΄μλ κΈλ¦¬μ₯μ€μ° νλ‘λ₯Ό μ κ΅νκ² μ‘°μ ν μ μκΈ° λλ¬Έμ΄λ©° λ³Έ μ°κ΅¬μμλ μλ‘μ΄ μ λ΅μ ν΅ν΄ λ€μν νν©λ¬Ό μμ°μ ν¨μ¨μ μΌλ‘ κΈλ¦¬μ₯μ€μ° νλ‘λ₯Ό νμ©ν΄λ³΄κ³ μ νμλ€.
νλμ νμ©μ μν΄μλ κΈλ¦¬μ₯μ€μ° νλ‘λ₯Ό μ κ΅νκ² μ‘°μ νλ κ²μ΄ νμμ μ΄λΌ νλ¨νμμΌλ©°, μ΄λ₯Ό μν΄μλ κΈλ¦¬μ₯μ€μ° νλ‘μ 첫 λ°μμ 맀κ°νλ μ΄μμνΈλ μ΄νΈ 리μμ μ λ°νμ μ κ΅νκ² μ‘°μ νκ³ μ νλ€. μ΄λ λ¨μν ν΄λΉ ν¨μκ° μ²« λ°μμ 맀κ°νλ κ² μ΄μΈμλ λλ¨Έμ§ λ°μμ λΉν΄ μ΄μνμ μΌλ‘ λΉνΈμμ μ΄λΌκ³ μλ €μ Έ μκΈ° λλ¬Έμ΄λ€. ν΄λΉ μ μ μμ μ λ° λ°ν μ‘°μ μ ν©μ±μλ¬Όνμ λꡬλ₯Ό νμ©νμ¬ μ μ¬ λ 벨μμ μ§νλμκ³ μ€μ λ‘ κΈλ¦¬μ₯μ€μ° νλ‘μ μ λ°μ μΈ νλ¦μ΄ ν΄λΉ ν¨μ λ°νμ λ°λΌ ν¨μ¨μ μΌλ‘ μ‘°μ λλ κ²μ νμΈν μ μμμΌλ©°, λ€μν νν©λ¬Ό μμ°μ μ§μ μ μ©ν΄λ³΄κ³ μ νλ€.
λ¨Όμ , μ°¨μΈλ νμμμΈ μμΈνΈμ°μ νμ©ν νν©λ¬Ό μμ°μ μ μ©ν΄λ³΄κ³ μ νλ€. μμΈνΈμ°μ κ΅μ₯ν νλΆνκ³ κ°μΈκ² μ»μ΄μ§ μ μλ νμμμΌλ‘ κ²½μ μ± λΆμΌμμ νΉν κ°κ΄λ°λ μ°¨μΈλ νμμμ΄λ λ―Έμλ¬Όμ μ±μ₯μ μ ν΄νλ€λ μ μ κ·Έ νμ©μ΄ μ νλμ΄ μλ€. λ³Έ μ°κ΅¬μμλ κΈλ¦¬μ₯μ€μ° νλ‘μ μ κ΅ν μ‘°μ λ° λ€μν λμ¬ νλ¦ μ΅μ νλ₯Ό ν΅ν΄ μμΈνΈμ°μ λμ¬λ₯μ ν° νμΌλ‘ ν₯μμν¬ μ μμμΌλ©°, μ΅μ’
μ μΌλ‘ κ³ λΆκ°κ°μΉλ¬Όμ§μΈ μ΄νμ½μ°κ³Ό νμ΄λ‘μ μμ°μ±μ κ°κ° ν¬κ² ν₯μμν¬ μ μμλ€. μ΄ κ³Όμ μμ μμΈνΈμ°μ μ μ¬μ νμ© κ°λ₯μ±μ λλ³΄μΌ μ μμμΌλ©°, νΉν νμ΄λ‘μ μ κ²½μ°μλ TCA νλ‘κ° μλ ν΄λΉ νλ‘(glycolytic pathway)μ μ€κ°μ²΄λ‘λΆν° νμλμ΄ μμ°λλ νν©λ¬Όμ΄λΌλ μ μμ λ³Έ μ°κ΅¬μ μ λ΅μ΄ ν λκ² νμ©λ μ μμμ 보μλ€.
λλΆμ΄, λ³Έ μ°κ΅¬μ μ λ΅μ κΈλ£¨μ½μ€μ€(glucose)λ‘λΆν° 5-μλ―Έλ
Έλ λΆλ¦°μ°(5-aminoluvulinic acid) μμ°μλ μ μ©μμΌ λ³΄κ³ μ νλ€. ꡬ체μ μΌλ‘, 5-μλ―Έλ
Έλ λΆλ¦°μ° μμ° κ³Όμ μμ μκΈ°λ TCA λΆκ· νμ κΈλ¦¬μ₯μ€μ°μ λμ
λ° μ΅μ νλ₯Ό ν΅ν΄ ν΄κ²°νκ³ μ νμμΌλ©°, μ΄λ₯Ό ν΅ν΄ μΈν¬ μ±μ₯κ³Ό 5-μλ―Έλ
Έλ λΆλ¦°μ° μμ°μ΄ ν° νμΌλ‘ ν₯μλλ κ²μ νμΈν μ μμλ€. μ΄λ¬ν κ²°κ³Όλ₯Ό ν΅ν΄ κΈλ¦¬μ₯μ€μ° νλ‘μ νμ©μ΄ μμΈνΈμ°κ³Ό κ°μ μμ μ κΈ°μ°μ λμ¬λΏ μλλΌ λ€μν νμμμΌλ‘λΆν°μ νν©λ¬Ό μμ°μ μν μ κ΅ν νμ νλ¦ μ¬λΆλ°°μλ μ μ©λ μ μμμ 보μλ€
Multiple approach based on syntetic biology for engineering Escherichia coli to produce coenzyme B12
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