Sustainable production of Bio-based Succinic acid by Engineered Actinobacillus succinogenes from Lignocellulosic biomass = Lignocellulosic biomass 로부터 대사공학적으로 설계된 Actinobacillus succinogenes 에 의한 바이오 기반 숙신산의 지속 가능한 생산

국문 초록 (Abstract)

숙신산은 식품 첨가물, 식이 보조제, 생분해성 고분자의 전구체로써 핵심적인 역할을
하는 산업적으로 중요한 성분이다. 환경 및 경제적 문제로 인해 바이오 기반 숙신산
생산은 더욱이 주목을 받고 있다. 숙신산을 생산하기 위해 많은 미생물이
사용되었는데 본 연구에서는 숙신산을 생산하는 천연 세균인 Actinobacillus
succinogenes 를 조작하고자 한다. 첫째, 숙신산 생합성 경로의 유전자를 가진
플라스미드를 A. succinogenes 130Z 에 도입하였다. 생성된 균주는 숙신산을 생산을
개선하고 야생형 균주보다 1.3 배 더 많은 숙신산을 생산된 것을 확인하였다. 둘째,
조작된 균주를 배치 및 유가식 발효를 수행하였다. 배치 발효를 통해 포도당을 이용한
발효 공정이 포도당-자일로스 혼합 발효공정보다 숙신산 생산이 향상되었다.
마지막으로 전처리된 보리짚 당화액으로부터 AS-PMF (MDH; malate
dehydrogenase, FUM; fumarase 및 PCK; phosphoenolpyruvate carboxykinase
유전자)에 의한 배치 발효를 수행하였다. 이는 22.3 g/L 숙신산 생산을 달성하였다

다국어 초록 (Multilingual Abstract)

Succinic acid is an industrially important component that plays a key role in food additives, dietary supplements, and precursors for biodegradable polymers. Due to environmental and economic issues, bio-based succinic acid production has become increasingly attractive. Many microorganisms have been utilized for succinic acid production. This study aims to enhance succinic acid production in Actinobacillus succinogenes through genetic modifications and fermentation strategies, particularly using lignocellulosic biomass and magnesium bicarbonate as a substrate. First, we confirmed the effects of overexpressing succinic acid-generating enzymes in batch fermentations of engineered A. succinogenes strains. The engineered A. succinogenes(AS-PMF) exhibited a 1.3-fold increase in succinic acid production compared to the wild-type strain. Subsequently, the batch fermentation were carried out with magnesium bicarbonate and lignocellulosic biomass by engineered A. succinogenes strain (AS-PMF). Fed- batch fermentation using magnesium bicarbonate, produced 50 g/L and 0.79 g/g succinic acid titer and yield. Also, batch fermentation was conducted by engineered strains (AS-PMF) using lignocellulosic biomass as a carbon source. We achieved 22.2 g/L, 0.64 g/g succinic acid production titers and yields. Keywords: succinic acid (SA), Actinobacillus succinogenes, lignocellulosic biomass, metabolically engineered, magnesium bicarbonate

목차 (Table of Contents)

• List of Figures 2
• List of Tables 3
• Abstract 5
• I. Introduction 7
• II. Materials and Methods 10
• 2.1. Strains and culture condition 10
• 2.2. Plasmid and strains construction 10
• 2.3. Electroporation of A. succinogenes 10
• 2.4. Fermentation experimentation 11
• 2.5. Methods for pretreating lignocellulosic biomass 12
• 2.6. Analytic method 13
• III. Results and Discussion 24
• 3.1. Construction succinic acid biosynthesis gene overexpression strains 24
• 3.2. Confirmation enhanced succinic acid production 25
• 3.3. Confirmation metabolites from glucose and xylose 28
• 3.4. Fed-batch fermentation using MgCO3 as pH buffer and controller by engineered
• strain (AS-PMF) 29
• 3.5. Investigation of bioenergy lignocellulosic-fermenting A. succinogenes strains 31
• IV. Conclusions 33
• V. Reference 41