Browsing by Author "Lan, Ethan I."

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A balanced ATP driving force module for enhancing photosynthetic biosynthesis of 3-hydroxybutyrate from CO2
(2018-03-01)
Ku, Jason T.
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Lan, Ethan I.
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生物科技學系
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分子醫學與生物工程研究所
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Department of Biological Science and Technology
Using engineered photoautotrophic microorganisms for the direct chemical synthesis from CO2 is an attractive direction for both sustainability and CO2 mitigation. However, the behaviors of non-native metabolic pathways may be difficult to control due to the different intracellular contexts between natural and heterologous hosts. While most metabolic engineering efforts focus on strengthening driving forces in pathway design to favor biochemical production in these organisms, excessive driving force may be detrimental to product biosynthesis due to imbalanced cellular intermediate distribution. In this study, an ATP-hydrolysis based driving force module was engineered into cyanobacterium Synechococcus elongatus PCC 7942 to produce 3-hydroxybutyrate (3HB), a valuable chemical feedstock for the synthesis of biodegradable plastics and antibiotics. However, while the ATP driving force module is effective for increasing product formation, uncontrolled accumulation of intermediate metabolites likely led to metabolic imbalance and thus to cell growth inhibition. Therefore, the ATP driving force module was reengineered by providing a reversible outlet for excessive carbon flux. Upon expression of this balanced ATP driving force module with 3HB biosynthesis, engineered strain produced 3HB with a cumulative titer of 1.2 g/L, a significant increase over the initial strain. This result highlighted the importance of pathway reversibility as an effective design strategy for balancing driving force and intermediate accumulation, thereby achieving a self-regulated control for increased net flux towards product biosynthesis.
Chemical Production from Methanol Using Natural and Synthetic Methylotrophs
(1970-01-01)
Chen, Arvin Y.
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Lan, Ethan I.
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生物科技學系
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分子醫學與生物工程研究所
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Department of Biological Science and Technology
Methanol as a chemical feedstock is becoming increasingly important as it is derived from natural gas and is a feasible end-product for captured carbon dioxide. Biological conversion of methanol through natural and synthetic methylotrophs increases the chemical repertoire and is an important direction for one carbon (C1) based chemical economy. Advances in the metabolic engineering and synthetic biology enable development of microbial cell factories for converting methanol into various platform chemicals. In this review, the current status of methanol utilizing microbial factory development is summarized. Also the development of synthetic methylotrophy and methanol-augmented bioproductions is discussed.
Cometabolic degradation of toluene and TCE contaminated wastewater in a bench-scale sequencing batch reactor inoculated with immobilized Pseudomonas putida F1
(2019-11-01)
Yang, Cai-En
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Wu, Chih-Yun
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Liu, Yu-Cheng
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Lan, Ethan I.
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Tsai, Shen-Long
Toluene and Trichloroethylene (TCE) are two of the most common organic contaminates at hazardous sites and contaminated water. In this study, a sequencing batch reactor (SBR) inoculated with PVA/alginate-immobilized Pseudomonas putida F1 was used for biological treatment of toluene and TCE contaminated wastewater. Moreover, a mathematical model was developed to simulate the dynamic behavior of a SBR treating competitive contaminates. Results showed high removal efficiencies of both toluene and TCE in the effluents. For the duration of filling time, an instantaneous fill was the optimum for moderate amount of toluene and TCE. Toluene concentration affected TCE degradation. The lower the toluene presence, the higher the removal efficiency of both toluene and TCE. Interestingly, the presence of 20 mg/L iron, or 1 mg/L nickel stimulated the degradation rates of both toluene and TCE. However, the degradation rate was reduced when both 20 mg/L iron and 1 mg/L nickel were presented simultaneously. The model developed in this research was able to simulate the kinetic behaviors of toluene and TCE removal in the SBR successfully. (C) 2019 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Escherichia coli as a host for metabolic engineering
(2018-11-01)
Pontrelli, Sammy
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Chiu, Tsan-Yu
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Lan, Ethan I.
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Chen, Frederic Y. -H.
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Chang, Peiching
Over the past century, Escherichia coli has become one of the best studied organisms on earth. Features such as genetic tractability, favorable growth conditions, well characterized biochemistry and physiology, and availability of versatile genetic manipulation tools make E. coli an ideal platform host for development of industrially viable productions. In this review, we discuss the physiological attributes of E. coli that are most relevant for metabolic engineering, as well as emerging techniques that enable efficient phenotype construction. Further, we summarize the large number of native and non-native products that have been synthesized by E. coli, and address some of the future challenges in broadening substrate range and fighting phage infection.
Metabolic Engineering Design Strategies for Increasing Acetyl-CoA Flux
(2020-04-01)
Ku, Jason T.
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Chen, Arvin Y.
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Lan, Ethan I.
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生物科技學系
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分子醫學與生物工程研究所
Acetyl-CoA is a key metabolite precursor for the biosynthesis of lipids, polyketides, isoprenoids, amino acids, and numerous other bioproducts which are used in various industries. Metabolic engineering efforts aim to increase carbon flux towards acetyl-CoA in order to achieve higher productivities of its downstream products. In this review, we summarize the strategies that have been implemented for increasing acetyl-CoA flux and concentration, and discuss their effects. Furthermore, recent works have developed synthetic acetyl-CoA biosynthesis routes that achieve higher stoichiometric yield of acetyl-CoA from glycolytic substrates.
Metabolic engineering of cyanobacteria for the photosynthetic production of succinate
(2016-11)
Lan, Ethan I.
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Wei, Crystal T.
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生物科技學系
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Department of Biological Science and Technology
Succinate is an important commodity chemical currently used in the food, pharmaceutical, and polymer industries. It can also be chemically converted into other major industrial chemicals such as 1,4-butanediol, butadiene, and tetrahydrofuran. Here we metabolically engineered a model cyanobacterium Synechococcus elongatus PCC 7942 to photosynthetically produce succinate. We expressed the genes encoding for aketoglutarate decarboxylase and succinate semialdehyde dehydrogenase in S. elongatus PCC 7942, resulting in a strain capable of producing 120 mg/L of succinate. However, this recombinant strain exhibited severe growth retardation upon induction of the genes encoding for the succinate producing pathway, potentially due to the depletion of a-ketoglutarate. To replenish alpha-ketoglutarate, we expressed the genes encoding for phosphoenolpyruvate carboxylase and citrate synthase from Corynebacterium glutamicum into the succinate producing strain. The resulting strain successfully restored the growth phenotype and produced succinate with a titer of 430 mg/L in 8 days. These results demonstrated the possibility of photoautotrophic succinate production.
Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria
(2019-04-01)
Lai, Martin J.
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Lan, Ethan I.
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生物科技學系
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Department of Biological Science and Technology
Direct conversion of carbon dioxide into chemicals using engineered autotrophic microorganisms offers a potential solution for both sustainability and carbon mitigation. Butyrate is an important chemical used in various industries, including fragrance, food, and plastics. A model cyanobacterium Synechococcus elongatus PCC 7942 was engineered for the direct photosynthetic conversion of CO2 to butyrate. An engineered Clostridium Coenzyme A (CoA)-dependent pathway leading to the synthesis of butyryl-CoA, the precursor to butyrate, was introduced into S. elongatus PCC 7942. Two CoA removal strategies were then individually coupled to the modified CoA-dependent pathway to yield butyrate production. Similar results were observed between the two CoA removal strategies. The best butyrate producing strain of S. elongatus resulted in an observed butyrate titer of 750 mg/L and a cumulative titer of 1.1 g/L. These results demonstrated the feasibility of photosynthetic butyrate production and expanded the chemical repertoire accessible for production by photoautotrophs.
Renewable synthesis of n-butyraldehyde from glucose by engineered Escherichia coli
(2017-12-04)
Ku, Jason T.
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Simanjuntak, Wiwik
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Lan, Ethan I.
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生物科技學系
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分子醫學與生物工程研究所
Background: n-Butyraldehyde is a high-production volume chemical produced exclusively from hydroformylation of propylene. It is a versatile chemical used in the synthesis of diverse C4-C8 alcohols, carboxylic acids, esters, and amines. Its high demand and broad applications make it an ideal chemical to be produced from biomass. Results: An Escherichia coli strain was engineered to produce n-butyraldehyde directly from glucose by expressing a modified Clostridium CoA-dependent n-butanol production pathway with mono-functional Coenzyme A-acylating aldehyde dehydrogenase (Aldh) instead of the natural bifunctional aldehyde/alcohol dehydrogenase. Aldh from Clostridium beijerinckii outperformed the other tested homologues. However, the presence of native alcohol dehydrogenase led to spontaneous conversion of n-butyraldehyde to n-butanol. This problem was addressed by knocking out native E. coli alcohol dehydrogenases, significantly improving the butyraldehyde-to-butanol ratio. This ratio was further increased reducing media complexity from Terrific broth to M9 media containing 2% yeast extract. To increase production titer, in situ liquid-liquid extraction using dodecane and oleyl alcohol was investigated. Results showed oleyl alcohol as a better extractant, increasing the titer of n-butyraldehyde produced to 630 mg/L. Conclusion: This study demonstrated n-butyraldehyde production from glucose. Through sequential strain and condition optimizations, butyraldehyde-to-butanol ratio was improved significantly compared to the parent strain. Results from this work may serve as a basis for further development of renewable n-butyraldehyde production.