A Bio-Catalytic Approach to Aliphatic Ketones

Xiong, Mei; Deng, Jin; Woodruff, Adam P.; Zhu, Minshan; Zhou, Jun; Park, Sun Wook; Li, Hui; Fu, Yao; Zhang, Kechun

doi:10.1038/srep00311

Cited by 35 publications

(22 citation statements)

References 35 publications

(39 reference statements)

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“…Recent efforts in microbial biofuel production hinge upon constructing efficient metabolic pathways to produce a variety of fatty acids (FA)-based fuels (17)(18)(19)(20)(21)(22)(23). To date, most of the work has taken a static perspective to coordinate the expression of enzymes and optimize production titer and yield, including modification of plasmid copy number (24), promoter strength (25,26), and combinations of these strategies (21,27).…”

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confidence: 99%

Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

Zhang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

440

358

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Global energy demand and environmental concerns have stimulated increasing efforts to produce carbon-neutral fuels directly from renewable resources. Microbially derived aliphatic hydrocarbons, the petroleum-replica fuels, have emerged as promising alternatives to meet this goal. However, engineering metabolic pathways with high productivity and yield requires dynamic redistribution of cellular resources and optimal control of pathway expression. Here we report a genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli. The engineered strains were able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis. Implementation of this metabolic control resulted in an oscillatory malonyl-CoA pattern and a balanced metabolism between cell growth and product formation, yielding 15.7-and 2.1-fold improvement in FA titer compared with the wild-type strain and the strain carrying the uncontrolled metabolic pathway. This study provides a new paradigm in metabolic engineering to control and optimize metabolic pathways facilitating the high-yield production of other malonyl-CoA-derived compounds.biofuels | dynamic metabolic control | transcriptional regulation A grand challenge in synthetic biology is to move the design of biomolecular circuits from purely genetic constructs toward systems that integrate different levels of cellular complexity, including regulatory networks and metabolic pathways (1). Despite the fact that a large volume of regulatory architectures and motifs has been discovered (2, 3), little has been accomplished in pathway engineering to improve cellular productivity and yield by exploiting dynamic pathway regulation and metabolic control (4). One essential part in implementing synthetic metabolic control in pathway engineering is to engineer novel metabolite sensors with desired input-output relationships. For example, have designed and applied a regulatory circuit that can sense the glycolytic pathway hallmark metabolite acetylphosphate to control the lycopene biosynthetic pathway (5) and generate oscillatory gene expression (6) as well as achieve artificial cell-cell communication (7). Dahl et al. (8) have used stressresponse promoters to improve farnesyl pyrophosphate production, and Tsao et al. (9) have rewired the Escherichia coli native quorumsensing regulon for autonomous induction of recombinant proteins.Traditional metabolic engineering is largely focused on the overexpression of rate-limiting steps (10

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confidence: 99%

Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

Zhang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

440

358

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“…Among all developed alternatives, microbial synthesis has been proven to be competent and promising. So far, a variety of chemicals, including biofuels, amino acids, and plant secondary metabolites, have been produced by metabolically engineered microorganisms (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

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A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

Sun

Lin

Huang

et al. 2013

Appl Environ Microbiol

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cMuconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ؎ 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid.

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“…Several key residues at the LeuA active site have been verified to play essential role in controlling the pocket size and hence substrate specifictity, in particular, H97, S139 and G462 shown in Fig. S2 (Chen et al, 2017; Xiong et al, 2012). The natural substrates of LeuA are 2-ketoisovalerate and 2-ketobutyrate (Shen and Liao, 2008).…”

Section: Resultsmentioning

confidence: 99%

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle

Cheng

Song²,

Wang³

et al. 2019

Preprint

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22Bioplastics produced from microbial source are promising green alternatives to 23 traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, 24 especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential 25 monomers for the synthesis of polymeric bioplastics as their primary amine and 26 carboxylic acid are ideal functional groups for polymerization. Previous pathways for 27 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from 28 L-lysine catabolism and citric acid cycle, respectively. Here, we show the construction 29 of an artificial iterative carbon-chain-extension cycle in Escherichia coli for 30 simultaneous production of a series of nonnatural amino acids with varying chain length. 31 Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate as a 32 non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-33 extended α-ketoacid is subsequently decarboxylated and oxidized by an α-ketoacid 34 decarboxylase and an aldehyde dehydrogenase, respectively, to yield the nonnatural 35 straight-chain amino acid products. The engineered system demonstrated simultaneous 36 in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate 37 and 4.78 mg/L of 7-aminoheptanoate after 8 hours of enzyme catalysis starting from 2-38 keto-6-aminocaproate as the substrate. Furthermore, simultaneous production of 2.15 39 g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-40 aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising 41 metabolic-engineering strategy to access other medium-chain organic acids with -NH2, 42 -SCH3, -SOCH3, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-43 elongation chemistry.44 Keywords: Nonnatural straight chain amino acid, 6-Aminocaproate, Carbon chain 45 elongation, Synthetic biology, Iterative cycle 46 3 47 Abbreviations: 48 NNSCAA, Nonnatural straight chain amino acid; 5AVA, 5-Aminovalerate; 6ACA, 6-49

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A Bio-Catalytic Approach to Aliphatic Ketones

Cited by 35 publications

References 35 publications

Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle

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