Multidimensional Engineering of Escherichia coli for Efficient Adipic Acid Synthesis From Cyclohexane

Abstract

Adipic acid (AA), a key aliphatic dicarboxylic acid, is conventionally manufactured through energy-intensive, multi-step chemical processes with significant environmental impacts. In contrast, biological production methods offer more sustainable alternatives but are often limited by low productivity. To overcome these challenges, this study reports the engineering of a single Escherichia coli for efficient biosynthesis of AA starting from cyclohexanol (CHOL), KA oil (mixture of CHOL and cyclohexanone (CHONE)), or cyclohexane (CH). To start with, a comprehensive screening of rate-limiting enzymes is conducted, particularly focusing on cytochrome P450 monooxygenase, followed by the optimization of protein expression using strategies such as protein fusion, promoter replacement, and genome editing. Consequently, an engineered E. coli capable of efficiently converting either KA oil or CH into AA is obtained, achieving remarkable product titers of 110 and 22.6 g L^-1, respectively. This represents the highest productivity record for the biological production of AA to date. Finally, this developed biocatalytic system is successfully employed to convert different cycloalkanes and cycloalkanols with varied carbon chain lengths into their corresponding dicarboxylic acids, highlighting its great potential as well as broad applicability for industrial applications.

Keywords: adipic acid; balance; biosynthesis; cascade pathway.

Figure 1

Industrial chemical and proposed biocatalytic processes for AA production. A) Current chemical industrial process for the synthesis of AA. B) Proposed biosynthesis route for AA production from KA oil. C) Designed biosynthesis route for AA production by a single engineered E. coli harboring corresponding enzymes.

Figure 2

Concept of Single E. coli construction for converting CH to AA with a bottom‐up approach. A) The biosynthesis route from CH to AA contains three basic modules respectively. Module 1(orange block): P450‐catalyzed hydroxylation of CH to CHOL; Module 2 (purple block): Alcohol dehydrogenase (ADH) and Baeyer‐Villiger monooxygenase (BVMO) for two‐step oxidation of CHOL to ε‐CL; Module 3 (blue block): Lactonase for hydrolysis of ε‐CL to 6‐HHA and alcohol dehydrogenase (ChnD) and aldehyde dehydrogenase (ChnE) for two sequential oxidation steps of 6‐HHA to AA. The expression of enzymes in Module 1 and Module 2 is upregulated, whereas the expression of enzymes in Module 3 is downregulated. B) Bottom‐up reverse strategy for the construction of single E. coli for converting CHOL or CH to produce AA. First, E. coli (Module 3) was constructed to produce AA from ε‐CL. Next, optimal enzymes in Module 2 were integrated to create E. coli (Module 2_3) for the biotransformation from CHOL to AA. Subsequently, selected enzymes in Module 1 were incorporated into E. coli (Module 2_3) with the enhancement of cofactor (NADPH/NADH) supply. Finally, the recombinant single E. coli (Module 1_2_3) capable of converting CH to AA was obtained.

Figure 3

Biotransformation of ε‐CL to AA by engineered E. coli (Module 3) with enzyme genes integrated into the genome. A) Scheme of conversion of ε‐CL to AA. B) Construction of single‐cell E. coli with genome integration approach. C) Biotransformation of ε‐CL to AA by engineered E. coli (Module 3) cells: E. coli (M3‐1) ∼ E. coli (M3‐8). As shown in the table, ldhA, ahr, pgi, adhE was selected as the integration sites (replaced by exogenous genes). P _T7 DRBSE: Under the control of the P _T7 promoter, genes encoding ChnD and ChnE were linked by the RBS sequence; P _T7 Lac: Gene encoding Lactonase was under the control of P _T7 promoter; P _T7 DE: Proteins ChnD and ChnE were fused with linker, and the resulting ChnDE fusion gene was driven by P _T7 promoter; Lac: Gene encoding Lactonase was under the control of the native promoter of the gene that was deleted at the integration site in the engineered strain. Reactions were conducted at 25 °C and 220 rpm using E. coli cells (16 g CDW L⁻¹) expressing target enzymes in 200 mm phosphate buffer (pH 8.0) with 100 mm ε‐CL. The error bars indicate the standard deviation of three biological replicates (n = 3).

Figure 4

Construction of recombinant E. coli catalyst for converting CHOL to ε‐CL. A) E. coli strains expressing various ADHs were evaluated for CHOL‐to‐CHONE biotransformation under the following conditions: 25 °C, 220 rpm, 8 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 50 mm CHOL. B) E. coli strains expressing BVMO fused different tags were evaluated for CHONE‐to‐ε‐CL biotransformation under the following conditions: 25 °C, 220 rpm, 8 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 50 mm CHONE; cofactor NAD(P)H was provided by the E. coli using 5% (w/v) glucose as an energy source. C) E. coli (Module 2) co‐expressing ChnA and BVMO were evaluated for CHOL‐to‐ε‐CL biotransformation under the following conditions: 25 °C, 220 rpm, 8 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 50 mm CHOL, with or without the addition of 5% (w/v) glucose. D) E. coli (Module 2) co‐expressing LbADH and BVMO were evaluated for CHOL‐to‐ε‐CL biotransformation under the following conditions: 25 °C, 220 rpm, 8 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 50 mm CHOL, with or without the addition of 5% (w/v) glucose. The error bars indicate the standard deviation of three biological replicates (n = 3).

Figure 5

Biotransformation of CHOL to AA with engineered E. coli (Module 2_3). A) Scheme of biotransformation from CHOL to AA. B) Construction of single E. coli containing enzymes of Module 2_3 for converting CHOL to AA. C) Different engineered E. coli (Module 2_3) cells were evaluated for CHOL‐to‐AA biotransformation under the following conditions: 25 °C, 220 rpm, 16 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 150 mm CHOL for 24 h. AB: Plasmid pRSFDuet‐1 which carries both ChnA and flag‐tagged BVMO; LB: Plasmid pRSFDuet‐1 which carries both LbADH and flag‐tagged BVMO. For engineered E. coli based on ChnA (E. coli (M23‐1‐A); E. coli (M23‐2‐A); E. coli (M23‐3‐A); E. coli (M23‐4‐A); E. coli (M23‐7‐A) and E. coli (M23‐8‐A)), 5% (w/v) glucose was added as an energy source to provide NAD(P)H; for engineered E. coli based on LbADH (E. coli (M23‐1‐L); E. coli (M23‐2‐L); E. coli (M23‐3‐L); E. coli (M23‐4‐L); E. coli (M23‐7‐L) and E. coli (M23‐8‐L)), no glucose was added to the reaction systems. D) Time course of recombinant E. coli (M23‐8T‐L) catalyzed CHOL‐to‐AA biotransformation under the following conditions: 25 °C, 220 rpm, 16 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 100 mm CHOL. The pH was maintained ≈8.0 through the addition of 10 m NaOH. The error bars indicate the standard deviation of three biological replicates (n = 3).

Figure 6

Biotransformation of CHOL to AA with engineered E. coli (M23‐8T‐L) in a 5‐L fermenter. A) Scheme of biotransformation from CHOL to AA by engineered E. coli (M23‐8T‐L); B) Biotransformation of CHOL into AA in a 5‐L fermenter; C) Biotransformation results for E. coli (M23‐8T‐L) catalyzed conversion of CHOL to AA. D) Biotransformation results for E. coli (M23‐8T‐L) catalyzed conversion of industrially produced KA oil into AA. Refer to the experimental section for detailed reaction conditions. Arrow: the addition of CHOL or KA oil for initiating the reactions.

Figure 7

Biotransformation of CH to CHOL by P450CHX in the presence of different redox pairs or fused with reductase domain derived from self‐sufficient P450s. A) Scheme of conversion of CH to CHOL catalyzed by P450CHX. B) The hydroxylation of CH catalyzed by P450 CHX when coupled with different pairs of redox partners or fused with the reductase domain from different sources. Redox partners: Fdr‐Fdx derived from Synechococcus elongatus PCC7942;^{[ 39 ]} Fpr‐YkuN derived from Bacillus subtilis;^{[ 40 ]} CamA‐CamB derived from Pseudomonas putida.^{[ 37 ]} Reductase domains were from self‐sufficient P450s: 116B46 was reductase domain of CYP116B46 from thermophilic Tepidiphilus thermophilus;^{[ 38 ]} BM3 was reductase domain of CYP102A1 from Bacillus megaterium;^{[ 18 ]} RhFRed was reductase domain of P450RhF from Rhodococcus sp. strain NCIMB 9784;^{[ 38 ]} 19A12 was reductase domain of CYP102A1 mutant 19A12.^{[ 18 ]} E. coli co‐expressing P450CHX and different redox partners or expressing the chimeric P450CHX were evaluated for CH‐to‐CHOL biotransformation under the following conditions for 20 h: 25 °C, 220 rpm, 16 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 100 mm CH and 5% (w/v) glucose added. The error bars indicate the standard deviation of three biological replicates (n = 3).

Figure 8

Biotransformation of CH to AA by engineered E. coli (Module1_2_3). A) Scheme of conversion from CH to AA. B) A single E. coli strain (Module1_2_3) was engineered to express all enzymes required for CH‐to‐AA conversion. C) Different engineered E. coli for biotransformation from CH by optimizing cofactor supply at the cellular level. D) Time course of recombinant E. coli (M123‐2‐A) catalyzed CH‐to‐AA biotransformation under the following conditions: 25 °C, 220 rpm, 16 g CDW L⁻¹ cell density in 200 mm phosphate buffer (pH 8.0) with 100 mm CH and 5% (w/v) glucose added. The pH was maintained ≈8.0 through the addition of 10 m NaOH. The error bars indicate the standard deviation of three biological replicates (n ≥ 3).

Figure 9

Biotransformation of CH to AA in a 5‐L fermenter with the best‐engineered E. coli (M123‐2‐A). A) Bioreactor setup for CH feed by the gas phase. B) Results of E. coli (M123‐2‐A) catalyzed biotransformation of CH to AA. Refer to the experimental section for detailed reaction conditions.

Figure 10

Substrate scope examination. A) The production of DCAs with engineered E. coli (M23‐8T‐L) from cycloalkanols. B) The production of DCAs with engineered E. coli (M123‐2‐A) from cycloalkanes. Reactions were performed in 3 mL of cell suspensions containing 100 mm substrates at 16 g CDW L⁻¹, 25 °C, and 220 rpm for 24 h. For E. coli (M23‐8T‐L), no glucose was added to the reaction systems. For E. coli (M123‐2‐A), 5% (w/v) glucose was added as an energy source to provide NAD(P)H. The errors indicate the standard deviation of three biological replicates (n = 3).