Next Article in Journal
A High-Throughput Absolute Abundance Quantification Method for the Characterisation of Daqu Core Fungal Communities
Previous Article in Journal
Colaconema formosanum, Sarcodia suae, and Nostoc commune as Fermentation Substrates for Bioactive Substance Production
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 8
Issue 7
10.3390/fermentation8070344
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

High-Level Production of Catechol from Glucose by Engineered Escherichia coli

by
Guotian Song
1,2,3,
Fengli Wu
1,2,
Yanfeng Peng
1,2,
Xiaolong Jiang
1,2 and
Qinhong Wang
1,2,*
1
CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
2
National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China
3
School of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China
*
Author to whom correspondence should be addressed.
Fermentation 2022, 8(7), 344; https://doi.org/10.3390/fermentation8070344
Submission received: 30 June 2022 / Revised: 14 July 2022 / Accepted: 19 July 2022 / Published: 21 July 2022
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
Browse Figures
Versions Notes

Abstract

:
Catechol (CA) is an aromatic compound with important applications in the fine chemical and pharmaceutical fields. As an alternative strategy to petroleum-based chemical synthesis, the production of catechol by using microbial cell factories has attracted great interest. However, the toxicity of catechol to microbial cells significantly limits the efficient production of bio-based catechol via one-step fermentation. Therefore, in this study, a two-step strategy for the efficient synthesis of CA was designed. Protocatechuic acid (PCA) was first efficiently produced by the engineered Escherichia coli strain AAA01 via fermentation, and then PCA in the fermentative broth was converted into CA by the whole-cell biocatalyst AAA12 with PCA decarboxylase. By optimizing the expression of flavin isoprenyl transferases and protocatechuic acid decarboxylases, the titer of CA increased from 3.4 g/L to 15.8 g/L in 12 h through whole-cell biocatalysis, with a 365% improvement; after further optimizing the reaction conditions for whole-cell biocatalysis, the titer of CA achieved 17.7 g/L within 3 h, which is the highest titer reported so far. This work provides an effective strategy for the green biomanufacturing of toxic compounds by Escherichia coli cell factories.

Graphical Abstract

1. Introduction

Catechol (1,2-dihydroxybenzene, CA) is an important aromatic compound with many applications. It can be used as a reagent in photography, dyes, electroplating, rubber, and plastic production, and also used as a precursor in the synthesis of fine chemicals such as cis and cis-muconic acid (ccMA) [1,2,3]. ccMA can be used to produce a variety of valuable polymers and drugs, including adipic acid and terephthalic acid [4,5]. In the past few decades, CA has continued to be obtained mainly via chemical conversion of petroleum derivatives, although some catechol is distilled from coal tar [6]. The manufacturing of CA or many other chemicals based on petroleum, a non-renewable resource, is increasingly constrained in many ways due to the growing scarcity of resources and the degradation of the natural environment. Therefore, it is urgently necessary to create a cost-effective and environmentally friendly method for producing CA.
Microbial cell factories have been created as a result of the ongoing development of biotechnology for the environmentally friendly production of organic alcohols, amino acids, organic acids, vitamins, and natural products made from renewable resources, including L-phenylalanine [7,8,9], p-coumaric acid [10,11] and p-hydroxybenzoic acid [12]. For instance, Draths et al. initially described the biomanufacturing of 2 g/L catechol from glucose using an engineered Escherichia coli strain in 1995 [6]. The precursor of catechol, 3-dehydroshikimic acid (DHS), is synthesized from glucose after three steps of catalytic phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) (which are precursors of DHS), and then protocatechuic acid (PCA) and CA are successively synthesized via exogenous DHS dehydratase and PCA decarboxylase. However, low titer, preventing biomanufacturing catechol from replacing chemical synthesis, has to be addressed as the main challenge, even though the research progress on this compound supports the feasibility of CA manufacture by E. coli cell factories.
Two main factors contribute to the low titer of CA production in microbial cell factories: One is the low activity of PCA decarboxylase; the other is the toxicity of CA to microbial cells. Despite several works successfully improving the production of the precursor, DHS, and PCA [13,14,15,16], the titer of CA remained less than 10 g/L, indicating a major limitation in the synthesis of CA from PCA catalyzed via nonoxidative PCA decarboxylases. These enzymes belong to the hydroxy aromatic decarboxylase/phenol carboxylase family and consist of three distinct subunits encoded by the kpdB, aroY, and kpdD genes [17]. Weber et al. expressed the three subunits of which only aroY optimized, achieving CA production by Saccharomyces cerevisiae under aerobic conditions [17]. Further research confirmed that the combined expression of kpdB and aroY resulted in a 14-fold increase in the activity of PCA decarboxylase compared to aroY alone [18]. However, the mechanism by which kpdB promotes aroY catalysis has not been elucidated. Recently, the resolution of KpdB protein function revealed that it is a highly homologous protein with ubix in E. coli, involved in the synthesis of a new cofactor-isoprenyl riboflavin cofactor, which plays an important role in the decarboxylation reaction of aroY [19,20]. Then, through a protein comparison, Johnson et al. combined the expression of aroY and ecdB (encodes flavin isoprenyl transferase) from Enterobacter cloacae and increased ccMA titer by more than threefold [21]. Taken together, these works demonstrated that optimization of flavin isoprenyl transferase and PCA decarboxylase is the key to further improving CA titer in microbial cell factories.
Moreover, the toxicity of CA on cells also affects the CA production in microbial cell factories. For E. coli cell factories, cell growth was significantly inhibited when the titer of CA reached 1 g/L [1]; when the CA titer was 0.275 g/L, no colony grew on LB plates; and when CA titer reached 2.75 g/L, the titer of DHS decreased almost fivefold compared with no CA added [6]. Therefore, addressing the toxicity of catechol in microbial cell factories is essential for efficient CA production. Recently, extensive research was reported to address the impact of targets toxicity on production, including enhancing the tolerance of microbial cell factories through adaptive laboratory evolution [22], selecting more tolerant microbial cell factories as chassis [14], isolating cell growth and targets production by using dynamic regulation [23], removing toxic targets from culture media [3], whole-cell biocatalysis [24], or cell-free bioconversion [25]. For example, to reduce the titer of CA in a culture medium by using in situ resin-based extraction, Li et al. increased the titer of CA from 4.2 g/L to 8.5 g/L [3]. However, this strategy increases the cost of large-scale production of CA. The approach of whole-cell biocatalysis has been widely used for efficient biosynthesis of fine and bulk chemicals due to its unique advantages [26]. For instance, Wang et al. created a cofactor self-sufficient whole-cell biocatalysis system, achieving the efficient production of 2-phenyl ethanol (9.14 g/L), which is toxic to cells [27]. Therefore, whole-cell biocatalysis will play an important role as an effective strategy for CA production in microbial cell factories.
To achieve high-level production of CA from glucose by engineered E. coli, an engineered E. coli strain AAA03 directly producing CA was constructed based on a PCA cell factory AAA01. However, under one-step fermentation conditions, the cell growth and the titer of CA were significantly affected due to the toxicity of CA. To address this issue, we designed a two-step strategy for improving CA production using different engineered E. coli strains at each step. Optimizing the expression of flavin isoprenyl transferase and PCA decarboxylase increased the activity of PCA decarboxylase. Finally, the high-level production of CA by optimizing whole-cell biocatalytic reaction conditions was achieved.

2. Materials and Methods

2.1. Strains and Plasmids

The strains and plasmids used in this study are listed in Figure 1 and Table 1.

2.2. Plasmid Construction

First, according to the codon bias of E. coli, ecdB and EcaroY from Enterobacter cloacae [21] and KpdB from Klebsiella pneumoniae [17] were codon-optimized and sent to Beijing Tsingke Biotechnology Co. for gene synthesis, generating three plasmids (pET30a-T7-kpdB/ecdB/EcaroY). PCR amplified the ubiX DNA fragment from E. coli and the aroY DNA fragment from Klebsiella pneumoniae and ligated them to the pET30a fragment containing the T7 promoter, generating pET30a-T7-ubiX and pET30a-T7-aroY. Subsequently, to evaluate the effect of flavin isoprenyl transferases on CA production, codon-optimized aroY (laboratory collection) with RBS was inserted into three plasmids (pET30a-T7-kpdB/ecdB/ubiX) and formed pET30a-T7-kpdB/ecdB/ubiX-RBS-aroY; similarly, EcaroY with RBS was inserted into pET30a-T7-kpdB, generating plasmid pET30a-T7-kpdB-RBS-EcaroY for optimizing CA production via two-step whole-cell biocatalysis.

2.3. Strain Construction

In order to construct the engineered strain for CA production via one-step fermentation, we selected the PCA-producing engineered E. coli AAA01 (laboratory collection) as a chassis. The targeting fragment (ptsI40-cat-sacB-ptsI40) with 40 bp upstream and downstream homologous arms of ptsI was amplified using cat-sacB (laboratory collection) as a template, and the other targeting fragment (ptsI40-M1-93 -aroY-ptsI40) was obtained by overlapping PCR M1-93 (laboratory collection) [28] fragment and aroY fragment, which amplified using pET30a-T7-kpdB-aroY as a template. Based on the λ-Red two-step homologous recombination method [29], the engineered E. coli strain AAA03 was successfully for CA production built by sequentially integrating two targeting fragments.

2.4. Media and Growth Conditions

E. coli BL21 (DE3) was used for genetic cloning and recombinant protein expression as well as whole-cell biocatalysis. Strains were cultured at 30 °C in LB liquid medium (10 g/L NaCl, 5 g/L yeast extract, and 10 g/L tryptone) or on agar plates. Corresponding antibiotics (34 μg/mL chloramphenicol, 50 μg/mL kanamycin, and 100 μg/mL ampicillin) were added to the culture medium to avoid plasmid loss.
For protein expression in a shake flask, the single colony of biocatalysts cells was picked from the plate in tubes containing 2 mL of LB medium (with kanamycin antibiotic) and incubated overnight at 30 °C, 250 rpm. The overnight seeds were then transferred to 1 L shake flasks containing 100 mL LB with kanamycin antibiotics and incubated at 30 °C, 250 rpm until OD600 was about 0.6, and then adding 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) to induce protein expression at 30 °C for 10 h at 250 rpm. Lastly, cells were harvested via centrifugation at 5000 g for 10 min at 4 °C.
For CA or PCA production using shake-flask fermentation, the engineered E. coli AAA01/AAA03 was cultured in tubes containing 2 mL of LB medium from their plate at 37 °C, 250 rpm. Then, the overnight seeds were transferred to 100 mL shake flasks containing 10 mL of NBS media (20 g/L glucose, 6.5 g/L K2HPO4·3H2O, 3.5 g/L KH2PO4, 3.5 g/L (NH4)2HPO4, 0.12 g/L MgSO4, 0.16 mg/L FeCl3·6H2O, 11 mg/L CaCl2, 0.02 mg/L CoCl2·6H2O, 5 mg/L thiamine-HCL, 0.015 mg/L CuSO4·5H2O, 0.02 mg/L Na2MoO4·2H2O, 0.02 mg/L ZnCl2 and 0.05 mg/L H3BO3) at a ratio of 1:100 and at 37 °C, 250 rpm for 48 h. All the inorganic salt reagents are purchased from Sinopharm Chemical Reagent Co., Shanghai, China.
For CA or PCA production through 5 L fed-batch fermentation, the single colony of E. coli AAA01 or AAA03 was cultured in tubes containing 2 mL of LB medium from their plate at 37 °C, 250 rpm. The overnight seeds were then transferred to 1 L shake flasks containing 200 mL LB and cultured for 10–12 h at 37 °C, 250 rpm. After this, the seed culture was totally transferred into a 5 L bioreactor (BIOTECH-5BG, Bxbio, Shanghai, China) containing 1.8 L fermentation medium (30 g/L glucose, 1.6 g/L (NH4)2SO4, 7.5 g/L K2HPO4·3H2O, 2 g/L citric acid, 2 g/L MgSO4·7H2O, 4 mg/L CoCl2·6H2O, 0.0075 g/L FeSO4·7H2O, 20 mg/L Na2SO4, 0.6 mg/L Cu2SO4·5H2O, 6.4 mg/L ZnSO4 and 4.525 mg/L MnSO4·H2O) at 37 °C. The agitation was changed to maintain the dissolved oxygen (DO) concentration above 30% saturation. The pH was controlled at 7.0 using 25% (w/v) NH3·H2O. The DO-stat feeding strategy was adopted to supply 600 g/L glucose to the bioreactor, keeping the residual glucose below 5 g/L.

2.5. Tolerance Assays of E. coli DSM 1576 to CA and PCA

The single colony of E. coli DSM 1576 was cultured in tubes containing 2 mL of LB medium from their plate at 37 °C, 250 rpm. Then, the overnight seeds were transferred to 100 mL shake flasks containing 10 mL of NBS media and different final concentrations of CA and PCA (0 g/L, 1 g/L, 2 g/L, and 5 g/L), with an initial OD600 of 0.1 at 37 °C and 250 rpm for 24 h. Samples were collected every 6 h to determine OD600.

2.6. Whole-Cell Biocatalysis Conditions

For whole-cell biocatalysis, the harvested cells expressing protein were added to the PCA fermentative broth, which was collected via centrifugation at 10,000 g for 15 min at room temperature (Avanti JXN-26, Beckman, Brea, CA, USA). The 10 mL reaction mixture consisting of 10–40 OD600 of whole-cell biocatalysts, the final titer of 9 g/L, or 45 g/L PCA (from PCA fermentative broth) were incubated at 20–37 °C and 3–12 h under resting or shaking conditions. Finally, the concentrations of CA and PCA were determined.

2.7. Crude-Extract Biocatalysis Conditions

For crude-extract biocatalysis, the 20 OD600 whole cells were resuspended in 0.9% (w/v) NaCl solution and crushed via sonication (25 W power, 10 s on, 10 s off for 10 minutes). Then, the supernatant was collected after centrifugation at 10,000 g for 15 min at 4 °C. The 10 mL reaction mixture consisting of crude extracts and the final titer of 45 g/L PCA (from PCA fermentative broth) were incubated at 30 °C and stewed at 12 h. Finally, the concentrations of CA and PCA were determined.

2.8. Analysis of OD600 and CA or PCA Accumulation

OD600 was measured using a spectrophotometer. The fermentation or biocatalysis broth of CA or PCA was diluted to the appropriate concentration. Additionally, 1 mL of the supernatant obtained via centrifugation at 12,000 rpm for 10 min at 4 °C was analyzed using HPLC (1200 series, Agilent Technologies, CA, USA) equipped with an Innoval C18 column (4.6 mm × 250 mm, 5 μm, Bonna-Agela, CA, USA). The ratio of phosphoric acid 0.1% (v/v) in phase A was 80% and methanol in phase B was 20%. The flow rate was 0.8 mL/min. The detection wavelength was 210 nm, and the column temperature was 30 °C.

3. Results

3.1. The Toxicity of CA Significantly Affects the CA Production via One-Step Fermentation

To achieve one-step fermentation for CA production in engineered E .coli, we first constructed an engineered E. coli AAA03 strain (Figure 2A) by integrating a strong artificial regulatory element M1-93 [28] and expressing aroY from Klebsiella pneumoniae at the ptsI locus on the chromosome of the engineered E. coli AAA01 strain. After 48 h of shake-flask fermentation, the engineered E. coli AAA01 strain produced 8.8 g/L PCA, accumulated 0.5 g/L DHS, and OD600 was 11.7, while the engineered E. coli AAA03 strain produced 2.2 g/L CA and accumulated 2.3 g/L PCA, but the OD600 was 50% lower than that of the engineered E. coli AAA01 strain (Figure 2B–E). After 42 h of fed-batch fermentation, a titer of PCA reached 53.6 g/L, and the highest OD600 was 41.9 (Figure 3A), while a titer of CA achieved 3.1 g/L, and OD600 was only 32.1 (Figure 3B). There was a significant decrease in the cell growth of the engineered E. coli AAA03 strain, compared with that of the engineered E. coli AAA01 strain using either fermentation method. These results suggested that, although the synthesis of CA was achieved in the engineered E. coli strains, the toxicity of CA significantly affects CA production through one-step fermentation.
To evaluate the effects of PCA and CA on cell growth, we investigated the tolerance of E. coli strain to different concentrations of PCA and CA. We found that when the concentration of PCA reached 5 g/L, the cell growth decreased by almost 50%, compared with that without PCA addition, while the cell growth was affected with the addition of 2 g/L PCA at 0–12 h (Figure 4A). For catechol, the cell growth of the E. coli strain was significantly inhibited (a 93.8% decrease compared with that without CA addition) when the concentration of CA in the culture medium was more than 1 g/L (Figure 4B). These results showed that CA was more toxic to E. coli cells than PCA, which led us to the conclusion that the engineered E. coli AAA01 strain could produce higher PCA, while the engineered E. coli AAA03 strain could only produce lower CA. Therefore, how to avoid the toxicity of CA should be addressed for improving CA production.

3.2. Introducing Flavin Isoprenyl Transferase Could Efficiently Improve the Conversion of PCA to CA

To address the toxicity of CA to microbial cells, we designed a two-step biosynthetic method for high-level production of CA. In detail, PCA was first efficiently produced by the engineered E. coli AAA03 strain in a 5 L bioreactor and then converted into CA using a whole-cell biocatalyst expressing PCA decarboxylase. As indicated in previous reports [19,20], the isoprenyl riboflavin cofactor (prFMN) is required to catalyze the PCA decarboxylation reaction. Based on this, we investigated the effects of different sources of flavin isoprenyl transferases (encoded by ecdB/kpdB/ubiX) on whole-cell biocatalysis for CA synthesis. The best engineered E. coli AAA09 co-expressing aroY and kpdB produced 5.42 g/L CA, a 70% increase, compared with engineered E. coli AAA04 expressing aroY alone, while both the engineered E. coli AAA10 and AAA11 strains produced 5.4 g/L CA. The PCA concentrations accumulated to about 0.1 g/L in engineered E. coli AAA09, AAA10, and AAA11, while it reached 3.8 g/L in engineered E. coli AAA04 (Figure 5 and Supplement Figure S1). To verify the maximum productive performance of the whole-cell catalytic system with different flavin isoprenyl transferases, the fermentative broth with 45 g/L PCA was used for the biocatalytic reaction. After 12 h of biocatalysis, the titer of CA reached 11.4 g/L in engineered E. coli AAA09, with a 235% increase compared with the CA titer in engineered E. coli AAA04 (Figure 5), and there was no significant difference between the three flavin isoprenyl transferases. These results indicated that the overexpression of kpdB or ecdB or ubiX increased the activity of PCA decarboxylase and enhanced the production of CA, and the three flavin isoprenyl transferases had similar enzyme activity. However, there was still about 11 g/L PCA in engineered E. coli AAA09 fermentative broth that could not be converted into CA, suggesting that high-level CA production was limited in whole-cell biocatalysis systems.

3.3. Optimization of Whole-Cell Biocatalysis Systems Could Improve the CA Production

To completely convert PCA into CA, we optimized and characterized the whole-cell and enzymatic biocatalysis. Through whole-cell biocatalysis, the engineered E. coli AAA12 constructed with a higher activity of EcaroY from Enterobacter cloacae, compared with aroY [21], produced 15.8 g/L CA, with a 38.6% increase compared with engineered E. coli AAA09, which produced 11.4 g/L CA (Figure 5 and Figure 6), with a 31.7% increase compared with engineered E. coli AAA08, indicating that flavin isoprenyl transferase promoted the decarboxylation reaction of PCA, which is also consistent with the previous results (Figure 5 and Figure 6). To verify the effect of increasing the concentrations of EcaroY and KpdB on CA synthesis, we further investigated the production of CA in crude extracts. The crude extracts of engineered E. coli AAA12 produced 14.5 g/L CA, which was 8.2% lower compared with that of whole-cell biocatalysts, probably due to enzyme loss during the crude extract preparation. Then, the titer of CA increased by 6.9% and 11% when the concentration of flavin isoprenyl transferase or PCA decarboxylase was further increased, indicating that both enzymes could promote the conversion of PCA into CA (Figure 6). However, compared with whole-cell biocatalysis, biocatalysis with crude extracts is more complex, and the enhancement of CA titer is not significant. Therefore, the strategy of whole-cell biocatalysis is more favorable for the large-scale production of CA.

3.4. Optimization of Whole-Cell Biocatalysis Reaction Conditions Could Further Improve the CA Production

To further improve CA production, we optimized the whole-cell biocatalytic conditions, such as time, cell concentration, temperature, and dissolved oxygen using engineered E. coli AAA12 as the biocatalyst. We found that the higher titer (17.0 g/L) of CA was achieved at 3 h, probably due to the degradation of CA after 3 h (Figure 7A). To reduce the CA toxicity on cells by increasing the cell concentration, the highest titer of CA achieved 17.7 g/L with 40 OD600 for 3 h, a 4.1% and 9.3% increase compared with 20 OD600 and 10 OD600, respectively (Figure 7B). The temperature had no significant effect on whole-cell biocatalysis, and the higher CA titer achieved 17.0 g/L at 30 °C (Figure 7C). The effect of different dissolved oxygen (DO) levels on CA production was investigated using resting or shaking biocatalysis. Only 7.3 g/L CA was produced under high DO level conditions (via shaking biocatalysis), a 57% reduction compared with that observed at low DO level (via resting biocatalysis) (Figure 7D). These results indicated that the high DO levels significantly affected whole-cell biocatalytic reactions. In summary, a titer of 17.7 g/L CA was produced by the AAA12 whole-cell biocatalyst under optimum conditions, which was the highest titer reported so far (Table 2).

4. Discussion

As we mentioned in this study, only 2.2 g/L (20 mmol/L) CA and 2.3 g/L PCA were produced by the engineered E. coli AAA03 strain via one-step fermentation. If there was no degradation of CA, only 5.4 g/L PCA (20 mmol/L PCA plus 2.3 g/L PCA) was theoretically produced by the engineered E. coli AAA03 strain, a 38.6% decrease compared with the engineered E. coli AAA01 strain (8.8 g/L PCA), indicating that CA toxicity inhibited cell growth (50% decrease in OD600) and thus affected PCA synthesis (Figure 2B–D). These results suggested that the toxicity of CA to cells is a key factor limiting CA bioproduction. According to a previous report [32], during the oxidation of CA to form quinone, large amounts of hydrogen peroxide are generated, which leads to cell death by oxidizing proteins as well as fragmenting DNA. In addition, more hydroxyl radicals can be generated by the intracellular Fenton reaction in the presence of CA [33]. Hydroxyl radical has a reduction potential greater than H2O2 and fragments DNA and proteins, resulting in cell death [34] (Supplementary Figure S2). These results explain the low titer of CA in one-step fermentation (aerobic fermentation) (Figure 3B) or two-step shaking biocatalysis (Figure 7D).
To reduce the toxicity of CA to microbial cells, we designed a two-step whole-cell biocatalytic approach to overproduce CA in a resting biocatalytic way and then optimized and characterized the key enzymes, flavin isoprenyl transferase, and PCA decarboxylase. We found that the co-expression of flavin isoprenyl transferases with PCA decarboxylase facilitated the efficient production of CA (Figure 6), but the effects of different sources of them on CA were marginal (Figure 5 and Figure 6). However, only the supply of isoprenyl riboflavin cofactor (prFMN) was enhanced in our work, and the recycling of prFMN was not actually achieved, which could be the reason for the highest titer of CA at 3 h via two-step whole-cell biocatalysis.
For PCA decarboxylase, we utilized EcaroY from Enterobacter cloacae instead of aroY from Klebsiella pneumoniae according to the literature [21], and the production of CA was increased by 38.6% (from 11.4 g/L to 15.8 g/L) using the two-step whole-cell biocatalysis (Figure 5 and Figure 6). However, we only characterized a reported PCA decarboxylase (EcaorY) of which the enzymatic activity is higher than aroY, and it might be very necessary to test more PCA decarboxylases.
Finally, we efficiently produced CA with a titer of 17.7 g/L based on the designed two-step whole-cell biocatalysis approach (Figure 7B), which is the highest titer reported so far. Furthermore, in-depth research will help to enhance the titer of CA. Further studies may include adding more antioxidants (Vitamin C) or biocatalysis under micro-oxygenated conditions to further reduce the toxicity of CA; constructing an isoprenyl riboflavin cofactor recycling system to maintain the efficiency of the whole-cell biocatalytic process; and screening PCA decarboxylase with higher enzyme activity from a larger database or modifying PCA decarboxylase based on protein structure/machine learning. In summary, using different strategies to solve the bottlenecks of CA production through two-step whole-cell biocatalysis will facilitate the large-scale production of bio-based CA and also provide effective guidance for the green biomanufacturing of high-value-added toxic chemicals.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation8070344/s1, Figure S1: The residual PCA after producing catechol via two-step whole-cell biocatalysis; Figure S2: The mechanism of CA toxicity to microbial cells.

Author Contributions

Conceptualization, G.S. and Q.W.; methodology, G.S., Y.P. and X.J.; validation, G.S., F.W., Y.P. and X.J.; investigation, G.S. and Y.P.; data curation, G.S.; F.W. and Q.W.; writing—original draft preparation, G.S.; writing—review and editing, G.S.; F.W. and Q.W.; supervision, Q.W.; project administration, Q.W.; funding acquisition, Y.P., F.W., X.J. and Q.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China (2018YFA0901400 and 2018YFA0903700) and the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-CXRC-057). Qinhong Wang was funded by Tianjin Industrial Synthetic Biology Innovation Team.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Pugh, S.; McKenna, R.; Osman, M.; Thompson, B.; Nielsen, D.R. Rational engineering of a novel pathway for producing the aromatic compounds p-hydroxybenzoate, protocatechuate, and catechol in Escherichia coli. Process Biochem. 2014, 49, 1843–1850. [Google Scholar] [CrossRef]
  2. Balderas-Hernández, V.E.; Treviño-Quintanilla, L.G.; Hernández-Chávez, G.; Martinez, A.; Bolívar, F.; Gosset, G. Catechol biosynthesis from glucose in Escherichia coli anthranilate-overproducer strains by heterologous expression of anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. Microb. Cell Factories 2014, 13, 136–147. [Google Scholar] [CrossRef] [PubMed]
  3. Li, W.; Xie, D.; Frost, J.W. Benzene-free synthesis of catechol interfacing microbial and chemical catalysis. J. Am. Chem. Soc. 2005, 127, 2874–2882. [Google Scholar] [CrossRef] [PubMed]
  4. Bentley, G.J.; Narayanan, N.; Jha, R.K.; Salvachúa, D.; Elmore, J.R.; Peabody, G.L.; Black, B.A.; Ramirez, K.; De Capite, A.; Michener, W.E.; et al. Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440. Metab. Eng. 2020, 59, 64–75. [Google Scholar] [CrossRef] [PubMed]
  5. Choi, S.; Lee, H.N.; Park, E.; Lee, S.J.; Kim, E.S. Recent advances in microbial production of cis,cis-muconic acid. Biomolecules 2020, 10, 1238. [Google Scholar] [CrossRef] [PubMed]
  6. Draths, D.K.; Frost, J.W. Environmentally compatible synthesis of catechol from D-glucose. J. Am. Chem. Soc. 1995, 117, 2395–2400. [Google Scholar] [CrossRef]
  7. Ding, D.; Li, J.; Bai, D.; Fang, H.; Lin, J.; Zhang, D. Biosensor-based monitoring of the central metabolic pathway metabolites. Biosens. Bioelectron. 2020, 167, 112456–112467. [Google Scholar] [CrossRef]
  8. Liu, X.; Niu, H.; Li, Q.; Gu, P. Metabolic engineering for the production of l-phenylalanine in Escherichia coli. 3 Biotech 2019, 9, 85–92. [Google Scholar] [CrossRef]
  9. Liu, Y.; Xu, Y.; Ding, D.; Wen, J.; Zhu, B.; Zhang, D. Genetic engineering of Escherichia coli to improve L-phenylalanine production. BMC Biotechnol. 2018, 18, 5–17. [Google Scholar] [CrossRef]
  10. Liu, Q.; Yu, T.; Li, X.; Chen, Y.; Campbell, K.; Nielsen, J.; Chen, Y. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals. Nat. Commun. 2019, 10, 4976–4989. [Google Scholar] [CrossRef] [Green Version]
  11. Rodriguez, A.; Kildegaard, K.R.; Li, M.; Borodina, I.; Nielsen, J. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis. Metab. Eng. 2015, 31, 181–188. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Kitade, Y.; Hashimoto, R.; Suda, M.; Hiraga, K.; Inui, M. Production of 4-hydroxybenzoic acid by an aerobic growth-arrested bioprocess using metabolically engineered Corynebacterium glutamicum. Appl. Environ. Microbiol. 2018, 84, 2587–2599. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Choi, S.S.; Seo, S.Y.; Park, S.O.; Lee, H.N.; Song, J.S.; Kim, J.Y.; Park, J.H.; Kim, S.; Lee, S.J.; Chun, G.T.; et al. Cell factory design and culture process optimization for dehydroshikimate biosynthesis in Escherichia coli. Front. Bioeng. Biotechnol. 2019, 7, 241–252. [Google Scholar] [CrossRef]
  14. Kogure, T.; Suda, M.; Hiraga, K.; Inui, M. Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways. Metab. Eng. 2021, 65, 232–242. [Google Scholar] [CrossRef] [PubMed]
  15. Li, K.; Mikola, M.R.; Draths, K.M.; Worden, R.M.; Frost, J.W. Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61–73. [Google Scholar] [CrossRef]
  16. Li, J.; Ye, B.C. Metabolic engineering of Pseudomonas putida KT2440 for high-yield production of protocatechuic acid. Bioresour. Technol. 2021, 319, 124239–124248. [Google Scholar] [CrossRef]
  17. Weber, C.; Bruckner, C.; Weinreb, S.; Lehr, C.; Essl, C.; Boles, E. Biosynthesis of cis,cis-muconic acid and its aromatic precursors, catechol and protocatechuic acid, from renewable feedstocks by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2012, 78, 8421–8430. [Google Scholar] [CrossRef] [Green Version]
  18. Sonoki, T.; Morooka, M.; Sakamoto, K.; Otsuka, Y.; Nakamura, M.; Jellison, J.; Goodell, B. Enhancement of protocatechuate decarboxylase activity for the effective production of muconate from lignin-related aromatic compounds. J. Biotechnol. 2014, 192, 71–77. [Google Scholar] [CrossRef] [Green Version]
  19. Payne, K.A.; White, M.D.; Fisher, K.; Khara, B.; Bailey, S.S.; Parker, D.; Rattray, N.J.; Trivedi, D.K.; Goodacre, R.; Beveridge, R.; et al. New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition. Nature 2015, 522, 497–501. [Google Scholar] [CrossRef] [Green Version]
  20. White, M.D.; Payne, K.A.; Fisher, K.; Marshall, S.A.; Parker, D.; Rattray, N.J.; Trivedi, D.K.; Goodacre, R.; Rigby, S.E.; Scrutton, N.S.; et al. UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis. Nature 2015, 522, 502–506. [Google Scholar] [CrossRef] [Green Version]
  21. Johnson, C.W.; Salvachua, D.; Khanna, P.; Smith, H.; Peterson, D.J.; Beckham, G.T. Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity. Metab. Eng. Commun. 2016, 3, 111–119. [Google Scholar] [CrossRef] [PubMed]
  22. Matson, M.M.; Cepeda, M.M.; Zhang, A.; Case, A.E.; Kavvas, E.S.; Wang, X.; Carroll, A.L.; Tagkopoulos, I.; Atsumi, S. Adaptive laboratory evolution for improved tolerance of isobutyl acetate in Escherichia coli. Metab. Eng. 2022, 69, 50–58. [Google Scholar] [CrossRef] [PubMed]
  23. Liang, C.; Zhang, X.; Wu, J.; Mu, S.; Wu, Z.; Jin, J.M.; Tang, S.Y. Dynamic control of toxic natural product biosynthesis by an artificial regulatory circuit. Metab. Eng. 2020, 57, 239–246. [Google Scholar] [CrossRef] [PubMed]
  24. Hou, F.; Feng, D.; Xian, M.; Huang, W. De novo biosynthesis and whole-cell catalytic production of 2-acetamidophenol in Escherichia coli. J. Agric. Food Chem. 2022, 70, 238–246. [Google Scholar] [CrossRef] [PubMed]
  25. Grubbe, W.S.; Rasor, B.J.; Kruger, A.; Jewett, M.C.; Karim, A.S. Cell-free styrene biosynthesis at high titers. Metab. Eng. 2020, 61, 89–95. [Google Scholar] [CrossRef] [PubMed]
  26. Lin, B.; Tao, Y. Whole-cell biocatalysts by design. Microb. Cell Factories 2017, 16, 106–118. [Google Scholar] [CrossRef] [Green Version]
  27. Wang, P.; Yang, X.; Lin, B.; Huang, J.; Tao, Y. Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol. Metab. Eng. 2017, 44, 143–149. [Google Scholar] [CrossRef]
  28. Lu, J.; Tang, J.; Liu, Y.; Zhu, X.; Zhang, T.; Zhang, X. Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization. Appl. Microbiol. Biotechnol. 2012, 93, 2455–2462. [Google Scholar] [CrossRef]
  29. Thomason, L.C.; Sawitzke, J.A.; Li, X.; Costantino, N.; Court, D.L. Recombineering: Genetic engineering in bacteria using homologous recombination. Curr. Protoc. Mol. Biol. 2014, 106, 1.16.1–1.16.39. [Google Scholar] [CrossRef]
  30. Weber, H.E.; Gottardi, M.; Brückner, C.; Oreb, M.; Boles, E.; Tripp, J.; Vieille, C. Requirement of a functional flavin mononucleotide prenyltransferase for the activity of a bacterial decarboxylase in a heterologous muconic acid pathway in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2017, 83, 3472–3488. [Google Scholar] [CrossRef] [Green Version]
  31. Brückner, C.; Oreb, M.; Kunze, G.; Boles, E.; Tripp, J. An expanded enzyme toolbox for production of cis, cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae. FEMS Yeast Res. 2018, 18, 17–29. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Chichirau, A.; Flueraru, M.; Chepelev, L.L.; Wright, J.S.; Willmore, W.G.; Durst, T.; Hussain, H.H.; Charron, M. Mechanism of cytotoxicity of catechols and a naphthalenediol in PC12-AC cells: The connection between extracellular autoxidation and molecular electronic structure. Free Radic. Biol. Med. 2005, 38, 344–355. [Google Scholar] [CrossRef] [PubMed]
  33. Lima, J.P.P.; Tabelini, C.H.B.; Ramos, M.D.N.; Aguiar, A. Kinetic evaluation of Bismarck Brown Y Azo dye oxidation by Fenton processes in the presence of aromatic mediators. Water Air Soil Pollut. 2021, 232, 1–16. [Google Scholar] [CrossRef]
  34. Zhao, Z. Iron and oxidizing species in oxidative stress and Alzheimer’s disease. Aging Med (Milton) 2019, 2, 82–87. [Google Scholar] [CrossRef] [PubMed]
Fermentation 08 00344 g001 550
Figure 1. The flowchart of engineered E. coli strains using in each step of this study.
Figure 1. The flowchart of engineered E. coli strains using in each step of this study.
Fermentation 08 00344 g001
Fermentation 08 00344 g002 550
Figure 2. One-step fermentative production of catechol from glucose in engineered E. coli strain by shaking flask: (A) the synthetic pathway of catechol in engineered E. coli AAA03 using glucose as a carbon source. Catechol production (B), cell growth (C), PCA titer (D), and DHS titer (E) in shake-flask fermentation. Glu, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose 1,6-diphosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; E4P, D-erythrose 4-phosphate; DHS, 3-dehydroshikimic acid; X5P, xylulose-5-phosphate; PCA, protocatechuic acid; CA, catechol; pgi, phosphohexose isomerase; pykF/A, pyruvate kinase; tktA, transketolase; aroF, 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase; aroB, 3-dehydroquinate synthase; aroE, shikimate dehydrogenase; quiC, 3-dehydroshikimic acid dehydratase; aroY, protocatechuate decarboxylase.
Figure 2. One-step fermentative production of catechol from glucose in engineered E. coli strain by shaking flask: (A) the synthetic pathway of catechol in engineered E. coli AAA03 using glucose as a carbon source. Catechol production (B), cell growth (C), PCA titer (D), and DHS titer (E) in shake-flask fermentation. Glu, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose 1,6-diphosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; E4P, D-erythrose 4-phosphate; DHS, 3-dehydroshikimic acid; X5P, xylulose-5-phosphate; PCA, protocatechuic acid; CA, catechol; pgi, phosphohexose isomerase; pykF/A, pyruvate kinase; tktA, transketolase; aroF, 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase; aroB, 3-dehydroquinate synthase; aroE, shikimate dehydrogenase; quiC, 3-dehydroshikimic acid dehydratase; aroY, protocatechuate decarboxylase.
Fermentation 08 00344 g002
Fermentation 08 00344 g003 550
Figure 3. One-step fermentative production of PCA and CA from glucose using engineered E. coli AAA01 (A) and AAA03 (B) in 5 L bioreactor. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Figure 3. One-step fermentative production of PCA and CA from glucose using engineered E. coli AAA01 (A) and AAA03 (B) in 5 L bioreactor. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Fermentation 08 00344 g003
Fermentation 08 00344 g004 550
Figure 4. Growth response of engineered E. coli strain to the exogenous addition of PCA (A) and CA (B) at 0 g/L (black), 1 g/L (pink), 2 g/L (green), and 5 g/L (purple). Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Figure 4. Growth response of engineered E. coli strain to the exogenous addition of PCA (A) and CA (B) at 0 g/L (black), 1 g/L (pink), 2 g/L (green), and 5 g/L (purple). Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Fermentation 08 00344 g004
Fermentation 08 00344 g005 550
Figure 5. The effects of different flavin isoprenyl transferases on catechol production with 9 g/L or 45 g/L PCA via whole-cell biocatalysis. E. coli BL21 (DE3) with pET30a-T7 plasmid was used as the blank control (CK). Biocatalytic conditions: Diluted PCA fermentative broth (final concentrations of 9 g/L and 45 g/L), 20 OD600 whole cells, 10 mL of total volume, at 30 °C and pH 7.0. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Figure 5. The effects of different flavin isoprenyl transferases on catechol production with 9 g/L or 45 g/L PCA via whole-cell biocatalysis. E. coli BL21 (DE3) with pET30a-T7 plasmid was used as the blank control (CK). Biocatalytic conditions: Diluted PCA fermentative broth (final concentrations of 9 g/L and 45 g/L), 20 OD600 whole cells, 10 mL of total volume, at 30 °C and pH 7.0. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Fermentation 08 00344 g005
Fermentation 08 00344 g006 550
Figure 6. The effects of flavin isoprenyl transferases, protocatechuate decarboxylase, and the different ways of biocatalysts on catechol production. Whole-cell biocatalytic conditions: 20 OD600 whole cells; crude extracts biocatalytic conditions: 20 OD600 prepared for crude extracts. All reactions were performed under 45 g/L PCA fermentative broth, total volume of 10 mL, at 30 °C, and pH 7.0. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Figure 6. The effects of flavin isoprenyl transferases, protocatechuate decarboxylase, and the different ways of biocatalysts on catechol production. Whole-cell biocatalytic conditions: 20 OD600 whole cells; crude extracts biocatalytic conditions: 20 OD600 prepared for crude extracts. All reactions were performed under 45 g/L PCA fermentative broth, total volume of 10 mL, at 30 °C, and pH 7.0. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Fermentation 08 00344 g006
Fermentation 08 00344 g007 550
Figure 7. Effects of time (A), OD600 (B), temperature (C), and dissolved oxygen (D) on CA production. All reactions were performed under 45 g/L PCA fermentative broth, total volume of 10 mL, and pH 7.0. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Figure 7. Effects of time (A), OD600 (B), temperature (C), and dissolved oxygen (D) on CA production. All reactions were performed under 45 g/L PCA fermentative broth, total volume of 10 mL, and pH 7.0. Data are the average mean of triplicate samples, and the error bars represent standard deviations.
Fermentation 08 00344 g007
Table
Table 1. Strains and plasmids used in this study.
Table 1. Strains and plasmids used in this study.
Name.DescriptionSource
Strains
E. coli DSM 1576Wild typeLab collection
E. coli AAA01DSM 1576 M1-12-aroETTG M1-30-aroFfbr ΔtyrR M1-93-tktA
ΔptsI M1-12-galP M1-93-glk M1-12-pykFTTG M1-12-pykATTG
M1-12-pgiTTG M1-93-aroBopt		Lab collection for PCA production
E. coli AAA03AAA01 ∆ ptsI :: M1-93 -aroYThis study
E. coli AAA04BL21 (DE3) with pET30a-T7-aroYThis study
E. coli AAA05BL21 (DE3) with pET30a-T7-kpdBThis study
E. coli AAA06BL21 (DE3) with pET30a-T7-ecdBThis study
E. coli AAA07BL21 (DE3) with pET30a-T7-ubiXThis study
E. coli AAA08BL21 (DE3) with pET30a-T7-EcaroYThis study
E. coli AAA09BL21 (DE3) with pET30a-T7-kpdB-RBS-aroYThis study
E. coli AAA10BL21 (DE3) with pET30a-T7-ecdB-RBS-aroYThis study
E. coli AAA11BL21 (DE3) with pET30a-T7-ubiX-RBS-aroYThis study
E. coli AAA12BL21 (DE3) with pET30a-T7-kpdB-RBS-EcaroYThis study
Plasmids
pET30a-T7-kpdBpET30a expressing kpdB with T7 promoterThis study
pET30a-T7-ecdBpET30a expressing ecdB with T7 promoterThis study
pET30a-T7-ubiXpET30a expressing ubiX with T7 promoterThis study
pET30a-T7-aroYpET30a expressing aroY with T7 promoterThis study
pET30a-T7-EcaroYpET30a expressing EcaroY with T7 promoterThis study
pET30a-T7-kpdB-RBS-aroYpET30a expressing kpdB and aroY with T7 promoterThis study
pET30a-T7-ecdB-RBS-aroYpET30a expressing ecdB and aroY with T7 promoterThis study
pET30a-T7-ubiX-RBS-aroYpET30a expressing ubiX and aroY with T7 promoterThis study
pET30a-T7-kpdB-RBS-EcaroYpET30a expressing kpdB and EcaroY with T7 promoterThis study
pET30a-T7-ecdB-RBS-EcaroYpET30a expressing ecdB and EcaroY with T7 promoterThis study
Table
Table 2. Production of CA by engineered microbial cell factories.
Table 2. Production of CA by engineered microbial cell factories.
StrainCarbon SourceSynthetic MethodTime (h)Titer (g/L)Reference
E. coliGlucoseResin-based extraction368.50[3]
E. coliGlucoseFed-batch fermentation482.00[6]
S. cerevisiaeGlucoseShake-flask fermentation1200.58[17]
E. coliGlucoseBatch fermentation844.47[2]
E. coliPCAWhole-cell biocatalysis30.08[18]
E. coliGlucoseBatch fermentation860.63[1]
S. cerevisiaePCAWhole-cell biocatalysis1680.09[30]
S. cerevisiaeGlucoseShake-flask fermentation960.62[31]
E. coliGlucoseFed-batch fermentation423.10This work
E. coliGlucoseWhole-cell biocatalysis317.70This work
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Song, G.; Wu, F.; Peng, Y.; Jiang, X.; Wang, Q. High-Level Production of Catechol from Glucose by Engineered Escherichia coli. Fermentation 2022, 8, 344. https://doi.org/10.3390/fermentation8070344

AMA Style

Song G, Wu F, Peng Y, Jiang X, Wang Q. High-Level Production of Catechol from Glucose by Engineered Escherichia coli. Fermentation. 2022; 8(7):344. https://doi.org/10.3390/fermentation8070344

Chicago/Turabian Style

Song, Guotian, Fengli Wu, Yanfeng Peng, Xiaolong Jiang, and Qinhong Wang. 2022. "High-Level Production of Catechol from Glucose by Engineered Escherichia coli" Fermentation 8, no. 7: 344. https://doi.org/10.3390/fermentation8070344

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop