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Acetate production from corn stover hydrolysate using recombinant Escherichia coli BL21 (DE3) with an EP-bifido pathway

Microbial Cell Factories volume 23, Article number: 300 (2024) Cite this article

Abstract

Background

Acetate is an important chemical feedstock widely applied in the food, chemical and textile industries. It is now mainly produced from petrochemical materials through chemical processes. Conversion of lignocellulose biomass to acetate by biotechnological pathways is both environmentally beneficial and cost-effective. However, acetate production from carbohydrate in lignocellulose hydrolysate via glycolytic pathways involving pyruvate decarboxylation often suffers from the carbon loss and results in low acetate yield.

Results

Escherichia coli BL21 (DE3) was confirmed to have high tolerance to acetate in this work. Thus, it was selected from seven laboratory E. coli strains for acetate production from lignocellulose hydrolysate. The byproduct-producing genes frdA, ldhA, and adhE in E. coli BL21 (DE3) were firstly knocked out to decrease the generation of succinate, lactate, and ethanol. Then, the genes pfkA and edd were also deleted and bifunctional phosphoketolase and fructose-1,6-bisphosphatase were overexpressed to construct an EP-bifido pathway in E. coli BL21 (DE3) to increase the generation of acetate from glucose. The obtained strain E. coli 5K/pFF can produce 22.89 g/L acetate from 37.5 g/L glucose with a yield of 0.61 g/g glucose. Finally, the ptsG gene in E. coli 5K/pFF was also deleted to make the engineered strain E. coli 6K/pFF to simultaneously utilize glucose and xylose in lignocellulosic hydrolysates. E. coli 6K/pFF can produce 20.09 g/L acetate from corn stover hydrolysate with a yield of 0.52 g/g sugar.

Conclusion

The results presented here provide a promising alternative for acetate production with low cost substrate. Besides acetate production, other biotechnological processes might also be developed for other acetyl-CoA derivatives production with lignocellulose hydrolysate through further metabolic engineering of E. coli 6K/pFF.

Introduction

Acetate is an important platform feedstock expansively applied in the production of bulk chemicals like vinyl acetate, monochloroacetic acid, acetate esters, and acetic anhydride [1,2,3,4,5,6,7,8]. It also has potential applications in the food preservation, adhesives and textile treatments [9,10,11,12]. Acetate is now mainly produced via petrochemical pathways including methanol carbonylation, ethylene oxidation, and acetaldehyde oxidation [13,14,15]. These chemical processes rely on non-renewable feedstocks under harsh reaction conditions, and thus are costly and environmentally unfriendly. Some biochemical processes have also been developed for acetate production from regenerable resources. However, only 10% of the world acetate demand is fulfilled by microbial fermentation up to now [16].

Currently, acetic acid bacteria are regarded as the most efficient acetate producers which mainly produce acetate from ethanol through two oxidization reactions for making vinegar [17,18,19,20,21]. Autotrophic acetogenic bacteria can also utilize syngas to produce acetate using the Wood–Ljungdahl pathway [22, 23]. Besides ethanol and syngas, lignocellulose is termed as another promising renewable resource for biological acetate production. Hydrolysis of lignocellulose results in a hydrolysate with glucose and xylose as the major utilizable sugars [24,25,26]. Most of the studies toward the conversion of lignocellulose biomass to acetate used heterotrophic acetogens species. Pyruvate produced from glucose and xylose is oxidatively decarboxylated to generate acetyl-CoA, which will be channeled into TCA cycle for energy generation, utilized for different chemicals production or transformed into acetate through phosphate acetyltransferase (Pta) and acetate kinase (AckA), with acetyl-phosphate (AcP) as the key intermediate. However, the pyruvate decarboxylation results in the carbon loss and decreases the theoretical yield of acetate from lignocellulose hydrolysate.

Non-oxidative glycolytic (NOG) pathway was created by Bogorad et al. to prevent CO2 release during C2 metabolites production from glucose [27]. The key enzyme in the NOG pathway was bifunctional phosphoketolase (Fxpk) [28], which splits fructose-6-phosphate (F6P) into AcP and erythrose-4-phosphate (E4P), as well as xylulose-5-phosphate (X5P) into glyceraldehyde-3-phosphate (G3P) and AcP in “bifid shunt” of bifidobacteria. E4P and G3P can also be transformed into AcP via carbon atom rearrangements, which was pushed by fructose-1,6-bisphosphatase (Fbp), another key enzyme in NOG pathway. Recently, Wang et al. deleted 6-phosphofructokinase (PfkA) and 6-phosphogluconate dehydratase (Edd), overexpressed Fxpk and Fbp, and successfully constructed an EP-bifido pathway combining Embden-Meyerhof-Parnas pathway, Pentose Phosphate pathway and bifid shunt in Escherichia coli [29]. The utilization of EP-bifido pathway can result in theoretical maximum production of C2 metabolites from glucose and generate enough NADPH to support growth of E. coli. Xylose can also be converted into X5P with endogenous xylose isomerase (XylA) and xylulokinase (XylB) [30,31,32]. Thus, C2 metabolites production from xylose in E. coli may also be improved via the EP-bifido pathway.

In this work, E. coli BL21 (DE3) was metabolically engineered to produce acetate from lignocellulose hydrolysate (Fig. 1). Byproduct-producing genes in E. coli BL21 (DE3), including frdA, ldhA, and adhE, were knocked out to decrease the generation of succinate, lactate, and ethanol. The EP-bifido pathway was introduced to increase the generation of acetate with glucose. The obtained strain E. coli 5K/pFF produced 22.89 g/L acetate from 37.5 g/L glucose with a yield of 0.61 g/g glucose. Then, the glucose-specific transporter EIICBGlc coding gene ptsG was also knockout and the strain E. coli 6K/pFF produced 20.09 g/L acetate from corn stover hydrolysate.

Fig. 1
figure 1

Metabolic engineering strategies for acetate production from corn stover hydrolysate by E. coli BL21 (DE3). PTS, phosphotransferase system; GalP, galactose permease; XylE, xylose transporter; XylFGH, xylose ABC transporter; ptsG, glucose-specific transporter EIICBGlc coding gene; glK, glucose kinase coding gene; xylA, xylose isomerase coding gene; xylB, xylulose kinase coding gene; zwf, glucose-6-phosphate dehydrogenase coding gene; pgl, 6-phosphogluconolactonase coding gene; gnd, 6-phosphogluconate dehydrogenase coding gene; rpe, ribulose-5-phosphate 3-epimerase coding gene; pgi, glucose-6-phosphate isomerase coding gene; edd, 6-phosphogluconate dehydratase coding gene; fxpk, phosphoketolase coding gene; fbp, fructose-1,6-bisphosphatase coding gene; tal, transaldolase coding gene; tkt, transketolase coding gene; rpi, ribulose-5-phosphate epimerase coding gene; pfkA/pfkB, 6-phosphofructokinase coding gene; fba, fructose-1,6-bisphosphate aldolase coding gene; tpiA, triosephosphate isomerase coding gene; ldhA, lactate dehydrogenase coding gene; adhE, acetaldehyde/alcohol dehydrogenase coding gene; frdA, fumarate reductase subunit A coding gene; pta, phosphate acetyltransferase coding gene; ackA, acetate kinase coding gene; poxB, pyruvate oxidase coding gene. Abbreviations for metabolites: G6P, glucose-6-phosphate; 6PGL, gluconolactone-6-phosphate; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; 2KDPG, 2-keto-3-deoxy-6-phosphogluconate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; AcP, acetyl-phosphate; E4P, erythrose-4-phosphate; S7P, sedoheptulose-7-phosphate; G3P, glyceraldehyde-3-phosphate; R5P, ribose-5-phosphate; DHAP, dihydroxyacetone phosphate

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Materials and methods

Chemicals and reagents

Glucose was supplied by Sinopharm Chemical Reagent Co. (Shanghai, China). DNA Restriction enzymes and T4 DNA ligase were purchased from Thermo Fisher Scientific (Waltham, USA). Polymerase chain reaction (PCR) primers were synthesized by Tsingke Biotechnology Co., Ltd. (Qingdao, China). DNA oligonucleotides and DNA polymerase were obtained from Vazyme Biotechnology Co., Ltd. (Nanjing, China). Corn stover hydrolysate containing glucose (411.0 g/L), xylose (140.8 g/L), and arabinose (5.0 g/L) was a kindly gift from Dacheng Group Co., Ltd. (Changchun, China). All other chemicals are commercially available.

Bacterial strains, plasmids and growth conditions

The bacterial strains and plasmids used in this work are listed in Table 1. E. coli BL21 (DE3) and its derivatives were generally cultivated in Lysogeny Broth (LB) medium at 37℃ and 180 rpm. Defined medium with 5 g/L yeast extract (DYM) containing 13.5 g/L KH2PO4, 4 g/L (NH4)2HPO4, 1.7 g/L citric acid, 1.4 g/L MgSO4·7H2O, 4.5 mg/L vitamin B1, 10% (v/v) trace metals solution and different carbon sources (glucose or corn stover hydrolysate) was used for acetate synthesis [33].

Table 1 Strains and plasmids used in this study
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DNA manipulation in E. coli BL21 (DE3)

Primers used in this work are listed in Additional file 1: Table S1. Gene knockout of E. coli BL21 (DE3) was conducted through CRISPR/Cas9 system containing the pEcCas and pEcgRNA plasmids as described previously [34, 35]. Herein, the knockout of frdA was taken as an example. Single-strand oligonucleotides (gRNA-frdA-F/gRNA-frdA-R) targeting frdA were annealed and inserted into pEcgRNA to obtain plasmid pEcgRNA-ΔfrdA. To obtain donor DNA, the upstream and downstream homologous arms of frdA were amplified from the genome of E. coli BL21 (DE3) by uf-frdA/ur-frdA and df-frdA/dr-frdA, and connected through recombinant PCR. Then, a mixture containing 250 ng of pEcgRNA-ΔfrdA and 1000 ng of donor DNA was co-transferred into E. coli BL21 (DE3) harboring pEcCas plasmid by electroporation. The correct colonies were screened out from LB plate containing 50 µg/mL spectinomycin and 50 µg/mL kanamycin, and then grown in LB supplemented with 10 mM rhamnose and 10% (w/v) sucrose to induce the curing of the pEcCas and pEcgRNA-ΔfrdA plasmids. Other E. coli BL21 (DE3) mutants were obtained using the same approach as described above.

Batch fermentation in shake flask for acetate production

E. coli BL21 (DE3) and its derivatives were cultured in 300 mL shake flask with 50 mL DYM containing 20 g/L glucose. Spectinomycin at a concentration of 50 µg/mL and isopropyl-β-d-1-thiogalactopyranoside (IPTG) at a concentration of 0.1 mM were supplemented into the medium when the engineered E. coli strains carrying plasmid pFF. Cultivations were conducted at 37℃ and 180 rpm. The pH of fermentation broth was adjusted to 6.8 before fermentation. Samples were taken periodically and the cell density, concentrations of glucose, acetate, and by-products were determined after an appropriate dilution.

Batch fermentation in a 1-L bioreactor for acetate production

Batch fermentation for acetate production was also conducted in a 1-L bioreactor (Infors AG, Bottmingen, Switzerland) with a working volume of 0.8 L. The seed culture (10%, v/v) was injected into DYM containing different carbon sources and 0.1 mM IPTG. For acetate production by E. coli 5K/pFF, DYM containing 40 g/L glucose was used. For acetate production by E. coli 6K/pFF, corn stover hydrolysate was added to DYM to make glucose at a concentration of about 30 g/L and xylose at a concentration of about 10 g/L. Batch fermentation was carried out at 37℃, with an airflow rate of 1.2 vvm and a stirrer speed of 400 rpm. The pH of the fermentation broth was automatically adjusted to 6.8 by the addition of 5 M NaOH via a peristaltic pump.

Analytical methods

Cell growth was measured through detecting the optical density at 600 nm by visible spectrophotometer (V5100H, METASH, China). Concentrations of acetate, lactate, succinate and ethanol in the fermentation broth were detected by high-performance liquid chromatography (HPLC) (Shimadzu LC-20AT) as described previously [29]. The glucose concentration was determined by SBA-40D bioanalyzer (Shandong Academy of Sciences, China).

Results

Tolerance to acetate of E. coli BL21 (DE3)

E. coli has fast growth rate, powerful genetic manipulation tools and mature fermentation techniques, and is generally considered as a suitable host for bulk chemicals production [36]. Acetate is an overflow metabolite secreted by E. coli to support fast cell growth. However, excessive accumulation of acetate may inhibit the growth of E. coli and conversely decreased its final titer and productivity [37,38,39]. Thus, an ideal recombinant strain for acetate production should be acetate-tolerant. In this study, the tolerance of different laboratory E. coli strains including E. coli BL21 (DE3), E. coli MG1655, E. coli W3110, E. coli TOP10, E. coli JM109, E. coli HB101, and E. coli S17-1 to acetate was firstly evaluated. These strains were cultured in DYM with 20 g/L glucose and various concentrations (0, 5, 10 g/L) of acetate for 12 h. As shown in Fig. 2A, E. coli BL21 (DE3) exhibited higher biomass in DYM with either 5 g/L or 10 g/L acetate than other E. coli strains. In addition, this strain can still slightly grow in the presence of 30 g/L acetate (Fig. 2B).

As shown in Fig. 2C, E. coli BL21 (DE3) (referred to as E. coli 0K) consumed 17.33 g/L glucose within 15 h and accumulated 1.25 g/L succinate, 6.91 g/L lactate, 1.84 g/L ethanol and 5.25 g/L acetate (Fig. 2D). The yield of acetate was 0.30 g/g glucose and it accounted for 34.43% (concentration ratio) of the total fermentation products (Fig. 2E). These results indicated that E. coli BL21 (DE3) had the potential for high acetate production and was metabolically engineered in subsequent experiments.

Fig. 2
figure 2

E. coli BL21 (DE3) has the potential for high acetate production. (A) The biomass of different laboratory E. coli strains after cultivation in DYM with 20 g/L glucose and 0 g/L, 5 g/L, or 10 g/L acetate for 12 h. (B) The biomass of E. coli BL21 (DE3) after cultivation in DYM with 20 g/L glucose and different concentrations of acetate. E. coli BL21 (DE3) was inoculated into DYM with 20 g/L glucose and different concentrations of acetate to reach an initial OD600nm of 0.08. (C) Time profiles of biomass, glucose consumption, and acetate production of E. coli BL21 (DE3) during shake flask fermentation. (D) The concentrations of accumulated metabolites in the fermentation broth by E. coli BL21 (DE3). (E) The proportion of different metabolites during shake flask fermentation of E. coli BL21 (DE3). The data are shown as averages of three independent experiments with standard deviations

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Inactivating by-products generation pathways in E. coli 0K to enhance acetate production

Besides acetate, succinate (8.20%), lactate (45.31%), and ethanol (12.07%) were also produced by E. coli 0K through the mixed acid fermentation pathway [40]. The generation of these by-products would decrease the metabolic flux toward acetate production and restrict the yield of acetate from glucose. Thus, the genes frdA, ldhA, and adhE in E. coli 0K responsible for succinate, lactate and ethanol production, were knocked out sequentially to construct the recombinant strains E. coli 1K, E. coli 2K, and E. coli 3K. The cell growth, glucose consumption, and acetate accumulation of E. coli 1K, E. coli 2K, and E. coli 3K in DYM supplemented with glucose were studied (Fig. 3A-C). As expected, deletion of frdA, ldhA, and adhE obviously decreased the generation of succinate, lactate and ethanol, respectively (Fig. 3D-F). The strain E. coli 3K produced 7.93 g/L acetate from 18 g/L glucose within 24 h. The proportion of acetate in the total fermentation products increased to 94.18% while the yield of acetate increased to 0.44 g/g glucose (Fig. 3G-I).

Fig. 3
figure 3

Inactivating by-products generation pathways to increase acetate production by recombinant E. coli BL21 (DE3). (A) Time profiles of biomass, glucose consumption, and acetate production by E. coli 1K during shake flask fermentation. (B) Time profiles of biomass, glucose consumption, and acetate production by E. coli 2K during shake flask fermentation. (C) Time profiles of biomass, glucose consumption, and acetate production by E. coli 3K during shake flask fermentation. (D) The concentrations of accumulated metabolites in the fermentation broth by E. coli 1K. (E) The concentrations of accumulated metabolites in the fermentation broth by E. coli 2K. (F) The concentrations of accumulated metabolites in the fermentation broth by E. coli 3K. (G) The proportion of different metabolites during shake flask fermentation of E. coli 1K. (H) The proportion of different metabolites during shake flask fermentation of E. coli 2K. (I) The proportion of different metabolites during shake flask fermentation of E. coli 3K. The data are shown as averages of three independent experiments with standard deviations

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Introducing an EP-bifido pathway to enhance acetate production

Acetate is generated from acetyl-CoA by Pta and AckA with AcP as the key intermediate during the mixed acid fermentation of E. coli. However, the acetyl-CoA production from pyruvate with pyruvate dehydrogenase complex involves the inevitable decarboxylation reaction and may lead to low yield of acetate. Recently, Wang et al. designed an EP-bifido pathway in E. coli by overexpressing Fxpk from B. adolescentis and Fbp from E. coli and deleting PfkA and Edd [29]. Glucose can be converted into AcP with theoretical maximum yield via the EP-bifido pathway and then transformed into acetate through AckA. Thus, the pfkA and edd were also deleted in E. coli 3K to generate E. coli 5K. Then, the plasmid pFF with fxpk and fbp was introduced into E. coli 5K to obtain the recombinant strain E. coli 5K/pFF with the EP-bifido pathway.

The cell growth, glucose consumption, and acetate production of E. coli 5K and E. coli 5K/pFF were studied in DYM medium supplemented with glucose (Fig. 4A and B). As shown in Fig. 4C and D, E. coli 5K produced 6.23 g/L acetate from 17.33 g/L glucose, while E. coli 5K/pFF produced 10.52 g/L acetate from 14.97 g/L glucose (Additional file 1: Figure S1). The yield of acetate produced by E. coli 5K/pFF (0.70 g/g glucose) was slightly higher than theoretical value of acetate produced via pyruvate decarboxylation (0.67 g/g glucose) (Fig. 4F), which may be attributed to introduction of EP-bifido pathway and bioconversion of yeast extract added in the fermentation medium.

Fig. 4
figure 4

Introduction of an EP-bifido pathway to increase the yield of acetate. (A) Time profiles of biomass, glucose consumption, and acetate production by E. coli 5K during shake flask fermentation. (B) Time profiles of biomass, glucose consumption, and acetate production by E. coli 5K/pFF during shake flask fermentation. (C) The concentrations of accumulated metabolites in the fermentation broth by E. coli 5K. (D) The concentrations of accumulated metabolites in the fermentation broth by E. coli 5K/pFF. (E) Comparison the concentrations of acetate produced by different recombinant E. coli strains from glucose during shake flask fermentation. (F) Comparison of the yields of acetate produced by different recombinant E. coli strains during shake flask fermentation. The data are shown as averages of three independent experiments with standard deviations

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Acetate production by E. coli 5K/pFF from glucose through batch fermentation in a 1-L bioreactor

Then, the performance of E. coli 5K/pFF with the EP-bifido pathway in acetate production was evaluated in a 1-L bioreactor with DYM containing glucose as the substrate. IPTG at a concentration of 0.1 mM was added into the fermentation broth at 6 h to induce the expression of Fxpk and Fbp. As shown in Fig. 5A, the strain E. coli 5K/pFF finally consumed 37.5 g/L glucose and produced 22.89 g/L acetate with a yield of 0.61 g/g glucose. Acetate is the major product in the fermentation broth and accounted for 98.67% of the total products (Additional file 1: Figure S2A). Acetate can be transformed to acetyl-CoA and then channeled into TCA cycle under aerobic conditions. Microaerobic or even anaerobic conditions are beneficial for fermentative acetate production. The lower acetate yield of E. coli 5K/pFF in 1-L bioreactor might be due to high dissolved oxygen level (1.2 vvm and 400 rpm) induced higher biomass generation and TCA cycle flux.

Fig. 5
figure 5

Batch fermentation of recombinant E. coli 5K/pFF for acetate production in a 1‑L bioreactor. (A) Time profiles of biomass, glucose consumption, and acetate production by E. coli 5K/pFF during batch fermentation in a 1-L bioreactor using glucose as the substrate. (B) Time profiles of biomass, glucose consumption, and acetate production by E. coli 6K/pFF during batch fermentation in a 1‑L bioreactor using corn stover hydrolysate as the substrate

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Acetate production by E. coli 6K/pFF from corn stover hydrolysate through batch fermentation in a 1-L bioreactor

Corn stover hydrolysate is a promising sustainable feedstock for chemicals manufacture. In this work, the utilization of corn stover hydrolysate as feedstock for acetate production was also investigated. Glucose and xylose are two principal sugars in the corn stover hydrolysate. Due to the presence of carbon catabolite suppression, E. coli is theoretically unable to simultaneously metabolize both glucose and xylose [41, 42]. Thus, ptsG gene responsible for the carbon catabolite suppression was deleted in the strain E. coli 5K. Then, the plasmid pFF was transformed into the obtained strain E. coli 6K to generate E. coli 6K/pFF. The acetate production by E. coli 6K/pFF was also studied in a 1-L bioreactor. The corn stover hydrolysate was added into the DYM to make the glucose at a concentration of about 30 g/L and xylose at a concentration of about 10 g/L. As shown in Fig. 5B, E. coli 6K/pFF could simultaneously consume both glucose and xylose in corn stover hydrolysate to produce acetate. Acetate at a concentration of 20.09 g/L was produced from 29 g/L glucose and 9.66 g/L xylose with a yield of 0.52 g/g sugar, accounting for 98.34% of the total products (Additional file 1: Figure S2B).

Acetate is an important feedstock in chemicals production. Different biotechnological processes like ethanol oxidation by Acetobacter [18], syngas fermentation via the Wood–Ljungdahl pathway [43], and bioconversion of lignocellulosic substrates have been developed for acetate production (Additional file 1: Table S2). Among the reported biotechnological routes, the group of Ravinder obtained the highest acetate concentration of 30.98 g/L with Clostridium lentocellum SG6 from 100 g/L alkali-extracted paddy straw within 14 days [44]. Karekar et al. reported acetate production with corn stover hydrolysate by Acetobacterium woodii [45]. However, A. woodii can not use xylose for growth and 7.83 g/L acetate was produced within about 20 days from glucose in corn stover hydrolysate. In this study, the genes frdA, ldhA, and adhE in E. coli BL21 (DE3) were inactivated to reduce succinate, lactate, and ethanol generation. Then, the ptsG gene responsible for the carbon catabolite suppression was knocked out for co-utilization of glucose and xylose. Finally, an EP-bifido pathway was introduced to increase the yield of acetate. Acetate production from corn stover hydrolysate was achieved with high concentration (20.09 g/L), productivity (0.21 g/L/h), and yield (0.52 g/g sugar).

Acetyl-CoA is the precursor of many industrially relevant biochemicals like 3-hydroxypropionate [46], 1-butanol [47], isopropanol [48,49,50,51,52], 1,3-butanediol [53], poly-β-hydroxybutyrate [54], mevalonate [55] and fatty acids [56]. Wang et al. firstly constructed the EP-bifido pathway in E. coli [29] and achieved efficient production of mevalonate from glucose through dynamically regulation of glycolysis flux [55]. Recently, Shi et al. also established the EP-bifido pathway in E. coli and efficient poly-β-hydroxybutyrate production was realized using xylose as the substrate [57]. Corn is one of the main food crops in major agricultural countries such as China and the United States. Corn stover hydrolysate has been used for carbon sources to produce bioproducts like poly(3-hydroxybutyrate-co-lactate), glycolate, and ethanol [58,59,60]. In this study, E. coli 6K/pFF containing the EP-bifido pathway was constructed to efficiently produce acetate with AcP as the key intermediate. Besides dephosphorylation to generate acetate by AckA, AcP can be catalyzed into acetyl-CoA by Pta and then utilized for acetyl-CoA derivatives synthesis. E. coli 6K/pFF thus may be used as a chassis strain for the production of derivatives of acetyl-CoA from corn stover hydrolysate through deleting AckA and further introducing suitable metabolic pathways.

Conclusion

Herein, the recombination strains E. coli 5K/pFF and E. coli 6K/pFF were constructed to produce acetate from glucose and corn stover hydrolysate, respectively. The recombination strain E. coli 5K/pFF can produce 22.89 g/L acetate from 29 g/L glucose with a yield of 0.61 g/g glucose and E. coli 6K/pFF can produce 20.09 g/L acetate from corn stover hydrolysate with a yield of 0.52 g/g sugar. This study provides a promising alternative for both fermentative acetate production and resource utilization of corn stover hydrolysate. In addition to acetate production, the recombinant strain E. coli 6K/pFF might also have the potential to be engineered for producing other acetyl-CoA derivatives with lignocellulose hydrolysate.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

AckA:

Acetate kinase

Pta:

Phosphate acetyltransferase

AcP:

Acetyl-phosphate

NOG:

Non-oxidative glycolytic

Fxpk:

Bifunctional phosphoketolase

Fbp:

Fructose-1,6-bisphosphatase

F6P:

Fructose-6-phosphate

X5P:

Xylulose-5-phosphate

E4P:

Erythrose-4-phosphate

G3P:

Glyceraldehyde-3-phosphate

PfkA:

6-phosphofructokinase

Edd:

6-phosphogluconate dehydratase

XylA:

Xylose isomerase

XylB:

Xylulokinase

IPTG:

Isopropyl-β-d-1-thiogalactopyranoside

References

  1. Kolter K, Dashevsky A, Irfan M, Bodmeier R. Polyvinyl acetate-based film coatings. Int J Pharm. 2013;457(2):470–9.

    Article  PubMed  CAS  Google Scholar 

  2. Morrison GO, Shaw TG. By-products of the carbide industry: the manufacture of ethylidene diacetate and vinyl acetate. Trans Electrochem Soc. 1933;63(1):425–47.

    Article  Google Scholar 

  3. Zoeller JR, Agreda VH, Cook SL, Lafferty NL, Polichnowski SW, Pond DM. Eastman chemical company acetic anhydride process. Catal Today. 1992;13(1):73–91.

    Article  CAS  Google Scholar 

  4. Zoeller JR. Evolving production of the acetyls (acetic acid, acetaldehyde, acetic anhydride, and vinyl acetate): a mirror for the evolution of the chemical industry. ACS symposium series. 2009. pp. 365–87.

  5. Haynes A, Maitlis PM, Morris GE, Sunley GJ, Adams H, Badger PW, Bowers CM, Cook DB, Elliott PIP, Ghaffar T, Green H, Griffin TR, Payne M, Pearson JM, Taylor MJ, Vickers PW, Watt RJ. Promotion of Iridium-catalyzed methanol carbonylation: mechanistic studies of the cativa process. J Am Chem Soc. 2004;126(9):2847–61.

    Article  PubMed  CAS  Google Scholar 

  6. Greeshma N, Shah BH, Patel NM. Simulation study of reactive distillation for monochloroacetic acid using ChemCAD. Simulation. 2014;1(2):1–11.

    Google Scholar 

  7. Lindeman MK. Vinyl acetate and the textile industry. Text Chem Color. 1989;21(1):21–8.

    Google Scholar 

  8. Maki-Arvela P, Salmi T, Paatero E. Kinetics of the chlorination of acetic acid with chlorine in the presence of chlorosulfonic acid and thionyl chloride. Ind Eng Chem Res. 1994;33(9):2073–83.

    Article  CAS  Google Scholar 

  9. Ho CW, Lazim AM, Fazry S, Zaki HH, Lim SJ. Varieties, production, composition and health benefits of vinegars: a review. Food Chem. 2017;221:1621–30.

    Article  PubMed  CAS  Google Scholar 

  10. Tout R. A review of adhesives for furniture. Int J Adhes Adhes. 2000;20(4):269–72.

    Article  CAS  Google Scholar 

  11. Mohammadzadeh-Aghdash H, Sohrabi Y, Mohammadi A, Shanehbandi D, Dehghan P, Dolatabadi JEN. Safety assessment of sodium acetate, sodium diacetate and potassium sorbate food additives. Food Chem. 2018;257:211–5.

    Article  PubMed  CAS  Google Scholar 

  12. Nagarkar R, Patel J. Polyvinyl alcohol: a comprehensive study. Acta Sci Pharm Sci. 2019;3:34–44.

    Google Scholar 

  13. Kalck P, Le Berre C, Serp P. Recent advances in the methanol carbonylation reaction into acetic acid. Coord Chem Rev. 2020;402:213078.

    Article  CAS  Google Scholar 

  14. Budiman AW, Nam JS, Park JH, Mukti RI, Chang TS, Bae JW, Choi MJ. Review of acetic acid synthesis from various feedstocks through different catalytic processes. Catal Surv Asia. 2016;20:173–93.

    Article  CAS  Google Scholar 

  15. Pal P, Nayak J. Acetic acid production and purification: critical review towards process intensification. Sep Purif Rev. 2017;46(1):44–61.

    Article  CAS  Google Scholar 

  16. Kiefer D, Merkel M, Lilge L, Henkel M, Hausmann R. From acetate to bio-based products: underexploited potential for industrial biotechnology. Trends Biotechnol. 2021;39(4):397–411.

    Article  PubMed  CAS  Google Scholar 

  17. Gullo M, Verzelloni E, Canonico M. Aerobic submerged fermentation by acetic acid bacteria for vinegar production: process and biotechnological aspects. Process Biochem. 2014;49(10):1571–9.

    Article  CAS  Google Scholar 

  18. Hutkins RW. Vinegar fermentation. In Microbiology and Technology of Fermented Foods. 2006;11:397–417.

  19. Li S, Li P, Feng F, Luo LX. Microbial diversity and their roles in the vinegar fermentation process. Appl Microbiol Biotechnol. 2015;99:4997–5024.

    Article  PubMed  CAS  Google Scholar 

  20. Sengun IY, Karabiyikli S. Importance of acetic acid bacteria in food industry. Food Control. 2011;22(5):647–56.

    Article  CAS  Google Scholar 

  21. Sakurai K, Arai H, Ishii M, Igarashi Y. Changes in the gene expression profile of Acetobacter aceti during growth on ethanol. J Biosci Bioeng. 2012;113(3):343–8.

    Article  PubMed  CAS  Google Scholar 

  22. Ragsdale SW, Pierce E. Acetogenesis and the Wood–Ljungdahl pathway of CO2 fixation. Biochim Biophys Acta. 2008;1784(12):1873–98.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Ragsdale SW. Enzymology of the Wood–Ljungdahl pathway of acetogenesis. Ann N Y Acad Sci. 2008;1125(1):129–36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Sun W, Li X, Zhao J, Qin Y. Pretreatment strategies to enhance enzymatic hydrolysis and cellulosic ethanol production for biorefinery of corn stover. Int J Mol Sci. 2022;23(21):13163.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Takkellapati S, Li T, Gonzalez MA. An overview of biorefinery-derived platform chemicals from a cellulose and hemicellulose biorefinery. Clean Technol Environ Policy. 2018;20:1615–30.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Liu G, Qu Y. Integrated engineering of enzymes and microorganisms for improving the efficiency of industrial lignocellulose deconstruction. Eng Microbiol. 2021;1:100005.

    Article  CAS  Google Scholar 

  27. Bogorad IW, Lin TS, Liao JC. Synthetic non-oxidative glycolysis enables complete carbon conservation. Nature. 2013;502(7473):693–7.

    Article  PubMed  CAS  Google Scholar 

  28. Meile L, Rohr LM, Geissmann TA, Herensperger M, Teuber M. Characterization of the d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis. J Bacteriol. 2001;183(9):2929–36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Wang Q, Xu J, Sun Z, Luan Y, Li Y, Wang J, Qi Q. Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO2 emission. Metab Eng. 2019;51:79–87.

    Article  PubMed  CAS  Google Scholar 

  30. Wovcha MG, Steuerwald DL, Brooks KE. Amplification of d-xylose and d-glucose isomerase activities in Escherichia coli by gene cloning. Appl Environ Microbiol. 1983;45:1402–4.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Briggs KA, Lancashire WE, Hartley BS. Molecular cloning, DNA structure and expression of the Escherichia coli d-xylose isomerase. EMBO J. 1984;3:611–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Lawlis VB, Dennis MS, Chen EY, Smith DH, Henner D. Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli. Appl Environ Microbiol. 1984;47:15–21.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Chen Y, Zhu Y, Wang H, Chen R, Liu Y, Zhang W, Mu W. De novo biosynthesis of 2′-fucosyllactose in a metabolically engineered Escherichia coli using a novel ɑ1, 2-fucosyltransferase from Azospirillum lipoferum. Bioresour Technol. 2023;374:128818.

    Article  PubMed  CAS  Google Scholar 

  34. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015;81(7):2506–14.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Li Q, Sun B, Chen J, Zhang Y, Jiang YU, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin. 2021;53(5):620–7.

    Article  PubMed  CAS  Google Scholar 

  36. Wang W, He P, Zhao D, Ye L, Dai L, Zhang X, Sun Y, Zheng J, Bi C. Construction of Escherichia coli cell factories for crocin biosynthesis. Microb Cell Fact. 2019;18(1):120.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Basan M, Hui S, Okano H, Zhang Z, Shen Y, Williamson JR, Hwa T. Overflow metabolism in Escherichia coli results from efficient proteome allocation. Nature. 2015;528(7580):99–104.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Thomas VC, Sadykov MR, Chaudhari SS, Jones J, Endres JL, Widhelm TJ, Bayles KW. A central role for carbon-overflow pathways in the modulation of bacterial cell death. PLoS Pathog. 2014;10(6):e1004205.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Bernal V, Castaño-Cerezo S, Cánovas M. Acetate metabolism regulation in Escherichia coli: carbon overflow, pathogenicity, and beyond. Appl Microbiol Biotechnol. 2016;100:8985–9001.

    Article  PubMed  CAS  Google Scholar 

  40. Sawers RG, Clark DP. Fermentative pyruvate and acetyl-coenzyme A metabolism. EcoSal plus. 2004;1(1):101128.

    Article  Google Scholar 

  41. Deutscher J. The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol. 2008;11(2):87–93.

    Article  PubMed  CAS  Google Scholar 

  42. Kremling A, Geiselmann J, Ropers D, De Jong H. Understanding carbon catabolite repression in Escherichia coli using quantitative models. Trends Microbiol. 2015;23(2):99–109.

    Article  PubMed  CAS  Google Scholar 

  43. Harahap BM, Ahring BK. Acetate production from syngas produced from lignocellulosic biomass materials along with gaseous fermentation of the syngas: a review. Microorganisms. 2023;11(4):995.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Ravinder T, Ramesh B, Seenayya G, Reddy G. Fermentative production of acetic acid from various pure and natural cellulosic materials by Clostridium lentocellum SG6. World J Microbiol Biotechnol. 2000;16:507–12.

    Article  CAS  Google Scholar 

  45. Karekar SC, Srinivas K, Ahring BK. Kinetic study on heterotrophic growth of Acetobacterium woodii on lignocellulosic substrates for acetic acid production. Fermentation. 2019;5(1):17.

    Article  CAS  Google Scholar 

  46. Guo L, Liu M, Bi Y, Qi Q, Xian M, Zhao G. Using a synthetic machinery to improve carbon yield with acetylphosphate as the core. Nat Commun. 2023;14(1):5286.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Ferreira S, Pereira R, Wahl SA, Rocha I. Metabolic engineering strategies for butanol production in Escherichia coli. Biotechnol Bioeng. 2020;117(8):2571–87.

    Article  PubMed  CAS  Google Scholar 

  48. Soma Y, Yamaji T, Matsuda F, Hanai T. Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli. J Biosci Bioeng. 2017;123(5):625–33.

    Article  PubMed  CAS  Google Scholar 

  49. Okahashi N, Matsuda F, Yoshikawa K, Shirai T, Matsumoto Y, Wada M, Shimizu H. Metabolic engineering of isopropyl alcohol-producing Escherichia coli strains with 13C‐metabolic flux analysis. Biotechnol Bioeng. 2017;114(12):2782–93.

    Article  PubMed  CAS  Google Scholar 

  50. Wang Y, Lu J, Xu M, Qian C, Zhao F, Luo Y, Wu H. Efficient biosynthesis of isopropanol from ethanol by metabolically engineered Escherichia coli. ACS Sustainable Chem Eng. 2022;10(41):13857–64.

    Article  CAS  Google Scholar 

  51. Jojima T, Inui M, Yukawa H. Production of isopropanol by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol. 2008;77:1219–24.

    Article  PubMed  CAS  Google Scholar 

  52. Liang L, Liu R, Garst AD, Lee T, Beckham GT, Gill RT. CRISPR EnAbled trackable genome Engineering for isopropanol production in Escherichia coli. Metab Eng. 2017;41:1–10.

    Article  PubMed  CAS  Google Scholar 

  53. Wang J, Zhang R, Zhang J, Gong X, Jiang T, Sun X, Yan Y. Tunable hybrid carbon metabolism coordination for the carbon-efficient biosynthesis of 1,3-butanediol in Escherichia coli. Green Chem. 2021;23(21):8694–706.

    Article  CAS  Google Scholar 

  54. Li Y, Sun Z, Xu Y, Luan Y, Xu J, Liang Q, Wang Q. Enhancing the glucose flux of an engineered EP-Bifido pathway for high poly(hydroxybutyrate) yield production. Front Bioeng Biotech. 2020;8:517336.

    Article  Google Scholar 

  55. Zhu Y, Li Y, Xu Y, Zhang J, Ma L, Qi Q, Wang Q. Development of bifunctional biosensors for sensing and dynamic control of glycolysis flux in metabolic engineering. Metab Eng. 2021;68:142–51.

    Article  PubMed  CAS  Google Scholar 

  56. Xiao Y, Ruan Z, Liu Z, Wu SG, Varman AM, Liu Y, Tang YJ. Engineering Escherichia coli to convert acetic acid to free fatty acids. Biochem Eng J. 2013;76:60–9.

    Article  CAS  Google Scholar 

  57. Shi LL, Zheng Y, Tan BW, Li ZJ. Establishment of a carbon-efficient xylulose cleavage pathway in Escherichia coli to metabolize xylose. Biochem Eng J. 2022;179:108331.

    Article  CAS  Google Scholar 

  58. Wu J, Wei X, Guo P, He A, Xu J, Jin M, Wu H. Efficient poly(3-hydroxybutyrate-co-lactate) production from corn stover hydrolysate by metabolically engineered Escherichia coli. Bioresour Technol. 2021;341:125873.

    Article  PubMed  CAS  Google Scholar 

  59. Yang H, He Y, Zhou S, Deng Y. Dynamic regulation and cofactor engineering of Escherichia coli to enhance production of glycolate from corn stover hydrolysate. Bioresour Technol. 2024;398:130531.

    Article  PubMed  CAS  Google Scholar 

  60. Zhao R, Li H, Li Q, Jia Z, Li S, Zhao L, Cao L. High titer (> 100 g/L) ethanol production from pretreated corn stover hydrolysate by modified yeast strains. Bioresour Technol. 2024;391:129993.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We are grateful to Chengjia Zhang and Nannan Dong from Core Facilities for Life and Environmental Sciences (State Key Laboratory of Microbial Technology, Shandong University) for giving assistance in microbial fermentation.

Funding

This work was supported by grants from the National Key R&D Program of China (2022YFC2106000), National Natural Science Foundation of China (32300045, 32300029), Youth Program of Natural Science Foundation of Shandong Province (ZR2022QC092, ZR2023QC237), China Postdoctoral Science Foundation (2023M742085), Youth Program of Natural Science Foundation of Qingdao City (23-2-1-31-zyyd-jch), Postdoctoral Innovation Program of Shandong Province (No. SDCX-ZG-202400150), Qingdao Postdoctoral Research Project (QDBSH20230201011), State Key Laboratory of Microbial Technology Open Projects Fund (M2023-02), and State Key Laboratory of Microbial Technology (SKLMTFCP-2023–03).

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Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao, 266237, P. R. China

    Jieni Zhu, Wei Liu, Leilei Guo, Xiaoxu Tan, Weikang Sun, Hongxu Zhang, Hui Zhang, Wenjia Tian, Wensi Meng, Yidong Liu, Zhaoqi Kang, Chao Gao, Chuanjuan Lü & Cuiqing Ma

  2. School of Municipal and Environmental Engineering, Shandong Jianzhu University, Jinan, 250101, China

    Tianyi Jiang

  3. State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, 200240, China

    Ping Xu

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  1. Jieni Zhu
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  2. Wei Liu
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Contributions

Jieni Zhu: Investigation, Writing original draft, Data curation. Wei Liu: Formal analysis, Software, Validation. Leilei Guo: Software, Validation. Xiaoxu Tan: Software, Investigation. Weikang Sun: Supervision, Formal analysis. Hongxu Zhang: Software, Validation. Hui Zhang: Software, Supervision. Wenjia Tian: Supervision. Tianyi Jiang: Supervision. Wensi Meng: Formal analysis. Yidong Liu: Supervision. Zhaoqi Kang: Formal analysis. Chuanjuan Lü: Writing, Supervision, Software, Formal analysis. Chao Gao: Supervision, Writing, Software, Funding acquisition. Ping Xu: Supervision. Cuiqing Ma: Conceptualization, Supervision, Writing, Funding acquisition. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Corresponding authors

Correspondence to Chuanjuan Lü or Cuiqing Ma.

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12934_2024_2575_MOESM1_ESM.docx

Supplementary Material 1: Additional file: Figure S1. Comparison of concentrations of the metabolites produced by different recombinant E. coli strains from glucose during shake flask fermentation. Figure S2. The proportion of different metabolites produced by E. coli 5K/pFF and E. coli 6K/pFF during batch fermentation in a 1-L bioreactor. Table S1. The primers used in this study. Table S2. Fermentative acetate production from different lignocellulose biomass.

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Zhu, J., Liu, W., Guo, L. et al. Acetate production from corn stover hydrolysate using recombinant Escherichia coli BL21 (DE3) with an EP-bifido pathway. Microb Cell Fact 23, 300 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-024-02575-y

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Keywords

Microbial Cell Factories

ISSN: 1475-2859

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