Engineered the high efficiency pathway for HAc utilization in E. coli

HAc is employed as a potential substrate for production of biomass, biofuels and value added chemicals. However, E. coli has a restricted ability to use HAc for biomass growth. The metabolic pathways which E. coli utilizes HAc are mainly via AMP-forming acetyl-CoA synthetase (the acs pathway) and phosphotransacetylase/acetate kinase (the reversible PTA-ACKA pathway) [9, 22]. In the previous study, it has proven that overexpressing the single acs gene along with maintaining the native HAc pathways was the best strategy for HAc assimilation in E. coli [23, 24]. A suitable method to optimize pathway efficiency may be to use genes from different organisms [25]. In this paper, the acetyl-CoA synthase enzymes from native E. coli, Salmonella typhimurium LT2 and Acetobacter pasteurianus were assessed to utilize HAc for cell growth.

Therefore, in this paper, to construct the acetate utilization pathway in E. coli, the different ACS genes were screened. The gene ACS EC from E. coli, ACS ST from S. typhimurium LT2 and ACS AP from A. pasteurianus were cloned into the plasmid pCOLDuet-1 forming the plasmids pYJM60, pYJM61 and pYJM62, respectively. The different strains containing pYJM60, pYJM61 and pYJM62 named as YJM60, YJM61 and YJM62, were cultured using M9 medium with different concentrations of sodium acetate. As shown in Fig. 2, the results showed that the strain containing ACS AP gene grow best among three strains, also suggested that the enzyme from A. pasteurianus was the most efficient in the conversion of acetate into acetyl-CoA.

Fig. 2 Comparative growths of the engineered strains using acetic acid. The engineered strains E. coli BL21(DE3)/pCOLADUet-1 (a), BL21(DE3)/pYJM60 (b), BL21(DE3)/pYJM62 (c) and BL21(DE3)/pYJM61 (d) grew on different concentrations of acetate. The OD 600 was measured spectrophotometrically. The experiment was performed in triplicate Full size image

Biosynthesis of β-caryophyllene from HAc using native MEP pathway

Farnesyl diphosphate (FPP), generated from either the DXP or MVA pathway [26, 27], could be catalyzed by the β-caryophyllene synthase into β-caryophyllene [28, 29]. Nevertheless, E. coli harbors MEP pathway, and is not capable of producing β-caryophyllene on its own due to the lack of β-caryophyllene synthase. In the study, to produce β-caryophyllene, the optimized QHS1 gene from Artemisia annua was overexpressed along with ACS AP enzyme from A. pasteurianus in the engineered E. coli (YJM63 containing pACY-QHS1/pCOL-ACS AP ). Firstly, the recombinant strain YJM63 was grown in the M9 medium with acetate as the carbon source, and the target product β-caryophyllene was not detected. While the engineered strain cultured in the M9 medium with 2 g/L glucose as the carbon source, it could produce 56 ± 6 μg/L β-caryophyllene. No β-caryophyllene production was detected in the control strain harboring the empty vector pACYCDUet-1/pCOL-ACS AP . Based on the results, using the native MEP pathway, QHS1 gene from A. annua and ACS AP gene from A. pasteurianus, a novel biosynthetic pathway for the β-caryophyllene production with acetate as the carbon source has been successfully established in the E. coli. However, the effectiveness of the whole metabolic pathway for β-caryophyllene production is too low. The main reason might be short of other important precursors such as DMAPP, IPP and GPP.

Geranyl diphosphate (GPP), the key precursor of sesquiterpene production, is catalyzed from the condensation of dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthase [30]. In this paper, to enhance the supply of GPP and accordingly to increase β-caryophyllene production, the GPP synthase of Abies grandis was co-expressed in the cell along with QHS1 and ACS AP genes and formed into the engineered strain YJM64 (E. coli BL21(DE3) containing pACY-QHS1-GPPS2/pCOL-ACS AP ). According to the GC analysis results, after 24 h of culture, 102 ± 9 μg/L β-caryophyllene was produced by the E. coli strain YJM64. The results demonstrated that the heterologous expression of GPP synthase (GPPS) is beneficial to the β-caryophyllene production. This finding is corresponding to previous studies on the production of other terpenes such as α-pinene and sabinene [31, 32].

Establishing a MVA-mediated biosynthetic pathway for β-caryophyllene production from acetate

Despite of progress made in the β-caryophyllene biosynthesis using the native MEP pathway, the yield of β-caryophyllene was so low that it is not economic and feasible for industrial application. In previous studies, the hybrid MVA pathway has proven to be efficient on the biosynthesis of DMAPP and IPP [33, 34] which are the precursors of all terpenes. Based on these findings, a hypothesis can be put forward that the MVA pathway might be more efficient than the MEP pathway in β-caryophyllene production using acetate as carbon source.

In an attempt to increase the production of β-caryophyllene, the hybrid MVA pathway was introduced into E. coli and overexpressed along with QHS1 from A. annua, GPPS2 gene from A. grandis and ACS AP gene from A. pasteurianus. As expected, the recombinant strain YJM66 (pACY-mvaE-mvaS-QHS1-GPPS2/pTrc-Low/pCOL-ACS AP ) carrying hybrid MVA pathway could accumulate 8 ± 0.75 mg/L β-caryophyllene, which is about eight-fold higher than that (102 ± 9 μg/L) produced by the control strain YJM64 (pACY-QHS1-GPPS2/pCOL-ACS AP ) without the hybrid MVA pathway. According to the data obtained, it may be easier to make a conclusion that the hybrid MVA pathway is conducive to the β-caryophyllene biosynthesis.

The effect of AACS on β-caryophyllene production from Acetate

In recent studies, acetoacetyl-CoA synthase (AACS) from Streptomyces sp. strain CL190 has been proven to catalyze a single condensation of acetyl-CoA and malonyl-CoA to form acetoacetyl-CoA [35]. Unlike acetoacetyl-CoA thiolase (EC 2.3.1.9), which also synthesize acetoacetyl-CoA by reversible nondecarboxylative condensation of two molecules of acetyl-CoA and prefers acetoacetyl-CoA thiolysis to acetoacetyl-CoA synthesis, NphT7 proves no thiolysis activity against acetoacetyl-CoA, and since NphT7-catalyzed acetoacetyl-CoA synthesis is essentially an energy-favored reaction, NphT7 could be an ideal enzyme to supply acetoacetyl-CoA in cells [36].

When the AACS-encoding gene (nphT7) was expressed combining with the HMG-CoA synthase gene and the HMG-CoA reductase gene in E.coli, the engineered strain could achieve 3.5-fold higher production of mevalonate than the control strain without nphT7 expression [36]. Based on these findings, it may be hypothesized that overexpression of acetoacetyl-CoA synthase (AACS) in β-caryophyllene-producing strain would enhance β-caryophyllene productivity.

In the paper, to further increase β-caryophyllene production, the AACS-encoding gene (nphT7) was overexpressed in the β-caryophyllene producing E. coli YJM67 (pACY-mvaE-mvaS-QHS1-GPPS2/pTrc-Low/pCOL-ACS AP -nphT7). After cultured in the M9 medium with 60 mM acetate as the carbon source for 24 h, the yield of β-caryophyllene reached 22 ± 1.8 mg/L, which is 2.75-fold to the control strain YJM66 without nphT7 gene expression (8 ± 0.75 mg/L). The results demonstrated that acetoacetyl-CoA synthase is helpful to increase acetoacetyl-CoA in cells and accordingly to enhance β-caryophyllene production.

Effect of the type and concentration of the nitrogen source on β-caryophyllene production

The source of the nitrogen in the medium plays an important role in improving the biosynthesis of desired product [37, 38]. To investigate the effect of type and concentration of organic nitrogen source on β-caryophyllene production, six different organic nitrogen sources were chosen and valuated (Fig. 3a). Among the organic nitrogen supplements tried, the Solarbio beef extract allowed a significantly higher β-caryophyllene production than the other organic nitrogen sources.

Fig. 3 The effects of types and concentrations of nitrogen source on β-caryophyllene production by YJM67. a The Effect of different organic nitrogen source on β-caryophyllene production by YJM67. a Beef extract (Aladdin, ○); b yeast extract powder (Beijing AoBoXing Bio-Tech Co., Ltd, ●); c beef extract powder (MDBio, Inc, ■); d beef extract (Beijing Shuangxuan Microbe Culture Medium Products Factory, ▼); e beef extract (Sinopharm Chemical Reagent Co., Ltd, ◄); f beef extract (solarbio, ►). a The Effect of concentration of nitrogen source on β-caryophyllene production by YJM67. When OD 600 reached 0.6–0.9, cultures were induced for 56 h using IPTG in shake-flasks. All the experiments were carried out in triplicates. Optimized conditions: Nitrogen sources, beef power; concentrations of nitrogen source, 5 g/L Full size image

Then to determine the optimum concentration of Solarbio beef extract, various beef extract concentrations, ranging from 1 to 15 g/L, were tested. According to the data shown in Fig. 3b, the maximum production of β-caryophyllene reached 106 ± 9.6 mg/L, which was approximately five times as much as the production before optimization (22 ± 1.8 mg/L).

Based on the above data, the most suitable types and concentration of nitrogen source for β-caryophyllene production using the engineered strain YJM67 were 5 g/L Solarbio beef extract.

Lab-scale batch production of β-caryophyllene

To scale up β-caryophyllene production from HAc, a pH-coupled HAc fed batch fermentation, which can gradually add HAc to the fermentation medium, was performed in a 5-L-scale laboratory batch reactor, in which was grown our most optimized strain, YJM67. Based on the flask condition, the final engineered strain YJM67 was cultured in the 5-L-scale laboratory batch reactor with the minimal medium plus 5 g/L beaf extract. As seen in the Fig. 4, the production of biomass and β-caryophyllene increased substantially without obvious lag phase and their maximum concentrations reached 12.6 and 1.05 g/L at 72 h, respectively, with a specific productivity of 1.15 mg h−1 g−1 dry cells, and the conversion efficiency of HAc to β-caryophyllene (gram to gram) reached 2.1 %. The yield of β-caryophyllene on HAc of this strain also reached approximately 5.6 % of the theoretical yield (of 37.81 %) based on the following formula: 9 HAc → 9 Acetyl-CoA → β-caryophyllene.

Fig. 4 The time course of β-caryophyllene production by YJM67. Biomass (Δ) and β-caryophyllene accumulation (■) in YJM67. Induction was carried out when OD 600 reached about 6 at 30 °C. Other experimental conditions are described in section “Fed-Batch Fermentation” Full size image

These results suggest great potential for this engineered E. coli strain in the production of β-caryophyllene on a large scale.