A microbial fermentation system and process for producing linalool in a nicotine-containing environment, and a method for recycling tobacco waste

Abstract

The present application relates to the technical field of microbial fermentation, and particularly relates to a microbial fermentation system and process for producing linalool in a nicotine-containing environment and a method for recycling tobacco waste. The fermentation system comprises a substrate and a microbial combination for fermentation using the substrate; the substrate contains nicotine; the microorganism is composed of Saccharomyces cerevisiae, Nicotinibacterium and linalool engineering bacteria, and the linalool engineering bacteria comprise an ethanol utilization module. The present application solves the problems of limited carbon source utilization and nicotine sensitivity of the linalool engineering bacteria by introducing Nicotinibacterium and co-culturing Saccharomyces cerevisiae and the linalool engineering bacteria, and can effectively produce linalool in a nicotine environment, and is suitable for recycling of tobacco waste.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a microbial fermentation system and process for producing linalool in a nicotine-containing environment, and a method for recycling tobacco waste. Background Technology

[0002] Tobacco is widely cultivated globally and is an important economic crop. However, the cultivation and processing of tobacco generate a large amount of waste, mainly including tobacco pesticide residues (such as tobacco stalks and substandard tobacco leaves) and cigarette processing by-products. Improper disposal of these tobacco wastes will cause serious waste of resources and environmental pollution, such as damaging soil structure and polluting water bodies.

[0003] However, current utilization of tobacco waste largely focuses on only one component. For example, while research on the chemical extraction and microbial degradation of nicotine is emphasized, the utilization of biomass is often overlooked; or the focus is placed on biomass pretreatment, such as acid-base pretreatment followed by fermentation by microorganisms like yeast to produce ethanol, but this has low end-use value and lacks practical application, failing to compete with corn stalk biomass ethanol fermentation, resulting in low resource utilization of waste tobacco; furthermore, without corresponding downstream treatment technologies, the generated wastewater, waste gas, and waste residue still cause environmental pollution. In summary, tobacco waste treatment technologies lag far behind the requirements of green and low-carbon development in the tobacco industry, making the development of systematic and innovative technologies particularly important. In addition, the incompatibility between different processes is also a major challenge. For example, nicotine, unique to tobacco waste, has high biotoxicity, and its inherent toxicity, if not removed, leads to low efficiency in downstream enzymatic hydrolysis or microbial fermentation. Therefore, how to organically integrate the treatment processes for various tobacco components, overcome the inherent toxicity problem, and maximize resource utilization is a key technical issue in the current utilization of tobacco waste.

[0004] Linalool is an important terpene compound widely used in fragrances, cosmetics, and pharmaceuticals. The production of linalool through microbial fermentation has been a research hotspot in recent years. For example, patent document CN120738086A discloses an engineered linalool-producing bacterium and its application in the fermentation production of linalool. However, the inventors discovered that the linalool synthesis capacity of this engineered bacterium is severely inhibited in the presence of high concentrations of nicotine, and the yield is extremely low regardless of the carbon source used, making it difficult to apply to the treatment and resource utilization of nicotine-containing waste. Summary of the Invention

[0005] The present invention aims to solve the above problems by providing a microbial fermentation system and process that can maintain a high linalool yield in the presence of high concentrations of nicotine and effectively reduce nicotine content, so as to realize the resource reuse of tobacco waste.

[0006] The technical solution to the problem solved by this invention is:

[0007] In a first aspect, a microbial fermentation system for producing linalool in a nicotine-containing environment is provided, comprising a substrate and a microbial combination for fermentation using the substrate; the substrate contains nicotine; the microorganisms consist of Saccharomyces cerevisiae, Arthrobacter nicotine-eating bacteria, and linalool-engineered bacteria, wherein the linalool-engineered bacteria include an ethanol utilization module.

[0008] This invention provides a three-strain synergistic fermentation system. By introducing *Arthrobacter nicotine-eating* and *Saccharomyces cerevisiae* into co-culture with linalool-engineered bacteria, it simultaneously solves the problems of limited carbon source utilization and nicotine sensitivity in the linalool-engineered bacteria. Specifically, *Arthrobacter nicotine-eating* is introduced to degrade nicotine, reducing the high concentration of nicotine to a low concentration that the linalool-engineered bacteria can tolerate, thus reducing the toxicity of nicotine to microbial growth and metabolism, and providing a foundation for subsequent fermentation processes. The linalool-engineered bacteria are introduced with an ethanol utilization module, enabling them to utilize ethanol produced by *Saccharomyces cerevisiae* metabolism as a precursor for linalool synthesis in this three-strain co-fermentation system. This achieves functional coupling between the two strains at the metabolic level, constructing a carbon flow conversion pathway of "tobacco waste-ethanol-linalool," further improving the biomass conversion efficiency and utilization rate of tobacco waste.

[0009] microorganism

[0010] The brewing yeast is not limited and can be obtained by converting sugars into ethanol. Preferably, the brewing yeast is brewing yeast BY4741, which is not limited in origin and can be purchased directly from the market.

[0011] The *Arthrobacter nicotinovorans* strain is not limited to any strain capable of degrading nicotine. Preferably, the *Arthrobacter nicotinovorans* strain is JCM 3874, a publicly available type strain that can be directly purchased through commercial channels.

[0012] The linalool engineered bacteria need to have an ethanol utilization module. As a preferred embodiment of the present invention, the linalool engineered bacteria is linalool engineered bacteria tMTScL.

[0013] The chassis host of the linalool engineered bacterium tMTScLS is Escherichia coli MG1655, and the recA, endA and tnaA genes of the chassis host have been knocked out.

[0014] The linalool engineered bacteria tMTScLS contains plasmids pMVA1 and pScLS.

[0015] The plasmid pMVA1 carries the mevalonate pathway enzyme gene, which is controlled by the T7 and / or tac promoters;

[0016] The plasmid pScLS carries a linalool synthesis module and an ethanol utilization module, controlled by the T7 and / or pLuxB and / or pGyrA promoters; the linalool synthesis module includes a geranyl diphosphate synthase gene and a linalool synthase gene, and the ethanol utilization module includes an acetaldehyde dehydrogenase gene and an alcohol dehydrogenase gene.

[0017] Specifically, the linalool engineered bacteria tMTScLS is obtained through the following steps:

[0018] (1) Construction of a mevalonate synthesis module: Acetyl-CoA thiolysis enzyme (EcatoB, sequence shown in SEQ ID NO:1, GenBank accession number OQ725958.1) from *Escherichia coli*, hydroxymethylpentadiene-CoA synthase (Saccharomyces cerevisiae HMG-CoA synthase, SchmgS, sequence shown in SEQ ID NO:2, GenBank accession number M22002.1) from *Saccharomyces cerevisiae*, hydroxymethylglutaryl-CoA reductase (SchmgR, sequence shown in SEQ ID NO:3, GenBank accession number M22002.1) from *Saccharomyces cerevisiae*, and mevalonate kinase (ScMK, sequence shown in SEQ ID NO:1) from *Saccharomyces cerevisiae* were constructed. As shown in NO:4, the following enzymes were identified: GenBank accession number OQ725958.1; S. cerevisiae phosphomevalonate kinase (ScPMK, sequence shown in SEQ ID NO:5, GenBank accession number OQ725958.1); S. cerevisiae phosphomevalonate decarboxylase (ScPMD, sequence shown in SEQ ID NO:6, GenBank accession number X97557.1); and E. coli isopentenyl diphosphate isomerases (EcIDI, sequence shown in SEQ ID NO:4). As shown in NO:7, the GenBank accession number is OQ725958.1), which is ligated into the expression plasmid pMVA1(SpecR+pAC) to obtain the upstream pathway gene expression plasmid. Its gene expression operon is controlled by the T7 and tac promoters, respectively (isopropyl β-D-thiogalactopyranoside as the inducer [Isopropyl β-D-thiogalactopyranoside, IPTG]).

[0019] (2) Construction of the linalool synthesis pathway: truncated Abies grandis geranylpyrophosphate synthase (trAgGPPS, sequence shown in SEQ ID NO:8, GenBank accession number MW753220.1), linalool synthase from Streptomyces clavuligerus (ScLs, sequence shown in SEQ ID NO:9, GenBank accession number PZ305631), acetaldehyde dehydrogenase from Dickeya parazeae (DpADA, sequence shown in SEQ ID NO:10, GenBank accession number PZ305632), and alcohol dehydrogenase 2 from S. cerevisiae (ScADH2, sequence shown in SEQ ID NO:8) were constructed. As shown in IDNO:11 (GenBank accession number PZ305633), the expression plasmid pScLS was ligated to obtain downstream pathway gene expression plasmids; among them, the expression of trAgGPPS was controlled by the T7 promoter (IPTG as an inducer); the expression of ScLs was controlled by the pLuxB promoter (N-3-oxohexanoyl-homoserine lactone as an inducer [3-oxohexanoyl-homoserine lactone, 3OC6-HSL]); the DpADA and SCADH2 genes constituted the ethanol utilization module and were constitutively expressed through the pGyrA promoter;

[0020] (3) Electroporate the upstream pathway gene expression plasmid pMVA1 and the downstream pathway gene expression plasmid pScLS into E. coli MG1655 ΔrecA ΔendA ΔtnaA strain to obtain linalool engineered strain tMTScLS.

[0021] As a preferred embodiment of the present invention, the specific operation for electroporation transformation of *E. coli* is as follows: The overnight cultured strain in a tray is transferred to 10 mL of LB liquid medium and cultured at 37 °C for 3 h, followed by centrifugation to remove the supernatant. 1 mL of sterile water is used to transfer the bacterial cells to a 1.5 mL centrifuge tube and placed on ice for 10 min. Then, the tube is centrifuged at 10000 rpm for 1 min, the supernatant is removed, and 1 mL of sterile water is added to resuspend the cells. Centrifugation at 10000 rpm for another 1 min is then performed. After removing the supernatant, 100 μL of sterile water and 1 μL of the required plasmid are added and mixed well, then placed on ice for 10 min. The BIO-RAD electroporator is set to the "E. coli" mode for electroporation. 600 μL of LB liquid medium is quickly added, mixed well, and transferred to a test tube. The tube is placed on a shaker and incubated at 30 °C and 250 rpm for 1 h. Subsequently, an appropriate amount of bacterial culture is taken, spread on LB solid medium with the corresponding antibiotic label, and cultured overnight until the colonies on the plate reach a suitable size.

[0022] Linalool can be obtained by fermenting a substrate with a mixture of Saccharomyces cerevisiae, Bacillus nicotine consuming and engineered linalool-producing bacteria, but the yield of linalool is affected by the ratio of the three.

[0023] As a preferred embodiment of the present invention, the volume ratio of the nicotine-eating *Arthrobacter*, *Saccharomyces cerevisiae*, and linalool-engineered bacteria is 1:(0.1-5):(0.1-5). For example, when the volume fraction of *Arthrobacter* is 1 part, the *Saccharomyces cerevisiae* can be 0.1, 0.5, 1, 2, 3, 4, or 5 parts, preferably 0.3-3 parts; the linalool-engineered bacteria can be 0.1, 0.5, 1, 2, 3, 4, or 5 parts, preferably 0.3-3 parts.

[0024] In some embodiments, to further improve the yield of linalool, the volume ratio of the nicotine-eating *Arthrobacter*, *Saccharomyces cerevisiae*, and the engineered linalool strain is preferably 1:(0.5–5):1; more preferably 1:3:1. With the same substrate, the yield of linalool is increased within this range.

[0025] In other embodiments, if the primary purpose is to degrade nicotine and the secondary purpose is to produce linalool, the volume ratio of the nicotine-degrading Arthrobacter, Saccharomyces cerevisiae and linalool engineered bacteria is preferably 1:(0.1-0.5):(0.1-0.5), and more preferably 3:1:1.

[0026] Substrate

[0027] Nicotine source: As a preferred embodiment of the present invention, the initial concentration of nicotine in the substrate is 20–450 mg / L. The microbial fermentation system of the present invention can produce linalool within a wide range of initial nicotine concentrations.

[0028] As a preferred embodiment of the present invention, the nicotine is derived from tobacco leaves, preferably tobacco waste. Therefore, the present invention also substantially provides a method for reusing tobacco waste, utilizing the aforementioned microbial fermentation system and tobacco waste to produce linalool.

[0029] As a preferred embodiment of the present invention, the nicotine source in the substrate is obtained through the following steps: adding 0.5–1.5 v / v% concentrated sulfuric acid to a 0.05–0.15 g / mL aqueous dispersion of tobacco leaves, treating at 115–130 °C for 15–25 min, and adjusting the pH to 6–7; then centrifuging to obtain the supernatant. Preferably, centrifuging at 3500–4500 rpm for 5–15 min. For example, the concentration of the tobacco leaf aqueous dispersion can be 0.05 g / mL, 0.075 g / mL, 0.1 g / mL, 0.125 g / mL, or 0.15 g / mL; the amount of concentrated acid can be 0.5 v / v%, 0.75 v / v%, 1 v / v%, 1.25 v / v%, or 1.5 v / v%; the treatment temperature can be 115℃, 120℃, 125℃, or 130℃; the treatment time can be 15 min, 17.5 min, 20 min, 22.5 min, or 25 min; the centrifugation rate can be 3500 rpm, 3750 rpm, 4000 rpm, 4250 rpm, or 4500 rpm; and the centrifugation time can be 5 min, 7.5 min, 10 min, 12.5 min, or 15 min.

[0030] In some preferred embodiments, the nicotine is derived from the supernatant of tobacco leaf acid hydrolysis, obtained by the following steps: adding 1 v / v% concentrated sulfuric acid to a 0.1 g / mL aqueous dispersion of tobacco leaves, treating at 121 °C for 20 min, adjusting the pH to 6-7 with NaOH solution, centrifuging at 4000 rpm for 10 min, to obtain a supernatant of tobacco leaf acid hydrolysis with a concentration of 100 g / L.

[0031] As a preferred embodiment of the present invention, the concentration of the tobacco leaf acid hydrolysis supernatant in the substrate is 5–30 g / L to control the nicotine concentration. For example, the concentration can be 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L.

[0032] In some preferred embodiments, when the volume ratio of the nicotine-eating bacillus, saccharitomyces, and linalool engineered bacteria is 1:(1-5):(0.3-1), as a preferred embodiment of the present invention, the concentration of the supernatant in the substrate is 20-30 g / L, at which point the linalool yield is increased.

[0033] In this invention, the yield of linalool actually increased under conditions of high concentration of supernatant in the substrate and high nicotine concentration. This is at least due to: First, at higher nicotine concentrations, *Arthrobacter nicotine-eating* exhibits a faster degradation rate, which can more quickly relieve the inhibition on the engineered bacteria, allowing the linalool engineered bacteria to remain in an uninhibited state earlier and for a longer period, thereby accumulating more linalool. Second, the tobacco leaf supernatant also contains a carbon source; a higher concentration of carbon source in the higher concentration of supernatant promotes the production of ethanol by *Saccharomyces cerevisiae*, providing more linalool synthesis precursors for the engineered bacteria, and promoting higher expression of geranyl diphosphate synthase and linalool synthase in the engineered bacteria.

[0034] More preferably, the volume ratio of the nicotine-eating bacillus, saccharitomyces, and linalool engineered bacteria is 1:(2-4):1, and the concentration of the supernatant in the substrate is 20-30 g / L, resulting in a high linalool yield.

[0035] Carbon source: Since the supernatant of tobacco leaves already contains a carbon source, the substrate of this invention may or may not require an additional carbon source, or an additional carbon source may be added to improve production efficiency.

[0036] As a preferred embodiment of the present invention, the substrate includes an external carbon source, which comprises a monosaccharide and glycerol. The use of a monosaccharide carbon source effectively increases ethanol yield, thereby enabling the synthesis of linalool from ethanol. The monosaccharide, for example, may be glucose.

[0037] As a preferred embodiment of the present invention, the concentration of the monosaccharide in the substrate is 30 to 50 g / L, for example, it can be 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L.

[0038] As a preferred embodiment of the present invention, the substrate further includes 5 to 15 g / L of glycerol. For example, the concentration of glycerol may be 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, or 15 g / L.

[0039] other: The substrate also adaptively contains components required for microbial fermentation, such as organic nitrogen sources, phosphorus sources, and vitamins.

[0040] As a preferred embodiment of the present invention, the substrate further includes 15 to 25 g / L of tryptone, for example, 15 g / L, 17.5 g / L, 20 g / L, 22.5 g / L, or 25 g / L;

[0041] It also includes 5 to 15 g / L of yeast extract, for example, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, or 15 g / L.

[0042] It also includes potassium dihydrogen phosphate at concentrations of 10–15 g / L, for example, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L.

[0043] It also includes 2 to 6 g / L diammonium hydrogen phosphate, for example, 2 g / L, 3 g / L, 4 g / L, 5 g / L, and 6 g / L.

[0044] Secondly, another objective of this invention is to provide a microbial fermentation process for producing linalool in a nicotine-containing environment, using the aforementioned microbial fermentation system, with a fermentation time of 24–120 h. For example, the fermentation time can be 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, or 120 h.

[0045] In some embodiments, to further improve the yield of linalool, the fermentation time is preferably 40-50 h, at which point the yield of linalool reaches its peak.

[0046] In other embodiments, if the primary objective is to degrade nicotine and the secondary objective is to produce linalool, preferably, the fermentation time is not less than 120 h, at which point the nicotine is completely degraded.

[0047] Thirdly, another objective of this invention is a method for reusing tobacco waste by degrading nicotine and simultaneously producing linalool, including the aforementioned process.

[0048] In this invention, the synergistic fermentation of three bacteria—Saccharomyces cerevisiae, Arthrobacter nicotine phage, and linalool engineered bacteria—not only achieves linalool production but also simultaneously completes nicotine degradation. It also involves ethanol production and utilization, realizing multi-functional metabolic transformation.

[0049] The beneficial effects of this invention are:

[0050] 1. This invention provides a microbial fermentation system and process that introduces nicotine-eating bacteria and Saccharomyces cerevisiae to co-culture with linalool engineered bacteria. This solves the problems of limited carbon source utilization and nicotine sensitivity of linalool engineered bacteria, and achieves the production of linalool in a nicotine-containing environment, thereby realizing the resource reuse of linalool production from tobacco waste.

[0051] 2. This invention provides a method for recycling tobacco waste. The microbial fermentation system and process can not only produce linalool, but also simultaneously degrade nicotine. Attached Figure Description

[0052] Figure 1This is a schematic diagram of the synergistic fermentation metabolism of three bacteria: Nicotine-eating Bacillus JCM 3874, Saccharomyces cerevisiae BY4741, and the engineered linalool strain tMTScLS.

[0053] Figure 2 In Example 1, the nicotine, ethanol, and linalool contents of the supernatant from tobacco leaf acid hydrolysis at concentrations of 10 g / L and 25 g / L under different inoculation ratios of the three bacteria were measured (Figure A: supernatant from tobacco leaf acid hydrolysis at a concentration of 10 g / L; Figure B: supernatant from tobacco leaf acid hydrolysis at a concentration of 25 g / L).

[0054] Figure 3 In Example 2, the time curves of *Arthrobacter japonicus* JCM3874: *Saccharomyces cerevisiae* BY4741: *Linol-producing* tMTScLS at inoculation ratios of 1:3:1 and 3:1:1 were obtained from the supernatant of tobacco leaf acid hydrolysis at concentrations of 10 g / L and 25 g / L (Figure A: 10 g / L, inoculation ratio 1:3:1; Figure B: 25 g / L, inoculation ratio 1:3:1; Figure C: 10 g / L, inoculation ratio 3:1:1; Figure D: 25 g / L, inoculation ratio 3:1:1).

[0055] Figure 4 This is a GC-MS qualitative chromatogram of linalool titer and linalool standard of the engineered linalool bacterium tMTScLS in Comparative Example 1 under different concentrations of tobacco leaf acid hydrolysis supernatant and carbon sources. Detailed Implementation

[0056] The following are specific embodiments of the present invention, and the technical solution of the present invention will be further described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0057] The bacterial strains, reagents, and metabolic methods involved in the examples and comparative examples are as follows:

[0058] Linalool engineered bacteria tMTScLS :

[0059] Plasmid construction: Upstream and downstream primers for the target gene were designed for PCR. The PCR reaction system was prepared according to the instructions for the KOD high-fidelity PCR enzyme. After PCR, agarose gel electrophoresis was performed. Agarose gel electrophoresis: 0.9 g of agarose was weighed into a beaker, 100 mL of 1×TAE solution was added, and 5 μL of 10× nucleic acid dye was added and mixed. The mixture was then heated in a microwave oven until completely dissolved. After shaking, the mixture was poured into an electrophoresis plate, and a comb of appropriate well size was inserted. The plate was allowed to solidify at room temperature. An appropriate amount of 10× DNA loading buffer was added to the PCR reaction solution and mixed. The comb was carefully removed from the electrophoresis plate, and the agarose gel was placed in the electrophoresis tank. An appropriate amount of the prepared sample was added to each well. Gel electrophoresis and imaging: The electrophoresis tank voltage was set to 180 V and electrophoresis was performed for approximately 20 min. The agarose gel was then placed in a gel imaging system for observation and photographic recording. The agarose gel extraction procedure was performed according to the Tiangen gel extraction kit. After DNA recovery, the purity and concentration of DNA were determined using a micro spectrophotometer. Based on the recovered DNA concentration, the required volumes of the added gene fragment and plasmid backbone fragment were calculated. 2× seamless cloning assembly reagent was added, and the reaction was carried out at 50 °C for 5–15 min, followed by an ice bath for 5 min, for plasmid transformation.

[0060] Electroporation of *E. coli*: The overnight cultured strain was transferred to 10 mL of LB liquid medium and incubated at 37 °C for 3 h, followed by centrifugation to remove the supernatant. 1 mL of sterile water was used to transfer the bacterial cells to a 1.5 mL centrifuge tube and placed on ice for 10 min. After centrifugation at 10000 rpm for 1 min, the supernatant was removed, and the cells were resuspended in 1 mL of sterile water and centrifuged again at 10000 rpm for 1 min. After removing the supernatant, 100 μL of sterile water and 1 μL of the required plasmid were added and mixed well, then placed on ice for 10 min. The BIO-RAD electroporator was set to the "E. coli" mode for electroporation. 600 μL of LB liquid medium was quickly added, mixed well, and transferred to test tubes. The tubes were placed on a shaker and incubated at 30 °C and 250 rpm for 1 h. A suitable amount of the bacterial culture was then plated onto LB solid medium labeled with the appropriate antibiotic and incubated overnight until the colonies reached the appropriate size.

[0061] DNA Maker was purchased from Tiangen Biotech (Beijing) Co., Ltd.; gel extraction kit and mini plasmid extraction kit were purchased from Tiangen Biotech (Beijing) Co., Ltd.; seamless cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; gene and primer synthesis and sequencing were performed by Shanghai Sangon Biotech Co., Ltd.

[0062] The relevant plasmid genotype information is shown in Table 1, the linalool synthesis-related genes and their GenBank accession numbers are shown in Table 2, and the relevant strain genotypes are shown in Table 3.

[0063] Table 1. pMVA SpecR-pAC-LacI-pT7_EcatoB-SchmgS-SchmgR-rrnBT1T-pTrc_ScMK-ScPMK-ScPMD-EcIDI-T7T pScLs AmpR-pMB1-LacI-pT7_RBS1_trAgGGPS2-T7T-pLuxB_RBS1_ScLs-L3S2P21T-LuxR-pGyrA_RBS1_DpADA_RBS2stop_ScADH2-T7T

[0064] Table 2. EcatoB OQ725958.1 Escherichia coli SchmgS M22002.1 Saccharomyces cerevisiae SchmgR M22002.1 Saccharomyces cerevisiae ScMK OQ725958.1 Saccharomyces cerevisiae ScPMK OQ725958.1 Saccharomyces cerevisiae ScPMD X97557.1 Saccharomyces cerevisiae EcIDI OQ725958.1 Escherichia coli trAgGPPS MW753220.1 Abies grandis ScLS PZ305631 Streptomyces clavuligerus DpADA PZ305632 Dickeya parazeae ScADH2 PZ305633 Saccharomyces cerevisiae

[0065] Table 3. wt E. coli MG1655 ΔrecA ΔendA ΔtnaA tMTScLS E. coli MG1655 ΔrecA ΔendA ΔtnaA contains plasmids pMVA and pScLs.

[0066] Saccharomyces cerevisiae BY4741 :

[0067] Saccharomyces cerevisiae BY4741, MATa his3Δ1 leu2 met15Δ ura3-52, laboratory collection.

[0068] Nicotine-eating Arthroblastus JCM 3874 :

[0069] Paenarthrobacter nicotinovorans JCM 3874, purchased from Mingzhou Biotechnology (product number BMZ012241).

[0070] YPD medium :

[0071] Weigh 10 g tryptone, 5 g yeast extract, and 20 g glucose, dissolve them in distilled water, and bring the volume to 1 L. Sterilize at 121 °C for 20 min. When preparing YPD solid medium, add 20 g / L agar powder before autoclaving. Tryptone and yeast extract were purchased from Oxoid, USA.

[0072] Lysobroth (LB) medium :

[0073] Weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride, dissolve in distilled water, and bring the volume to 1 L. Sterilize at 121 °C for 20 min. When preparing LB solid medium, add 20 g / L agar powder before autoclaving. Tryptone and yeast extract were purchased from Oxoid Inc. (USA).

[0074] LB solid culture medium containing the antibiotics spectinomycin hydrochloride and ampicillin :

[0075] These two antibiotics were added to LB solid medium at a volume ratio of 1 / 1000.

[0076] Metabolite analysis methods :

[0077] High-performance liquid chromatography (HPLC) analysis of metabolites: 800 μL of fermentation broth was transferred to a 1.5 mL centrifuge tube and centrifuged at 13000 rpm for 2 min. The supernatant was aspirated using a 1 mL sterile syringe and filtered through a 0.22 μm nylon filter, avoiding aspiration of bacterial cells. 500 μL of the filtered supernatant was transferred to a clean 2 mL injection bottle, with an injection volume of 10 μL. The sample was analyzed using a Shimadzu LC-20ADXR HPLC system equipped with an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad) and a refractive index detector. The mobile phase was 5 mM sulfuric acid at a flow rate of 0.7 mL / min, and the column temperature was set to 40 °C. A differential detector was used, and the elution conditions were isocratic elution with 5 mM H₂SO₄ solution for 20 min. Standard solutions of different concentrations of glucose, xylose, glycerol, and ethanol were prepared to quantitatively analyze the components of the culture medium and the fermentation metabolites of the strain. These routine biochemical reagents were domestically produced analytical grade.

[0078] Metabolite analysis using gas chromatography-mass spectrometry (GC-MS): 500 µL of fermentation broth was transferred to a 1.5 mL centrifuge tube, and 500 μL of chloroform was added. The tube was placed in a heated constant-temperature mixer and vortexed at 800 rpm and 30 °C to extract nicotine. After 30 min, the extract was removed, centrifuged at 13000 rpm for 5 min, and 100 μL of the lower organic phase was transferred to an inner tube compatible with a 2 mL sample vial, which was then inserted into the vial. Nicotine standards were prepared for qualitative and quantitative analysis of residual nicotine in the fermentation broth. The samples were analyzed using an Agilent GC-MS system 7890B-5977B with an Agilent HP-5MS column (30 m × 0.25 mm × 0.25 μm). Temperature program: Initial temperature 50 °C, hold for 1 minute; then increase to 100 °C at 5 °C / min, hold for 1 minute; then increase to 200 °C at 5 °C / min, hold for 1 minute; then increase to 300 °C at 5 °C / min, hold for 1 minute. Inlet temperature 230 °C; split ratio 10:1; carrier gas: helium (purity >99.999%), flow rate 1.0 mL / min; injection volume 2 μL; mass spectrometry scan range 10–500 m / z. Nicotine was qualitatively analyzed based on ion information from the nicotine mass spectrometry library and retention times of standards. Quantitative analysis of nicotine content in fermentation samples was performed by preparing nicotine standard solutions of different concentrations. Nicotine (L-(−)-Nicotine) was purchased from Sigma-Aldrich, USA.

[0079] Metabolite analysis by gas chromatography: 1 mL of the fermentation-completed sample phase was centrifuged in a 1.5 mL centrifuge tube at 13000 rpm for 5 min. Then, 10 μL of the upper organic phase was dissolved in 90 μL of ethyl acetate and analyzed using a GC9720Plus gas chromatograph (Zhejiang Fuli Instruments Co., Ltd.) with an Agilent HP-5 column (30 m × 0.25 mm × 0.25 μm). Temperature program: initial temperature 50 °C, held for 1 min; then increased to 100 °C at 5 °C / min, held for 1 min; then increased to 200 °C at 5 °C / min, held for 1 min; then increased to 300 °C at 5 °C / min, held for 1 min. Injector temperature 230 °C, flow rate 1.0 mL / min; injection volume 2 μL. Quantitative analysis of fermentation yield was performed by preparing linalool standard solutions of different concentrations. Linalool (purity ≥98%) was purchased from Shanghai Aladdin Co., Ltd.

[0080] Example 1

[0081] A microbial fermentation system and process for producing linalool in a nicotine-containing environment, specifically a method for reusing tobacco waste to degrade nicotine and simultaneously produce linalool, as detailed below:

[0082] (1) Preparation of substrate: such as Figure 1 As shown, 20 g of tobacco powder was weighed, 200 mL of water was added, and 1% (v / v) concentrated sulfuric acid was added. The mixture was autoclaved at 121 °C for 20 min. After sterilization, 400 g / L NaOH solution was added to adjust the pH to the range of 6-7. The sterilized solution was then cooled and centrifuged at 4000 rpm for 10 min to obtain a 100 g / L supernatant of tobacco acid hydrolysis.

[0083] Prepare a low-nicotine concentration substrate containing 20 g / L tryptone, 10 g / L yeast extract, 10 g / L glycerol, 40 g / L glucose, 13.3 g / L potassium dihydrogen phosphate, 4 g / L diammonium hydrogen phosphate, and 10 g / L tobacco leaf acid hydrolysis supernatant.

[0084] Prepare a high-nicotine-concentration substrate containing 20 g / L tryptone, 10 g / L yeast extract, 10 g / L glycerol, 40 g / L glucose, 13.3 g / L potassium dihydrogen phosphate, 4 g / L diammonium hydrogen phosphate, and 25 g / L tobacco leaf acid hydrolysis supernatant.

[0085] (2) Inoculation and fermentation: Saccharomyces cerevisiae BY4741 was streaked onto YPD solid medium plates and incubated in an incubator (30℃, 48 h); Nicotine-eating Arthrobacter JCM 3874 was streaked onto LB solid medium plates and incubated in an incubator (30 ℃, 48 h); Linalool engineered bacteria tMTScLS was streaked onto LB solid medium plates containing the antibiotics spectinomycin hydrochloride and ampicillin and incubated in an incubator (37 ℃, 24 h). A single colony of *Saccharomyces cerevisiae* BY4741 was inoculated into 1 mL of YPD liquid medium and cultured on a shaker (30 ℃, 24 h, 250 rpm). A single colony of *Bacillus nicotine-eating* JCM 3874 was inoculated into 1 mL of LB liquid medium and cultured on a shaker (30 ℃, 24 h, 250 rpm). A single colony of the engineered linalool bacterium tMTScLS was inoculated into 1 mL of LB liquid medium containing the antibiotics spectinomycin hydrochloride and ampicillin and cultured on a shaker (37 ℃, 24 h, 250 rpm).

[0086] Seven combinations of nicotine-eating Artemisia japonica JCM 3874, Saccharomyces cerevisiae BY4741 and linalool engineered bacteria tMTScLS were set up with inoculation ratios of 1:1:1, 1:1:2, 1:1:3, 1:2:1, 1:3:1, 2:1:1 and 3:1:1. These combinations were inoculated into the low-nicotine-concentration substrate and the high-nicotine-concentration substrate in step (1), for a total of 14 groups.

[0087] Specifically, overnight seed cultures of three bacteria (Nicotin-eating Arthrobacter OD600≈4.8, Saccharomyces cerevisiae OD600≈4.4, and linalool-engineered strain tMTScLS OD600≈3.8) were inoculated into the substrate according to the specified ratio. The total inoculation volume was 2% (v / v) of the fermentation system, which was 2 mL, and the total inoculation volume of the three bacteria was 40 μL. The cultures were incubated at 30 ℃ on a shaker until the bacterial OD600 reached 1. Simultaneously, 0.05 mM IPTG and 0.01 μM OC6 were added as inducers, and 400 μL of hexadecane was added at a 20% (v / v) volume ratio. The cultures were then transferred to a shaker at 30 ℃ (250 rpm) for biphasic fermentation for 96 h.

[0088] (3) Detection and analysis: The contents of nicotine, ethanol, and linalool under different inoculation ratios of the three bacteria in different concentrations of tobacco leaf acid hydrolysis supernatant are as follows: Figure 2 As shown. Figure 2 In the figure, Figure A shows the fermentation results of a low-nicotine-concentration substrate (tobacco leaf acid hydrolysis supernatant concentration of 10 g / L), and Figure B shows the fermentation results of a high-nicotine-concentration substrate (tobacco leaf acid hydrolysis supernatant concentration of 25 g / L).

[0089] Depend on Figure 2It is evident that in a 2 mL test tube mixed fermentation system with three bacteria, the inoculation ratio of microorganisms and the concentration of tobacco acid hydrolysis supernatant affect the metabolites.

[0090] From the perspective of increasing linalool yield, the optimal linalool synthesis efficiency was achieved when the inoculation ratio of Nicotine-eating Artemisia japonica JCM 3874, Saccharomyces cerevisiae BY4741, and the engineered linalool strain tMTScLS was 1:3:1. At an inoculation ratio of 1:3:1, the linalool titer was 190 mg / L when the concentration of the tobacco leaf acid hydrolysis supernatant was 25 g / L, and 170 mg / L when the concentration of the tobacco leaf acid hydrolysis supernatant was 10 g / L.

[0091] Considering the degradation of nicotine and simultaneous production of linalool, the optimal inoculation ratio of *Arthrobacter nicotine consuming strain JCM 3874*, *Saccharomyces cerevisiae* BY4741, and the engineered linalool strain tMTScLS was 3:1:1. When the concentration of the supernatant from tobacco leaf acid hydrolysis was 10 g / L, almost no nicotine residue remained after fermentation, while the linalool titer still reached 90 mg / L. When the concentration of the supernatant from tobacco leaf acid hydrolysis was 25 g / L, the degradation efficiency in the culture medium reached 51%, while the linalool titer still reached 40 mg / L.

[0092] Example 2

[0093] This embodiment is basically the same as Embodiment 1, except that:

[0094] Four groups were established, with four inoculation ratios of *Arthrobacter nicotine-eating* JCM 3874, *Saccharomyces cerevisiae* BY4741, and *tMTScLS* (a linalool-producing strain) at 1:3:1 and 3:1:1, respectively, inoculated into substrates with either low or high nicotine concentrations. The total fermentation time was set at 120 h, with samples taken every 24 h to compare the effects of different fermentation times on the results.

[0095] The results are as follows Figure 3 As shown, Figure 3 In the diagram, Figure A shows a concentration of 10 g / L and an inoculation ratio of 1:3:1; Figure B shows a concentration of 25 g / L and an inoculation ratio of 1:3:1; Figure C shows a concentration of 10 g / L and an inoculation ratio of 3:1:1; and Figure D shows a concentration of 25 g / L and an inoculation ratio of 3:1:1.

[0096] Depend on Figure 3 It is evident that glycerol, glucose, and xylose were almost completely consumed as fermentation time increased. The ethanol titer showed a trend of first increasing and then decreasing, which is presumably related to ethanol volatilization and / or the linalool-producing engineered bacteria tMTScLS consuming ethanol to synthesize linalool through ethanol utilization pathway.

[0097] From the perspective of increasing linalool yield: the highest linalool titer was observed at a concentration of 25 g / L in the supernatant of tobacco acid hydrolysis, after 48 h of fermentation, under the condition of *Arthrobacter nicotine-eating* JCM 3874: *Saccharomyces cerevisiae* BY4741: linalool engineered strain tMTScLS = 1:3:1, reaching 118 mg / L. It should be noted that because samples were taken every 24 h in this example, changes occurred due to oxygen intake, carbon dioxide and ethanol loss, and temperature fluctuations, resulting in a change in yield compared to Example 1. This example aims to obtain the optimal fermentation time through comparison of different fermentation times.

[0098] Considering the degradation of nicotine and the simultaneous production of linalool: after 120 h of fermentation, nicotine was undetectable in the group with a concentration of 10 g / L in the tobacco leaf acid hydrolysis supernatant, while a certain amount of linalool was still obtained; in the group with a concentration of 25 g / L in the tobacco leaf acid hydrolysis supernatant, the nicotine degradation rate also reached 94%, while a certain amount of linalool was still obtained. It is evident that after 120 h of fermentation, nicotine can be effectively degraded, and linalool can be obtained simultaneously.

[0099] Comparative Example 1

[0100] Fermentation was carried out using only the engineered linalool strain tMTScLS.

[0101] Six control substrates were prepared: each substrate contained 10 g / L tryptone, 5 g / L yeast extract, 13.3 g / L potassium dihydrogen phosphate, and 4 g / L diammonium hydrogen phosphate. Three of these substrates also contained 10 g / L of tobacco leaf acid hydrolysis supernatant, and these three substrates were respectively free of exogenous carbon source, contained 10 g / L glucose, and contained 10 g / L glycerol. The other three substrates also contained 25 g / L of tobacco leaf acid hydrolysis supernatant, and these three substrates were respectively free of exogenous carbon source, contained 10 g / L glucose, and contained 10 g / L glycerol.

[0102] The linalool-engineered strain tMTScLS was streaked onto LB solid medium plates containing the antibiotics spectinomycin hydrochloride and ampicillin, and incubated in an incubator (37 ℃, 24 h). A single colony of the linalool-engineered strain tMTScLS was picked and inoculated into 1 mL LB liquid medium containing the antibiotics spectinomycin hydrochloride and ampicillin, and incubated on a shaker (37 ℃, 24 h, 250 rpm).

[0103] The overnight cultured linalool-engineered bacterium tMTScLS seed culture was inoculated into 2 mL portions of the above six substrates at a 2% (v / v) inoculation rate. The cultures were incubated at 37 °C and 250 rpm in a shaker. Once the OD600 of the bacterial culture reached approximately 1, 0.05 mM IPTG, 0.01 μM OC6, and 20% (v / v) hexadecane were added to initiate biphasic fermentation for 48 h. The results are as follows: Figure 4 As shown.

[0104] Depend on Figure 4 As shown in Figure A, when the concentration of the supernatant from tobacco acid hydrolysis was 10 g / L and the nicotine concentration was low, the linalool titer with the addition of 10 g / L glycerol reached 0.6 g / L, which was significantly higher than that with the addition of 10 g / L glucose and the group without exogenous carbon source (the latter two were almost undetectable). This indicates that the engineered linalool bacterium tMTScLS has a preference for glycerol, and the linalool yield is significantly reduced when monosaccharides and / or tobacco waste are used as carbon sources.

[0105] When the concentration of the supernatant from tobacco acid hydrolysis is 25 g / L and the nicotine concentration is high, the linalool content is almost zero regardless of whether a carbon source is added. This indicates that the linalool synthesis ability of the engineered linalool bacterium tMTScLS is severely inhibited in the presence of high concentrations of nicotine, and the yield is extremely low regardless of the carbon source used.

[0106] Therefore, by comparing Comparative Example 1 and Example 1, it can be seen that in this invention, by introducing Nicotine-eating Bacillus JCM 3874 and Saccharomyces cerevisiae BY4741 and co-culturing them with the linalool engineered strain tMTScLS, the problems of limited carbon source utilization and nicotine sensitivity of the linalool engineered strain tMTScLS are solved, and the effect of producing linalool in a nicotine-containing environment is achieved. At the same time, the resource reuse of tobacco waste in the production of linalool is also achieved.

[0107] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A microbial fermentation system for producing linalool in a nicotine-containing environment, characterized in that: Includes the substrate and the microorganisms that ferment the substrate; The substrate contains nicotine; The microorganisms consist of Saccharomyces cerevisiae, Nicotine-eating Arthrobacter, and linalool-engineered bacteria, wherein the linalool-engineered bacteria include an ethanol utilization module.

2. The microbial fermentation system for producing linalool in a nicotine-containing environment according to claim 1, characterized in that: The linalool engineered bacteria is the linalool engineered bacteria tMTScLS; The chassis host of the linalool engineered bacterium tMTScLS is Escherichia coli MG1655, and the recA, endA and tnaA genes of the chassis host have been knocked out. The linalool engineered bacteria tMTScLS contains plasmids pMVA1 and pScLS. The plasmid pMVA1 carries the mevalonate pathway enzyme gene, which is controlled by the T7 and / or tac promoters; The plasmid pScLS carries a linalool synthesis module and an ethanol utilization module, controlled by the T7 and / or pLuxB and / or pGyrA promoters; the linalool synthesis module includes a geranyl diphosphate synthase gene and a linalool synthase gene, and the ethanol utilization module includes an acetaldehyde dehydrogenase gene and an alcohol dehydrogenase gene.

3. The microbial fermentation system for producing linalool in a nicotine-containing environment according to claim 2, characterized in that: The linalool engineered bacteria tMTScLS was obtained through the following steps: (1) Construction of mevalonate synthesis module: Acetyl coenzyme A thiolysis enzyme EcatoB from Escherichia coli, hydroxymethylpentadiene coenzyme A synthase SchmgS from Saccharomyces cerevisiae, hydroxymethylglutaryl coenzyme A reductase SchmgR, mevalonate kinase ScMK, phosphate mevalonate kinase ScPMK, phosphate mevalonate decarboxylase ScPMD, and isopentenyl pyrophosphate isomerase EcIDI from Escherichia coli were ligated into expression plasmid pMVA1 to obtain upstream pathway gene expression plasmid; (2) Construction of linalool synthesis pathway: The truncated geranium diphosphate synthase trAgGPPS from fir, the linalool synthase ScLs from Streptomyces rota, the acetaldehyde dehydrogenase DpADA from Diggia zeylans, and the ethanol dehydrogenase ScADH2 from Saccharomyces cerevisiae were ligated into the expression plasmid pScLS to obtain the downstream pathway gene expression plasmid. (3) The upstream pathway gene expression plasmid pMVA1 and the downstream pathway gene expression plasmid pScLS were electroporated into E.coli MG1655 ΔrecA ΔendA ΔtnaA strain to obtain linalool engineered strain tMTScLS.

4. A microbial fermentation system for producing linalool in a nicotine-containing environment according to claim 1 or 2, characterized in that: The volume ratio of the nicotine-eating bacillus, saccharitomyces, and linalool-engineered bacteria is 1:(0.1-5):(0.1-5).

5. The microbial fermentation system for producing linalool in a nicotine-containing environment according to claim 4, characterized in that: The initial concentration of nicotine in the substrate is 20–450 mg / L.

6. The microbial fermentation system for producing linalool in a nicotine-containing environment according to claim 5, characterized in that: The nicotine is derived from tobacco leaves and is obtained through the following steps: 0.5–1.5 v / v% concentrated sulfuric acid is added to a 0.05–0.15 g / mL aqueous dispersion of tobacco leaves, and the mixture is treated at 115–130 °C for 15–25 min, then the pH is adjusted to 6–7, and the supernatant is obtained by centrifugation; the concentration of the supernatant in the substrate is 5–30 g / L.

7. The microbial fermentation system for producing linalool in a nicotine-containing environment according to claim 6, characterized in that: The nicotine is derived from the supernatant of tobacco leaf acid hydrolysis and is obtained through the following steps: 1 v / v% concentrated sulfuric acid is added to a 0.1 g / mL aqueous dispersion of tobacco leaves, and the mixture is treated at 121 °C for 20 min. The pH is adjusted to 6-7 with NaOH solution, and the mixture is centrifuged at 4000 rpm for 10 min to obtain a supernatant of tobacco leaf acid hydrolysis with a concentration of 100 g / L.

8. The microbial fermentation system for producing linalool in a nicotine-containing environment according to claim 1, characterized in that: The substrate includes an external carbon source, which includes monosaccharides and glycerol.

9. A microbial fermentation process for producing linalool in a nicotine-containing environment, characterized in that: The fermentation is carried out using the microbial fermentation system as described in any one of claims 1 to 8, and the fermentation time is 24 to 120 h.

10. A method for reusing tobacco waste by degrading nicotine and simultaneously producing linalool, characterized in that: Includes the process described in claim 9.

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