| Diacetyl is a spice with strong butter flavor, which widely used in cream, wine, cosmetics and tobacco industries. To date, diacetyl produced by microorganism was very low product and the primary research goal is to improve the diacetyl product. Bacillus is a food safety strain, which metabolic pathways of diacetyl is glucose as substrate converted to pyruvate via glycolysis, pyruvate via a-acetolactate synthase (ALS) catalytic generate (S)-α-acetolactate, followed to diacetyl by non-enzymatic oxidative decarboxylation; diacetyl reduced to (S)-acetoin by meso-2,3-butanediol dehydrogenase (BDH), finally reduced to (2S, 3S)-or meso-2,3-butanediol; Meanwhile, (S)-α-acetolactate via a-acetolactate decarboxylase (ALDC) direct decarboxylated to (R)-acetoin, and (R)-acetoin reducted to (2R,3R)-or meso-2, 3-butanediol by BDH. This means that acetoin and 2,3-butanediol are competitive byproduct of diacetyl; they have a common precursor a-acetolactate. In this study, a strain of Bacillus subspecies DL01 (B. sp. DL01) with high acetoin yield was used to study the structure-function relationship and metabolic regulation mechanism of key enzyme in the metabolic pathway of diacetyl. At the same time, using metabolic engineering techniques, the biosynthetic pathway of this strain was modified to obtain an efficient production strain of diacetyl, which was combined with fermentation control and optimization of fermentation process to realize the green manufacturing of diacetyl, as follows:1. Characterization of the key enzyme BDH in diacetyl metabolic pathwaysBDH gene budC of B. sp. DL01 was cloned, when compared with the budC gene from Enterobacter aerogenes CICC10293, two base mutantions (g.27A/T and g.581A/G) were discovered, in which the g.581A/G leads to an amino acid mutation (p.D194G). These two budC genes were expressed in E.coli BL21(DE3). The obtained BDHs, named as D194G-BDH and BDH, were purified, characterized and compared in vitro. BDHs are homologous tetramers. Enzymatic properties and kinetic parameters study of BDHs found that D194G-BDH exhibited only 2.3% activity of BDH. Acetoin/NADH were the optimal substrates of these two BDHs; however, Km of D194G-BDH is 5.63 times greater than that of BDH. Circular dichroism and native-PAGE revealed that D194G-BDH and BDH possessed similar protein secondary structures and were homologous tetramers. However, D194G-BDH was very sensitive to trypsin cleavage, indicating that Gly mutated to Asp caused the conformational change of D194G-BDH, and weakened its activity. Using X-ray crystal structure of Klebsiella pneumoniae-BDH (PDB ID:1GEG) as template, homology modelings of three dimentional structurcs of D194G-BDH and BDH with substrates and coenzyme indicated that the mutated Asp194 located far away from the catalytic tetrad (Asn-Ser-Tyr-Lys) of the enzyme. The Gly substitution of Asp at position 194 breaks all the hydrogen-bond and electrostatic interactions with the nitrogen atoms of Gly206, Gly208 and Thr209, since the side chain of glycine is too short as well as nonpolar to communicate with surrounding environment. Recombinant expression the active BDH from Enterobacter aerogenes in B. sp. DL01 found that B. sp. DL01 produced 2,3-butanediol. So, the 194 non-conservative amino acid Asp of BDH substituted by Gly, which is the cause of D194G-BDH activity loss, resulting in B. sp. DL01 high acetoinyield, and do not produce 2, 3-butanediol.2. Regulation of diacetyl biosynthesis metabolic fluxThe ALDC encoding gene alsD was knockouted with homologous recombination method, and ALDC knockout strain was constructed and named as B. sp. DL01-ΔalsD. ALDC of B. sp. DL01-AalsD had not activity, and 0.41 g/L diacetyl was accumulated, which is 8.2-fold of that of the B. sp. DL01. The recombinant plasmid pHY300PLK-alsS to overexpress ALS gene alsS was constructed, and transformed to alsD knockout strain B. sp. DLOl-ΔalsD, resulting a recombinant strain B. sp. DL01-ΔalsD-alsS. ALS activity of B. sp. DLOl-ΔalsD-alsS was 0.68 U/mg, which is about 2.5-fold of the B. sp. DL01.0.53 g/L diacetyl was accumulated, which is 10.6-fold of the B. sp. DL01, and 1.3-fold of the B. sp. DL01-ΔalsD. a-Acetolactate production also increased from 0.58 g/L to 1.19 g/L, an increase of 2.1-fold, the amount of a-acetolactate accumulation was 2.3-fold of diacetyl. To promote the non-enzymatic oxidation of a-acetolactate,20 mM Fe3+ was added into the culture medium after 12 h of fermentation, leading to diacetyl product increased 2.3-fold greater, from 0.53 g/L to 1.22 g/L.3. Optimization of the fermentation process to improve the diacetyl productEnvironmental parameters of B. sp. DL01-ΔalsD-alsS during fermentation and medium components were optimized in flasks. The optimum culture conditions were as follows: fermentation temperature 37℃, agitation speed 100 r/min, inoculation amount of 15%, initial pH 6.6. The optimum medium component:40 g/L glucose,5 g/L yeast extract,5 g/L (NH4)2SO4,0.3 g/L MgSO4·7H2O,0.05 g/L MnSO4,0.05 g/L FeSO4, and 0.05 g/L CaCl2. With optimal conditions and optimized media and the addition of 20 mM Fe3+ after mentation time point of 12 h, diacetyl production reached 3.98 g/L after 72 h. Comparing the fixed pH 6.6 fermentaiton process and floating pH (initial pH 6.6) fermentation process in fermenter, no significant changes of the yield and the efficiency of diacetyl fermentation were observed. Aeration rate at 0.6 vvm and agitation speed at 200 r/min were the optimum dissolved oxygen conditions. Using the constant substrate concentration strategy, the glucose concentration was maintained at 10 g/L by continuously adjusting the flow rate of the glucose, diacetyl concentration of 8.69 g/L, yield of 0.15 g/g glucose and productivity of 0.18 g/(L·h) was obtained with the total consumption of 58 g/L glucose.This study discovered and proved that the BDH enzyme activity loss is the cause of B. sp. DL01 high acetoin productwithout producing 2,3-butanediol, and constructed a recombinant strain with high diacetyl product, and finally diacetyl production reached 8.69 g/L by the constant substrate batch fermentation. |
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