(R-R)2,3-butanediol(BDO)is a chemical compound of significant medical value,and there is a desire to biologically produce high-purity(R-R)2,3-BDO instead of a mixture of various configurations.Acetoin,a key downstream...(R-R)2,3-butanediol(BDO)is a chemical compound of significant medical value,and there is a desire to biologically produce high-purity(R-R)2,3-BDO instead of a mixture of various configurations.Acetoin,a key downstream metabolite,gains attention for food industry applications.Simultaneous production through a single fermentation holds great potential,but the absence of a convenient and efficient separation strategy remains a source of disappointment.Here,we meticulously chose non-pathogenic Bacillus licheniformis as cell factories with specific genetic regulation on this metabolic pathway.Firstly,genome engineering was performed to attain 100%purity in the production of(R-R)2,3-BDO.Subsequently,by harnessing the inherent regulation of the glyoxylate cycle within the strain,a pioneering two-stage production strategy was developed.This strategy enables selective production of high-purity(R-R)2,3-BDO or acetoin based on production preferences,simply by terminating fermentation at the appropriate time.When using 80 g/L of glucose as the substrate,it is feasible to obtain 22.0 g/L of(R-R)2,3-BDO with 100%purity or 26.0 g/L of acetoin with 90.0%purity.In summary,this work presents an inspiring strategy that addresses dual production for a better product separation,which can serve as a source of inspiration for other cell factories in the field of biomanufacturing.展开更多
Acetoin is a natural flavor compound widely present in foods,but its cytotoxicity limits microbial production.Here,a multi-level metabolic engineering strategy was developed in Bacillus subtilis to reconstruct the ace...Acetoin is a natural flavor compound widely present in foods,but its cytotoxicity limits microbial production.Here,a multi-level metabolic engineering strategy was developed in Bacillus subtilis to reconstruct the acetoin pathway.Expression of key genes in the phosphotransferase system and glycolysis was enhanced,and pyruvate overflow was blocked to direct carbon flux toward acetoin precursors.Expression of a Streptococcus pneumoniae NADH oxidase restored shake-flask growth,increasing OD600 from 29.74 to 35.6.Fusion of acetolactate synthase(ALS)and acetolactate decarboxylase(ALDC)improved pathway efficiency,resulting in an 11.5%increase in titer to 57.89 g/L.Combinatorial CRISPR interference of six targets in competing pathways further increased the titer by 26.2%,reaching 73.2 g/L.Introduction of the polyhydroxybutyrate(PHB)biosynthetic pathway enhanced cellular tolerance,allowing robust growth under 80 g/L stress.The engineered strain ultimately achieved an acetoin titer of 87.9 g/L,a productivity of 1.45 g/L/h,and a yield of 0.45 g/g in a 3-L bioreactor,providing an effective framework for industrial acetoin production.展开更多
Acetoin is an important platform chemical,which has a wide range of applications in many industries.Halomonas bluephagenesis,a chassis for next generation of industrial biotechnology,has advantages of fast growth and ...Acetoin is an important platform chemical,which has a wide range of applications in many industries.Halomonas bluephagenesis,a chassis for next generation of industrial biotechnology,has advantages of fast growth and high tolerance to organic acid salts and alkaline environment.Here,α-acetolactate synthase andα-acetolactate decarboxylase from Bacillus subtilis 168 were co-expressed in H.bluephagenesis to produce acetoin from pyruvate.After reaction condition optimization and further increase ofα-acetolactate decarboxylase expression,acetoin production and yield were significantly enhanced to 223.4 mmol·L^(-1) and 0.491 mol·mol^(-1) from 125.4 mmol·L^(-1) and 0.333 mol·mol^(-1),respectively.Finally,the highest titer of 974.3 mmol·L^(-1)(85.84 g·L^(-1))of acetoin was accumulated from 2143.4 mmol·L^(-1)(188.6 g·L^(-1))of pyruvic acid within 8 h in fed-batch bioconversion under optimal reaction conditions.Moreover,the reusability of the cell catalysis was also tested,and the result illustrated that the whole-cell catalysis obtained 433.3,440.2,379.0,442.8 and 339.4 mmol·L^(-1)(38.2,38.8,33.4,39.0 and 29.9 g·L^(-1))acetoin in five repeated cycles under the same conditions.This work therefore provided an efficient H.bluephagenesis whole-cell catalysis with a broad development prospect in biosynthesis of acetoin.展开更多
基金funded by the National Key Research&Develop-ment Program of China(2020YFA0907700,2018YFA0900504 and 2018YFA0900300)the National Natural Foundation of China(32172174,31401674)the National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-22).
文摘(R-R)2,3-butanediol(BDO)is a chemical compound of significant medical value,and there is a desire to biologically produce high-purity(R-R)2,3-BDO instead of a mixture of various configurations.Acetoin,a key downstream metabolite,gains attention for food industry applications.Simultaneous production through a single fermentation holds great potential,but the absence of a convenient and efficient separation strategy remains a source of disappointment.Here,we meticulously chose non-pathogenic Bacillus licheniformis as cell factories with specific genetic regulation on this metabolic pathway.Firstly,genome engineering was performed to attain 100%purity in the production of(R-R)2,3-BDO.Subsequently,by harnessing the inherent regulation of the glyoxylate cycle within the strain,a pioneering two-stage production strategy was developed.This strategy enables selective production of high-purity(R-R)2,3-BDO or acetoin based on production preferences,simply by terminating fermentation at the appropriate time.When using 80 g/L of glucose as the substrate,it is feasible to obtain 22.0 g/L of(R-R)2,3-BDO with 100%purity or 26.0 g/L of acetoin with 90.0%purity.In summary,this work presents an inspiring strategy that addresses dual production for a better product separation,which can serve as a source of inspiration for other cell factories in the field of biomanufacturing.
基金supported by the National Key Research and Development Program for Food Nutrition and Safety(2023YFF1104100)the National Natural Science Foundation of China(32300064,32570077)+1 种基金the Jiangsu Basic Research Center for Synthetic Biology(BK20233003)the Natural Science Foundation of Jiangsu Province(BK20202002).
文摘Acetoin is a natural flavor compound widely present in foods,but its cytotoxicity limits microbial production.Here,a multi-level metabolic engineering strategy was developed in Bacillus subtilis to reconstruct the acetoin pathway.Expression of key genes in the phosphotransferase system and glycolysis was enhanced,and pyruvate overflow was blocked to direct carbon flux toward acetoin precursors.Expression of a Streptococcus pneumoniae NADH oxidase restored shake-flask growth,increasing OD600 from 29.74 to 35.6.Fusion of acetolactate synthase(ALS)and acetolactate decarboxylase(ALDC)improved pathway efficiency,resulting in an 11.5%increase in titer to 57.89 g/L.Combinatorial CRISPR interference of six targets in competing pathways further increased the titer by 26.2%,reaching 73.2 g/L.Introduction of the polyhydroxybutyrate(PHB)biosynthetic pathway enhanced cellular tolerance,allowing robust growth under 80 g/L stress.The engineered strain ultimately achieved an acetoin titer of 87.9 g/L,a productivity of 1.45 g/L/h,and a yield of 0.45 g/g in a 3-L bioreactor,providing an effective framework for industrial acetoin production.
基金supported by the National Key Research and Development Program of China (Grant No.2018YFA0900200)the National Natural Science Foundation of China (Grant No.NSFC-21621004).
文摘Acetoin is an important platform chemical,which has a wide range of applications in many industries.Halomonas bluephagenesis,a chassis for next generation of industrial biotechnology,has advantages of fast growth and high tolerance to organic acid salts and alkaline environment.Here,α-acetolactate synthase andα-acetolactate decarboxylase from Bacillus subtilis 168 were co-expressed in H.bluephagenesis to produce acetoin from pyruvate.After reaction condition optimization and further increase ofα-acetolactate decarboxylase expression,acetoin production and yield were significantly enhanced to 223.4 mmol·L^(-1) and 0.491 mol·mol^(-1) from 125.4 mmol·L^(-1) and 0.333 mol·mol^(-1),respectively.Finally,the highest titer of 974.3 mmol·L^(-1)(85.84 g·L^(-1))of acetoin was accumulated from 2143.4 mmol·L^(-1)(188.6 g·L^(-1))of pyruvic acid within 8 h in fed-batch bioconversion under optimal reaction conditions.Moreover,the reusability of the cell catalysis was also tested,and the result illustrated that the whole-cell catalysis obtained 433.3,440.2,379.0,442.8 and 339.4 mmol·L^(-1)(38.2,38.8,33.4,39.0 and 29.9 g·L^(-1))acetoin in five repeated cycles under the same conditions.This work therefore provided an efficient H.bluephagenesis whole-cell catalysis with a broad development prospect in biosynthesis of acetoin.
