A Study Of Construction Of A Metabolic Pathway For Lactic Acid Production By Fixed CO₂ In E.coli And Screening Of Rubisco With High Carboxylation Activity

Calvin-Benson-Bassham?CBB?is the main way to produce organic biomass from inorganic carbon.Ribulose-1,5-diphosphate carboxylation/oxygenase?Rubisco?is a key and rate-limiting enzyme in the first step of CBB to fix CO₂.The carboxylation activity of Rubisco directly affects the ability of fixing CO₂ through the CBB,and thus Rubisco plays a key role in crop production and the global carbon cycle.Rubisco's catalytic efficiency and selectivity of CO₂ are extremely low.Moreover,the active center of Rubisco is highly conservative,and it has little effect on transforming it by rational design or directed evolution.Therefore,designing an efficient screening system suitable for higher carboxylation activity and exploring and discovering unknown Rubisco₂are the current research hotspots.In addition,the use of Rubisco's carbon-fixing carboxylation activity to synthesize bio-based chemical products has become an important direction for microbial carbon fixation research.Although some chemical products,such as ethanol,isobutyraldehyde,L-butanol,and glycerol,have been produced in cyanobacteria,Saccharomyces cerevisiae and other microorganisms using Rubisco to fix CO₂,studies on using Rubisco to fix CO₂ for producing lactic acid in Escherichia coli have not been reported.Therefore,this study attempts to establish a Rubisco-fixed CO₂ lactic acid production pathway in E.coli.The main results are following:?1?The Glycerol-Xylose-Rubisco-PRK lactic acid metabolism pathway was constructed in E.coli.The theoretical yield of lactic acid was 107.8%,and the theoretical yield of ¹³C-labeled lactic acid was 25%.Through optimization of fermentation conditions and comparison of fermentation strains,the optimal fermentation conditions were determined as:under anaerobic conditions,M9 was the fermentation medium,the buffer was HEPES/Na HCO₃,and the induction temperature was 22°C;the optimal fermentation strain was E.coli BW25113?frd?pfl B::ldh A.The lactic acid yield of this strain was 2.51 g/L,the yield of lactic acid was 92.04%,and the net yield of lactic acid labeled with ¹³C was 19.66%.?2?Constructed a selection system suitable for screening efficient Rubisco carboxylation activity by preliminary screening of cell growth status and rescreening of the production of lactic acid and ¹³C-labeled lactic acid.Compared with the original screening system,this system has the advantages of high throughput,high accuracy,and simplicity.?3?BW-RBC9 strain was screened from 23 E.coli strains with different sources of Rubisco by using our constructed selection system for Rubisco with high carboxylation activity.The production of lactic acid was 3.1 g/L and the yield of lactic acid was 93.46%,which was close to the theoretical yield of 107.8%.The net yield of lactic acid labeled with ¹³C was 21.83%,which was close to the theoretical yield of25%.In summary,our project realized Rubisco's fixation of CO₂ to produce lactic acid in E.coli by the constructed glycerol-xylose-Rubisco-PRK metabolism pathway.Also,we established a screening system suitable for high-throughput screening of Rubisco and found a Rubisco with high-carboxylase activity.The study is a useful exploration of using Rubisco to fix CO₂ for producing lactic acid in E.coli and realizing industrial production in the future.