Development A CO₂ Fixing Cell Factory In Ralstonia Eutropha

Carbon,the building block of life,is the fourth most abundant element in atmosphere.the carbon cycle,which circulates back and forth between life forms and the environment through various biogeochemical cycles,maintains the stability and balance of earth.Since the industrial revolution began,the amount of carbon dioxide has been increasing continuously,and it is expected to reach 40.3Gt by 2030 and to 50Gt by 2050.Human urgently need to take efficient measures to avoid occurrence of the crisis of environment and resource caused by the increased carbon dioxide accumulation and reduction of fossil fuels stocks.Ralstonia eutropha?R.eutropha?was selected as the chassis for its CO2 fixing ability.This research is to develop a more efficient method for biosynthesis from carbon dioxide to organic chemicals.Fatty acids have a high energy density,which also play an important role in industry,pharmaceuticals,nutrition,cosmetics and daily necessities,as well as cellular structures construction and promote cell membranes flow.Therefore,related genes for fatty acid synthesis was firstly introduced through heterogenous or autogenous gene to modify its synthetic pathway.The resulting strain B2?pCT,pFP?was able to produce 124,48 mg/g?cell dry weight?free fatty acids with fructose as carbon source,a 4-fold increase over the parent strain H16.To develop a truly autotrophic fermentation technique with H2,CO2 and O2 as substrates,we assembled a relatively safe,continuous,lab-scale gas fermentation system by using micro fermentation tanks,H2 supplied by a hydrogen generator,and keeping the H2 to O2 ratio at 7:1.The system was equipped with an H2 gas alarm,rid of heat sources and placed into a fume hood to further improve the safety.With this system,the best strain B2?pCT,pFP?produced 60.64 mg free fatty acids per g biomass within 48 hours,growing in minimal medium supplemented with 9*103 mL/L/h hydrogen gas.To explore which of two copies of the CBB operon system and hydrogenase system on CO2 fixing growth is the primary one,we respectively constructed strains carrying only a single-copy of CBB operon or Hydrogenase operon.The results showed that CBB operon has similar function on autotrophic growth,but SH hydrogenase operon was more important on autotrophic ability compared with MBH hydrogenase.Then,to further explore the mechanism of optimizing CO2 fixing mechanism of R.eutropha,we tried to divide it into three modules:Rubisco carbon dioxide fixation module,hydrogenase energy module,and precursor material supply module.All three modules were evaluated in term of growth rate.The results showed that strain H16?pRub_Cynao-Groesl_R?was the only modification with positive results in the carbon dioxide fixation module,and its growth rate increased 177.1%compared with the control bacteria under the maximum growth difference.In hydrogenase module optimization experiment,we overexpressed four kinds of hydrogenases of R.eutropha membrane bound hydrogenase?MBH?,soluble hydrogenase?SH?,regulatory hydrogenase?RH?,and hypothetical hydrogenase?Hy4?individually.The results showed that SH hydrogenase and MBH hydrogenase enhanced the autotrophic growth of the engineered bacteria,while Hy4 overexpression was basically consistent with the control group.RH hydrogenase overexpression was found to inhibit the autotrophic growth.Subsequently,to reduce the influence of exogenous plasmids on bacteria,we modified the operon of MBH hydrogenase and SH hydrogenase at the genome level,and the promoter BBaJ₂3119 from our promoter library showed the strongest enhancement of autotrophic growth ability.The modified strain C5M119S119 was selected as the optimized chassis strain of autotrophic growth.Then,the final strain C5M119S119?pRub_Cynao-GroESL_R?achieved a significant improvement in OD600 and the autotrophic fermentation production of PHB increased from 0.17 g/g to 0.34 g/g.In this paper,we developed a true syngas fermentation system and a CO2 fixing fatty acids cell factories through molecular modification.Meantime,we also increased R.eutropha CO2 fixing ability by engineering of three modules.The engineered strain had increased growth rate in syngas fermentation system and improved PHB accumulation level.This work provides an alternative method to solve energy crisis and carbon dioxide accumulation.