Metabolic Engineering Of Cupriavidus Necator For Biosynthesis Of 2,3-butanediol From CO₂

Currently,sugars are used as carbon sources for the production of most biochemicals via biotechnology,which is often largely affected by the price fluctuation of sugars.With the development of national economy,the cost of sugar preparation gradually increases.Therefore,it is necessary to find alternative carbon sources.Considering the abundant availability,carbon dioxide is considered as one of the most ideal carbon sources.In this case,the use of CO2 as a carbon source to produce biochemicals and fuels has become a research hotspot.2,3-Butanediol(BDO)is an emerging energy compound with high added value,and has important applications in aerospace,food,and medicine fields as well.It is an important platform compound widely used in industry.Cupriavidus necator(also known as Ralstonia eutropha)is a chemoautotrophic strain with natural carbon fixation ability.Therefore,the purpose of this study is to engineer C.necator cell factories capable of synthesizing BDO from CO2 via synthetic biology approaches.There was a pathway(acoXABC)that could metabolize acetoin,the biosynthetic precursor of BDO in C.necator.Firstly,we knocked out the key genes(acoXABC operon)involved in this pathway use genome editing methods,resulting in the construction of strain C5-1(C5-ΔacoXABC).Subsequently,we cloned the BDO biosynthetic pathway genes(BudB from Bacillus licheniformis,BudA from Enterobacter cloacae,and BudC from Enterobacter cloacae)on the shuttle plasmid,which was transformed into the C5-1 to evaluate the BDO producing capability.In addition,we optimized the genetic elements for BDO production in C5-1,with pBBR-MCS1 and Pj5 chosen as the expression vector and promoter for the expression of BDO pathway genes.We then tried to increase the production of BDO by reducing the consumption of pyruvate,the precursor for BDO synthesis.The knockout of PHA biosynthesis related genes(phaCAB)failed to further increase the production of BDO in LB medium;while the knock out of the lactate dehydrogenase gene(ldhA)resulted in an increase in the production of BDO by 0.927 times.Before attempting to use CO2 as the sole carbon source to synthesize BDO by C.necator,we first optimized the BDO fermentation conditions in the inorganic salt medium(MM).In MM medium with 10 g/L fructose,compared with the original strain C5-1-1(C5-△acoXABC/pBBR-Pj5-BDO,0.145 g/L/CDW),the metabolically engineered strain C5-3-1(C5-ΔacoXABC-ΔphaCAB-ΔldhA/pBBR-Pj5-BDO,1.931 g/L/CDW)increased the BDO yield of by 13.32 times.Then,the fermentation conditions of BDO were further optimized:by adjusting the pH of the medium,the yield of BDO was the highest at pH=6.5;through controlling dissolved oxygen,the yield of BDO was the highest under the lower sealed condition,reaching 2.473 g/L.Subsequently,we tested the CO2/H2/O2 mixed gas fermentation,leading to a BDO yield of 0.384 g/L.In addition,we established a microbial electrochemical system(MES)that can be used for the C.necator strains.In MES,we control the voltage and current at 4 V and 18 mA,respectively.The modified C5-3-2 strain(C5-ΔacoXABC-ΔphaCAB-ΔldhA/pBBR-Pj5-RBS-BDO)was inoculated into MES.After 120 h of fermentation,the OD value reached 0.647,and the yield of BDO reached 0.1829 g/L.In summary,an engineered strain of C.necator that can produce BDO stably was constructed,and BDO biosynthesis was optimized through metabolic engineering and bioprocess engineering.For the first time,we achieved the production of BDO from CO2 in MES using metabolically engineered C.necator strains.In addition,this research lays the foundation for the construction of efficient C.necator cell factories and the development for synthetic biology tools for non-model microorganisms.