DataSheet1_Poly-3-hydroxybutyrate production from acetate by recombinant Pseudomonas stutzeri with blocked L-leucine catabolism and enhanced growth in acetate.docx

Acetate is a low-cost feedstock for the production of different bio-chemicals. Electrochemical reduction of CO2 into acetate and subsequent acetate fermentation is a promising method for transforming CO2 into value-added chemicals. However, the significant inhibitory effect of acetate on microbial growth remains a barrier for acetate-based biorefinery. In this study, the deletion of genes involved in L-leucine degradation was found to be beneficial for the growth of Pseudomonas stutzeri A1501 in acetate. P. stutzeri (Δpst_3217), in which the hydroxymethylglutaryl-CoA lyase catalyzing β-hydroxy-β-methylglutaryl-CoA into acetyl-CoA and acetoacetate was deleted, grew faster than other mutants and exhibited increased tolerance to acetate. Then, the genes phbCAB from Ralstonia eutropha H16 for poly-3-hydroxybutyrate (PHB) biosynthesis were overexpressed in P. stutzeri (∆pst_3217) and the recombinant strain P. stutzeri (∆pst_3217-phbCAB) can accumulate 0.11 g L−1 PHB from commercial acetate. Importantly, P. stutzeri (∆pst_3217-phbCAB) can also use CO2-derived acetate to produce PHB and the accumulated PHB accounted for 5.42% (w/w) of dried cell weight of P. stutzeri (∆pst_3217-phbCAB).

乙酸盐（Acetate）是制备多种生物化学品的低成本原料。将二氧化碳（CO₂）电化学还原为乙酸盐并结合后续乙酸盐发酵，是将CO₂转化为高附加值化学品的极具前景的方法。然而，乙酸盐对微生物生长的显著抑制效应，仍是制约乙酸基生物炼制的核心瓶颈。本研究发现，敲除参与L-亮氨酸降解的相关基因，可提升施氏假单胞菌A1501（Pseudomonas stutzeri A1501）在乙酸盐培养基中的生长性能。其中，敲除催化β-羟基-β-甲基戊二酸单酰辅酶A裂解为乙酰辅酶A（acetyl-CoA）与乙酰乙酸的羟甲基戊二酸单酰辅酶A裂解酶编码基因的突变株施氏假单胞菌（Δpst_3217），其生长速率快于其他突变株，且乙酸盐耐受性显著提升。随后，研究人员将真养产碱杆菌Ralstonia eutropha H16中负责聚-3-羟基丁酸酯（poly-3-hydroxybutyrate, PHB）生物合成的phbCAB基因，在施氏假单胞菌（Δpst_3217）中进行过表达，所得重组菌株施氏假单胞菌（Δpst_3217-phbCAB）可利用商用乙酸盐积累0.11 g·L⁻¹的PHB。尤为关键的是，该重组菌株还可利用CO₂衍生的乙酸盐生产PHB，其积累的PHB占该菌株干细胞重量的5.42%（w/w）。