Biosynthesis Of Diamines In Escherichia Coli

Diamines,as important monomers for the production of nylon,an important engineering plastic,are currently produced mainly by petroleum-based chemical methods.However,chemical methods suffer from harsh reaction conditions,high equipment and operational requirements,and many by-products.With the resource depletion,climate change,and development of carbon neutrality,the establishment of bioconversion pathways for synthesis of diamines from renewable feedstocks has become a preferred option for achieving economic and environmental sustainability.In this paper,we constructed an engineered strain for the biosynthesis of medium-chain diamines such as putrescine and 1,6-hexanediamine using Escherichia coli as hosts.The efficient synthesis of putrescine in E.coli was achieved through strategies such as engineering to improve the neutral p H adaptation of inducible arginine decarboxylase,enhancing endogenous synthesis pathway,blocking degradation metabolism and fermentation optimization.Meanwhile,the 1,6-hexanediamine synthesis pathway was heterologously constructed in E.coli,and its titer was increased based on strategies of enzyme element aptamer assembly and cofactor regulation.Furthermore,1,6-hexanediamine synthesis mechanism was resolve by in vitro enzyme cascade,identifying 1,6-hexanediamine synthesis metabolic flow nodes.Finally,the 1,6-hexanediamine synthesis pathway was applied successfully to C7-C10 diamine synthesis.（1）Neutral p H adaptation optimization of inducible arginine decarboxylase Adi A.There is an obvious neutral p H inhibition effect in Adi A,which limits its industrial application.In this paper,we constructed E445K mutant to release electrostatic repulsion at the interface by introducing a basic amino acid mutation in acidic amino acid-enriched region at the interface of Adi A pentameric rings,which increased the enzyme activity of Adi A at neutral p H by 3.2-fold.Then,by introducing acidic amino acids in the substrate channel to improve the affinity of Adi A for alkaline substrates,the enzyme activity of Adi A was also increased by 3-fold at neutral p H using H736E mutant.Further,the E467K＿H736E（Adi A-M2）was constructed,and its k_cat value was 4.5-fold higher than that of the wild-type Adi A,indicating that substrate turnover ability of Adi A was significantly enhanced.In addition,Cad A also suffers from enzyme activity inhibition at neutral p H,and the E445K＿T691D（Cad A-M2）was constructed using the same mutation strategy as Adi A,and its enzyme activity was increased by 2.16-fold at neutral p H than that of Cad A.It demonstrated that this mutation strategy has an ability to be extended in the neutral p H adaptation modification of this type of decarboxylase.（2）The efficient synthesis of putrescine in E.coli.The ADC synthesis pathway of putrescine was enhanced by simultaneously overexpressing the spe A,spe B and adi A genes in E.coli BL21（DE3）,obtaining putrescine producing strain PUT3.Then,the degradation of putrescine was blocked by knocking out the spe E,spe G,puu A and puu P genes（ΔEGAP）,and the yield of putrescine in theΔEGAP PUT3 strain was increased by 30%,and the Adi A-M2 mutant was applied to the synthesis of putrescine to construct the PUT6 strain,and the molar conversion rate of putrescine in PUT6 was increased to 0.99 mol·mol^-1 arginine,realizing the efficient synthesis of putrescine.Finally,by optimizing initial arginine addition concentration,Mg²⁺and Mn²⁺concentration,the PUT6 strain synthesized 217.3 mmol·L^-1(～19.1 g·L^-1)putrescine in batch fermentation when added 230 mmol·L^-1 initial arginine,2 mmol·L^-1 Mg²⁺and 1.5 mmol·L^-1 Mn²⁺.In the fed-batch fermentation,the production of putrescine reached 586 mmol·L^-1(～51 g·L^-1),which was 2.67 folds higher than that of batch fermentation.It has laid the foundation for the industrialization of bio-based putrescine.（3）The 1,6-hexanediamine biosynthetic pathway was constructed in E.coli BL21（DE3）.Firstly,the 1,6-hexanediamine synthesis pathway was successfully constructed in E.coli using carboxylic acid reductase MAB CAR and transaminase CVTA,and whole-cell biosynthesis of1,6-hexanediamine(0.42 mg·L^-1)was achieved.Then,the transaminase isozyme optimization and its aptamer assembly module design were carried out,increasing the titer of 1,6-hexanediamine to22 mg·L^-1 in DAH10 strain containing MAB CAR-Pat A-Gab T cascade module.Based on CAR-CAR-TA-TA cascade module optimization and expression intensity regulation,the titer of 1,6-hexanediamine was increased to 53.11 mg·L^-1 in DAH86 strain containing MSM CAR-MAB CAR-Pat A-SPTA cascade module.Finally,by knocking out the sth A gene to regulate of cofactor NADPH balance,theΔsth A-DAH86 was able to transform 6 g·L^-1 adipic acid to synthesize 238.5mg·L^-1 1,6-hexanediamine.（4）Analysis of the synthesis mechanism of 1,6-hexanediamine artificial pathway and its successful application in synthesis of medium-chain diamines.Based on enzymatic activity data of carboxylic acid reductase and transaminase,the carboxylic acid reduce of 6-aminohexanoic acid was identified as one of the rate-limiting steps of 1,6-hexanediamine synthesis.Based on in vitro enzymatic experiments,it was found that the conversion of adipic acid to adipaldehyde and the conversion of adipaldehyde to 1,6-hexanediamine was completed successfully,while the conversion of 6-aminohexanoic acid to 1,6-hexanediamine could not be achieved,confirming that the synthesis of 1,6-hexanediamine was mainly dependent on the adipaldehyde branch.By comparing the metabolic constituents in the fermentation broth fermented by stains BL21Δsth A andΔsth A-DAH86,it was found that introduction of exogenous pathway of 1,6-hexanediamine led to the accumulation of a large amount of medium and long chain fatty alcohols,aldehydes and esters,and the titer of 1,6-hexanediol reached 254 mg·L^-1,which was one of by-products for the synthesis of 1,6-hexanediamine.Eventually,1,7-heptanediamine(21.89 mg·L^-1),1,8-octanediamine(0.67 mg·L^-1),1,9-nonediamine(9.34 mg·L^-1)and 1,10-decanediamine(18.81mg·L^-1)were successfully synthesized using the artificial pathway of 1,6-hexanediamine.It provides an alternative direction for the development of biosynthetic pathways for medium-chain diamines.