Promoting Rate Of Microbial Extracellular Electron Transfer By Synthetic Biology

Microbial electrocatalytic systems based on electroactive microbesare emerging as a new type of production method of new green energy.However,the low efficiency in the extracellular electron transfer(EET)of exoelectrogens and electrotrophic microbes limited their industrial applications.In this paper,we systematically reconstructed the intracellular metabolism and extracellular electron transport pathway of Shewanella oneidensis MR-1 from the view of broadening the spectrum available substrate,inceaseing intracellular NADH regeneration,improving the intracellular releasable electronic pool size,and expanding the electron transfer shuttle with the aid of synthetic biology and metabolic engineering.The main research results are as follows:First,to enable S.oneidensis to directly utilize xylose as the sole carbon source for bioelectricity production in microbial fuel cells(MFCs),we used synthetic biology strategies to successfully construct four genetically engineered S.oneidensis by assembling the xylose transporters with intracellular xylose metabolic pathways,respectively.We found the strain GS was able to generate the highest power density,enabling a maximum electricity power density of 2.1±0.1 mW/m~2.The synthetic biology strategies could be further extended to rationally engineer other exoelectrogens for lignocellulosic biomass utilization to generate electricity power.Second,to incraese NADH regernation,we first adopted a modular metabolic engineering strategy to engineer and drive the metabolic flux toward the enhancement of intracellular NADH regeneration.We systematically studied 16 genes related to the NAD~+-dependent oxidation reactions for strengthening NADH regeneration in the four metabolic modules of S.oneidensis MR-1,i.e.,glycolysis,C1 metabolism,pyruvate fermentation,and tricarboxylic acid cycle.Then,we selected and assembled four most relative genes in S.oneidensis MR-1,?1.2-fold increase in intracellular NADH pool was obtained under anaerobic conditions without discharge,which elicited?3.0-fold increase in the maximum power output in microbial fuel cells.This modular engineering method in controlling the intracellular reducing equivalents would be a general approach in tuning the EET efficiency of exoelectrogens.Third,the slow rate of extracellular electron transfer(EET)of electroactive microorganisms remains a primary bottleneck that restricts the practical applications of bioelectrochemical systems.Intracellular NAD(H/~+)(i.e.,the total level of NADH and NAD~+)is a crucial source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways.However,how the total level of intracellular NAD(H/+)impacts the EET rate in Shewanella oneidensis has not been established.Here,we used a modular synthetic biology strategy to redirect metabolic flux towards NAD~+biosynthesis via three modules:de novo,salvage,and universal biosynthesis modules in S.oneidensis MR-1.Upon assemblage and simultaneous expression of five relative genes(ycel,pncB,nadM,nadD~*,and nadE~*)in the recombinant S.oneidensis strain SN5,the intracellular total NAD(H/~+)level was enhanced by 2.1-folds,which consequently enabled a 5.4-folds increase in the EET rate.Synthetic biology approaches to increase the level of the intracellular total NAD(H/~+)is a promising strategy in promoting the EET rate of other exoelectrogens.Last,the synthetic capacity of electron transport shuttle(ribflavin)is extremely limited and biofilm formed on the electrode surface is relatively thin,which seriously limits the practical application process of the microbial electrocatalytic system.Herein,a synthetic phenazine biosynthetic pathway encoding a conserved set of core biosynthetic genes consisting of(phzABCDEFG)responsible for synthesis of phenazine-1-carboxylic acid(PCA)was heterologously expressed in S.oneidensis to strengthen the bidirectional electron conduits of S.oneidensis.Further,graphene oxide was subsequently used to construct an engineered Shewanlla-reduced GO(rGO)3D self-assembled biohybrid,which dramatically enhanced the thickness and cell numbers in the electroactive biofilm.As a result,the maximum output density reached3.68 W/m~2 and(~210-folds higher than that of widl-type S.oneidensis).Meanwhile,the inward current density increased?68.1 times.The results show that the bidirectional EET efficiency can be enormously improved by synthetic PCA and 3D self-assembled electroactive biofilms.