Engineering an electroactive Escherichia coli for the microbial electrosynthesis of succinate from glucose and CO2.

BACKGROUND: Electrochemical energy is a key factor of biosynthesis, and is necessary for the reduction or assimilation of substrates such as CO2. Previous microbial electrosynthesis (MES) research mainly utilized naturally electroactive microbes to generate non-specific products. RESULTS: In this research, an electroactive succinate-producing cell factory was engineered in E. coli T110(pMtrABC, pFccA-CymA) by expressing mtrABC, fccA and cymA from Shewanella oneidensis MR-1, which can utilize electricity to reduce fumarate. The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism (CCM). This strain was fermented in an MES system with neutral red as the electron carrier and supplemented with HCO3+, which produced a succinate yield of 1.10 mol/mol glucose-a 1.6-fold improvement over the parent strain T110. CONCLUSIONS: The strain T110(pMtrABC, pFccA-CymA, pBTCA) is to our best knowledge the first electroactive microbial cell factory engineered to directly utilize electricity for the production of a specific product. Due to the versatility of the E. coli platform, this pioneering research opens the possibility of engineering various other cell factories to utilize electricity for bioproduction.

Mixed fermentation of Aspergillus niger and Candida shehatae to produce bioethanol with ionic-liquid-pretreated bagasse.

In this study, bagasse was pretreated with ionic liquid (IL) 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) and 1% NaOH solution for initial activation of bagasse. A mixed fermentation of treated bagasse by Aspergillus niger and Candida shehatae showed the optimal conditions with the addition of C. shehatae 12 h later at a 1:1 proportion to A. niger. To further improve the ethanol production and obtain optimal fermentation conditions, a Plackett-Burman design was applied to screen the significant formulation and process variables. The optimal ethanol fermentation conditions with IL pretreated bagasse were determined using response surface methodology by Box-Behnken design. Three variables "initial pH, (NH4)2SO4, fermentation time" were regarded as significant factors in the optimization study. The resulting optimum fermentation conditions for bioethanol was identified as: initial pH of 5.89, (NH4)2SO4 concentration of 0.40 g/50 mL, and fermentation time of 3.60 days. The verification experimental ethanol concentration was 8.14 g/L, which agreed with the predicted value. An enhancement of approximately 153.58% compared with initial fermentation conditions in ethanol production was found using optimized conditions. It demonstrated that optimization methodology had a positive effect on the improvement of ethanol production. Under the optimal fermentation medium and conditions, the ethanol production with IL-pretreated bagasse and untreated bagasse was 8.14 g/L and 5.03 g/L, respectively, which exhibited 62% increase, compared to initial conditions with production of 3.21 g/L and 2.67 g/L, respectively, which displayed 20% increase. Both under optimal and original fermentation conditions, compared to the fermentation medium with untreated bagasse, all the results indicated that IL-pretreated bagasse resulted in higher ethanol production than untreated bagasse, demonstrating that IL-pretreated bagasse successfully increased the ethanol production in the mixed fermentation by A. niger and C. shehatae.

Combinatorial modulation of initial codons for improved zeaxanthin synthetic pathway efficiency in Escherichia coli.

A balanced and optimized metabolic pathway is the basis for efficient production of a target metabolite. Traditional strategies mostly involve the manipulation of promoters or ribosome-binding sites, which can encompass long sequences and can be complex to operate. In this work, we found that by changing only the three nucleotides of the initiation codons, expression libraries of reporter proteins RFP, GFP, and lacZ with a large dynamic range and evenly distributed expression levels could be established in Escherichia coli (E. coli). Thus, a novel strategy that uses combinatorial modulation of initial codons (CMIC) was developed for metabolic pathway optimization and applied to the three genes crtZ, crtY, and crtI of the zeaxanthin synthesis pathway in E. coli. The initial codons of these genes were changed to random nucleotides NNN, and the gene cassettes were assembled into vectors via an optimized strategy based on type II restriction enzymes. With minimal labor time, a combinatorial library was obtained containing strains with various zeaxanthin production levels, including a strain with a titer of 6.33 mg/L and specific production value of 1.24 mg/g DCW-a striking 10-fold improvement over the starting strain. The results demonstrated that CMIC was a feasible technique for conveniently optimizing metabolic pathways. To our best knowledge, this is the first metabolic engineering strategy that relies on manipulating the initiation codons for pathway optimization in E. coli.

Not only osmoprotectant: betaine increased lactate dehydrogenase activity and L-lactate production in lactobacilli.

Lactobacilli are commonly used for industrial production of polymer-grade L-lactic acid. The present study tested the Tween 80 alternative betaine in L-lactate production by several industrial lactobacilli. In flask fermentation of Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactis and Lactobacillus rhamnosus, the betaine addition (2g/l) had similar osmoprotectant effect with Tween 80 but had increased the lactate dehydrogenase activities and L-lactate production than Tween 80 control. In fed-batch fermentation of L. casei, betaine supplementation improved the L-lactic acid titer to 190 g/l, the yield to 95.5% (g L-lactic acid/g glucose), the productivity to 2.6g/lh, and the optical purity to 97.0%. The results demonstrated that supplementation of Tween 80 alternative - betaine in the fermentation medium is feasible for industrial l-lactic acid fermentation by lactobacilli, which will improve the lactate production but will not increase the process costs and modify any process conditions.