BackgroundPseudomonas putida is a promising host for the bioproduction of chemicals, but its industrial applications are significantly limited by its obligate aerobic character. The aim of this paper is to empower the anoxic metabolism of wild-type Pseudomonas putida to enable bioproduction anaerobically, with the redox power from a bioelectrochemical system (BES).ResultsThe obligate aerobe Pseudomonas putida F1 was able to survive and produce almost exclusively 2–Keto-gluconate from glucose under anoxic conditions due to redox balancing with electron mediators in a BES. 2-Keto-gluconate, a precursor for industrial anti-oxidant production, was produced at an overall carbon yield of over 90 % based on glucose. Seven different mediator compounds were tested, and only those with redox potential above 0.207 V (vs standard hydrogen electrode) showed interaction with the cells. The productivity increased with the increasing redox potential of the mediator, indicating this was a key factor affecting the anoxic production process. P. putida cells survived under anaerobic conditions, and limited biofilm formation could be observed on the anode’s surface. Analysis of the intracellular pools of ATP, ADP and AMP showed that cells had an increased adenylate energy charge suggesting that cells were able to generate energy using the anode as terminal electron acceptor. The analysis of NAD(H) and NADP(H) showed that in the presence of specific extracellular electron acceptors, the NADP(H) pool was more oxidised, while the NAD(H) pool was unchanged. This implies a growth limitation under anaerobic conditions due to a shortage of NADPH and provides a way to limit biomass formation, while allowing cell maintenance and catalysis at high purity and yield.ConclusionsFor the first time, this study proved the principle that a BES-driven bioconversion of glucose can be achieved for a wild-type obligate aerobe. This non-growth bioconversion was in high yields, high purity and also could deliver the necessary metabolic energy for cell maintenance. By combining this approach with metabolic engineering strategies, this could prove to be a powerful new way to produce bio-chemicals and fuels from renewables in both high yield and high purity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0452-y) contains supplementary material, which is available to authorized users.

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para-Hydroxy benzoic acid (PHBA) is the key component for preparing parabens, a common preservatives in food, drugs, and personal care products, as well as high-performance bioplastics such as liquid crystal polymers. Pseudomonas putida KT2440 was engineered to produce PHBA from glucose via the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from Escherichia coli and a feedback resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase encoded by gene aroGD146N were overexpressed individually and simultaneously. In addition, genes related to product degradation (pobA) or competing for the precursor chorismate (pheA and trpE) were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor hexR was knocked out in order to increase erythrose 4-phosphate and NADPH supply. The best strain achieved a maximum titer of 1.73 g L−1 and a carbon yield of 18.1% (C-mol C-mol−1) in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase.

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It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm 2 , 0.27 ± 0.02 mmol/g CDW /hr and 0.25 ± 0.02 mmol/ g CDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.anode respiration, bioelectrochemical system, membrane-bound dehydrogenase, microbial electrosynthesis, Pseudomonas putida

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Research studies on NAD + have proven its crucial role in aging and disease. Nicotinamide mononucleotide (NMN), as the key intermediate of NAD + , plays a significant role in supplying and maintaining NAD + levels. In the present study, a biocatalytic method for the efficient synthesis of NMN was established. First, Escherichia coli was systematically modified to make it more conducive to the biosynthesis and accumulation of NMN. Next, the performance of nicotinamide phosphoribosyltransferase from Vibrio bacteriophage KVP40 (VpNadV) was determined, which has the best catalytic activity to produce NMN from nicotinamide. The accumulation of extracellular NMN was further increased after the introduction of an NMN transporter. Fine-tuning of gene expression and copy number led to the synthesis of NMN at the yield of 2.6 g/L at the shake flask level. The introduction of a nicotinamide transporter, BcniaP, could not obviously increase the production of NMN at the shake flask level, but it decreased the production of NMN at the bioreactor level. Finally, the titer of NMN reached 16.2 g/L with a conversion ratio of 97.0% from nicotinamide, both of which are highest according to currently available reports. The fed-batch fermentation with direct supplementation of nicotinamide could facilitate the industrialscale production of NMN compared to that achieved by the whole-cell catalysis process. These results also represent the highest reported yield of NMN synthesized from nicotinamide in E. coli.

The (2S)-naringenin is an important natural flavonoid with several bioactive effects on human health. It is also a key precursor in the biosynthesis of other high value compounds. The production of (2S)-naringenin is significantly influenced by the acetyl-CoA available in the cytosol. In this study, we increased the acetyl-CoA supply via the β-oxidation of fatty acids in the peroxisomes of Saccharomyces cerevisiae. Several lipases from different sources and PEX11, FOX1, FOX2, and FOX3, the key genes of the fatty acid β-oxidation pathway, were overexpressed during the production of (2S)-naringenin in yeast. The level of acetyl-CoA was 0.205 nmol higher than that in the original strain and the production of (2S)-naringenin increased to 286.62 mg/g dry cell weight when PEX11 was overexpressed in S. cerevisiae strain L07. Remarkable (2S)-naringenin production (1129.44 mg/L) was achieved with fed-batch fermentation, with the highest titer reported in any microorganism. Our results demonstrated the use of fatty acid β-oxidation to increase the level of cytoplasmic acetyl-CoA and the production of its derivatives.