Metabolic engineering of Escherichia coli for the production of L-malate from xylose.

Malate is regarded as one of the key building block chemicals which can potentially be produced from biomass at a large scale. Although glucose has been extensively studied as the substrate for malate production, its high price and potential competition with food production are serious limiting factors. In this study, Escherichia coli was metabolically engineered to effectively produce malate from xylose, the second most abundant sugar component of lignocellulosic biomass. First, the biosynthetic route of malate was constructed by overexpressing D-tagatose 3-epimerase, L-fuculokinase, L-fuculose-phosphate aldolase, and aldehyde dehydrogenase A. Second, genes encoding malic enzyme, malate dehydrogenase, and fumarate hydratase were knocked out to eliminate malate consumption, resulting in a titer of 1.99 g/l malate and a yield of 0.47 g malate/g xylose. Third, glycolate oxidase and malate synthase were overexpressed to strengthen the conversion of glycolate to malate, which led to a titer of 4.33 g/l malate and a yield of 0.83 g malate/g xylose, reaching 93% of the theoretical yield. Finally, catalase HPII was overexpressed to decompose H2O2 and alleviate its toxicity, which improved cell growth and further boosted malate titer to 5.90 g/l with a yield of 0.80 g malate/g xylose. To the best of our knowledge, this is the first study to report efficient malate production from xylose as the carbon source.

Microbial production and characterization of poly-3-hydroxybutyrate by Neptunomonas antarctica.

Considering the industrial interest of biodegradable polymer poly-3-hydroxybutyrate (PHB), the marine bacteria Neptunomonas antarctica was studied for its ability to accumulate PHB. The extracted polymer was confirmed to be PHB by nuclear magnetic resonance analysis. In shake flask cultures using natural seawater as medium components, PHB was produced up to 2.12 g/L with a yield of 0.18 g PHB/g fructose. In the presence of artificial seawater, the PHB titer and yield reached 2.13 g/L and 0.13 g PHB/g fructose, respectively. The accumulated polymer gradually decreased when fructose was exhausted, indicating that intracellular PHB was degraded by N. antarctica. The weight-average and number-average molecular weights of PHB produced within natural seawater were 2.4 × 10(5) g/mol and 1.7 × 10(5) g/mol, respectively. Our results highlight the potential of N. antarctica for PHB production with seawater as a nutrient source.

Effect of NADH kinase on poly-3-hydroxybutyrate production by recombinant Escherichia coli.

The cofactor NADPH participates in a variety of anabolic reactions and its availability is considered to play a critical role in biotransformation processes. NADH kinase (Pos5) from Saccharomyces cerevisiae catalyzes the phosphorylation of NADH to generate NADPH. To investigate the effect of NADH kinase on poly-3-hydroxybutyrate (PHB) production, pos5 was co-expressed with PHB synthetic operon phbCAB in Escherichia coli. The recombinant strain carrying pos5 and phbCAB co-expression plasmid reached 5.96 g/L cell dry weight with 64.1% PHB accumulation in 72 h shake flask cultivation, while the control strain without pos5 yielded 3.93 g/L cell dry weight with 58.5% PHB content. PHB production titer was enhanced from 2.30 g/L to 3.82 g/L. Intracellular cofactor concentration analysis revealed that the ratio of NADP/NAD in pos5 overexpression strain was two times more compared with that of the control without pos5. The results showed that NADH kinase could be employed as an effective metabolic manipulation target to improve PHB synthesis.

Enhanced production of poly-3-hydroxybutyrate by Escherichia coli over-expressing multiple copies of NAD kinase integrated in the host genome.

OBJECTIVES: With the help of attB-attP recombination technique, multiple copies of yfjB gene encoding the NAD kinase of Escherichia coli were inserted into the host chromosome to promote NADPH-dependent poly-3-hydroxybutyrate (PHB) production. RESULTS: The yfjB integration mutant E. coli T2 harbored a similar metabolic profile to that of the wild type control. When PHB biosynthesis operon was introduced, the yfjB integration mutant produced 3 g PHB l(-1) from 18.2 g glucose l(-1), while the wild type consumed 15.7 g glucose l(-1) to afford 2.34 g PHB l(-1) in 48 h of shake-flask cultivation. Transcriptional analysis showed that the transcription levels of genes within the PHB biosynthesis operon were increased by six to eightfold with yfj Bover-expression, which may be the primary reason for the improved PHB production. CONCLUSION: A practical method is demonstrated to construct genetically-stable strains harboring extra copies of NAD kinase to enhance NADPH-dependent reactions.