Metabolic Engineering toward Sustainable Production of Nylon-6.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.

Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase.

Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 µmol mg protein(-1)min(-1)). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (µ(aerobic) 0.09 ± 0.03 h(-1)). Co-expression of Lactococcus lactis ß-phosphoglucomutase accelerated maltose utilization via this route (µ(aerobic) 0.21 ± 0.01 h(-1), µ(anaerobic) 0.10 ± 0.00 h(-1)). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.

Catalase overexpression reduces lactic acid-induced oxidative stress in Saccharomyces cerevisiae.

Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2Delta reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h(-1) in anaerobic batch cultures containing lactic acid but only 0.16 h(-1) in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.

Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae.

Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the haa1Delta mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of S. cerevisiae to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.