Metabolic engineering of Escherichia coli for efficient production of L-alanyl-L-glutamine.

BACKGROUND: L-Alanyl-L-glutamine (AQ) is a functional dipeptide with high water solubility, good thermal stability and high bioavailability. It is widely used in clinical treatment, post-operative rehabilitation, sports health care and other fields. AQ is mainly produced via chemical synthesis which is complicated, time-consuming, labor-intensive, and have a low yield accompanied with the generation of by-products. It is therefore highly desirable to develop an efficient biotechnological process for the industrial production of AQ. RESULTS: A metabolically engineered E. coli strain for AQ production was developed by over-expressing L-amino acid α-ligase (BacD) from Bacillus subtilis, and inactivating the peptidases PepA, PepB, PepD, and PepN, as well as the dipeptide transport system Dpp. In order to use the more readily available substrate glutamic acid, a module for glutamine synthesis from glutamic acid was constructed by introducing glutamine synthetase (GlnA). Additionally, we knocked out glsA-glsB to block the first step in glutamine metabolism, and glnE-glnB involved in the ATP-dependent addition of AMP/UMP to a subunit of glutamine synthetase, which resulted in increased glutamine supply. Then the glutamine synthesis module was combined with the AQ synthesis module to develop the engineered strain that uses glutamic acid and alanine for AQ production. The expression of BacD and GlnA was further balanced to improve AQ production. Using the final engineered strain p15/AQ10 as a whole-cell biocatalyst, 71.7 mM AQ was produced with a productivity of 3.98 mM/h and conversion rate of 71.7%. CONCLUSION: A metabolically engineered strain for AQ production was successfully developed via inactivation of peptidases, screening of BacD, introduction of glutamine synthesis module, and balancing the glutamine and AQ synthesis modules to improve the yield of AQ. This work provides a microbial cell factory for efficient production of AQ with industrial potential.

Enhanced productivity of gamma-amino butyric acid by cascade modifications of a whole-cell biocatalyst.

We previously developed a gamma-amino butyric acid (GABA)-producing strain of Escherichia coli, leading to production of 614.15 g/L GABA at 45 °C from L-glutamic acid (L-Glu) with a productivity of 40.94 g/L/h by three successive whole-cell conversion cycles. However, the increase in pH caused by the accumulation of GABA resulted in inactivation of the biocatalyst and consequently led to relatively lower productivity. In this study, by overcoming the major problem associated with the increase in pH during the production process, a more efficient biocatalyst was obtained through cascade modifications of the previously reported E. coli strain. First, we introduced four amino acid mutations to the codon-optimized GadB protein from Lactococcus lactis to shift its decarboxylation activity toward a neutral pH, resulting in 306.65 g/L of GABA with 99.14 mol% conversion yield and 69.8% increase in GABA productivity. Second, we promoted transportation of L-Glu and GABA by removing the genomic region encoding the C-plug of GadC (a glutamate/GABA antiporter) to allow its transport path to remain open at a neutral pH, which improved the GABA productivity by 16.8% with 99.3 mol% conversion of 3 M L-Glu. Third, we enhanced the expression of soluble GadB by introducing the GroESL molecular chaperones, leading to 20.2% improvement in GABA productivity, with 307.40 g/L of GABA and a 61.48 g/L/h productivity obtained in one cycle. Finally, we inhibited the degradation of GABA by inactivation of gadA and gadB from the E. coli genome, which resulted in almost no GABA degradation after 40 h. After the cascade system modifications, the engineered recombinant E. coli strain achieved a 44.04 g/L/h productivity with a 99.6 mol% conversion of 3 M L-Glu in a 5-L bioreactor, about twofold increase in productivity compared to the starting strain. This increase represents the highest GABA productivity by whole-cell bioconversion using L-Glu as a substrate in one cycle observed to date, even better than the productivity obtained from the three successive conversion cycles.

Distribution and transport of selenium in Yutangba, China: impact of human activities.

Yutangba, one of the typical high-Se areas where a sudden incidence of Se poisoning occurred in 1963, is located in the northern part of Shuanghe town about 81 km SE of Enshi, Hubei Province, China. In this area, a comprehensive investigation was conducted on the distribution of Se in soils, plant species, stream water and sediment. The mean concentrations of Se were: total soil, 4.75+/-7.43 mg/kg (n=150); Corn seeds, 1.48+/-1.41 mg/kg (n=20); Agry wormwood, 1.68+/-1.27 mg/kg (n=30); Bracken fern, 0.63+/-1.61 mg/kg (n=57), and Central China dryoathyrium, 0.48+/-0.72 mg/kg (n=39); Stream water, 58.4+/-16.8 microg/L (n=12); stream sediment, 26.6+/-26.8 mg/kg (n=11). The spatial distribution of Se in soils and plants is significantly uneven and higher Se samples mainly distributed in the croplands and northwest Yutangba, while almost all the lower Se samples are located in undisturbed areas. 11 samples contained extremely high concentrations of Se, ranging from 346 to 2018 mg/kg with an average of 899+/-548 mg/kg, were found at croplands and discarded coal spoils in Yutangba. The distribution of Se in Yutangba is related to the pathways of Se transport, which was caused by human activities such as stone coal conveyance by local villagers, mining of stone coal for use as a fuel or fertilizer, and discharging lime into cropland to improve soil. These activities caused variable addition of Se to the soil and further accumulation of Se in food chain. Therefore, human activities have played an important role in the distribution, transport, and bioavailability of Se. Yutangba is still a high risk area where Se poisoning may occur again, and so are almost all high-Se areas in Enshi Prefecture.