Xixian Xie

Improved Production of Tryptophan in Genetically Engineered Escherichia coli with TktA and PpsA Overexpression

Intracellular precursor supply is a critical factor for amino acid productivity. In the present study, ppsA and tktA genes were overexpressed in genetically engineered Escherichia coli to enhance the availability of two precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate. The engineered strain, TRTH0709 carrying pSV709, produced 35.9 g/L tryptophan from glucose after 40 h in fed-batch cultivation. The two genes were inserted, independently or together, into a low-copy-number expression vector (pSTV28) and transferred to TRTH0709. Fed-batch fermentations at high cell densities of the recombination strains revealed that overexpression of the ppsA gene alone does not significantly increase tryptophan yield. On the other hand, overexpression of the tktA gene, alone or with the ppsA gene, could further improve tryptophan yield to a final tryptophan titer of 37.9 and 40.2 g/L, respectively. These results represent a 5.6% and 11.9% enhancement over the titer achieved by TRTH0709. No evident genetic modifications leading to growth impairment were observed.

Modification of tryptophan transport system and its impact on production of L-tryptophan in Escherichia coli.

The production of L-tryptophan through chemical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of transport system for aromatic amino acids in L-tryptophan producing strains has not been fully explored. In this study, the fact was revealed that L-tryptophan production and cell growth were affected by the modification of transport systems based on YddG functioning as aromatic amino acid excretion and AroP functioning as general aromatic amino acid permease. Through comparing glucose conversion rates of recombinant strains such as Escherichia coli TRTH ΔaroP, E. coli TRTH-Y, and E. coli TRTH ΔaroP-Y, the moderate modification of transport system resulted in the metabolic flux redistribution of L-tryptophan biosynthesis pathway. In the fed-batch fermentation by E. coli TRTH and E. coli TRTH-Y in 30-liter fermentor, the final production of L-tryptophan fermented by E. coli TRTH-Y was 36.3 g/L, which was 12.6% higher than fermentation by E. coli TRTH.

Low-molecular-mass purine nucleoside phosphorylase: characterization and application in enzymatic synthesis of nucleoside antiviral drugs

Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillussubtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni2+-NTA chromatography. Compared with other rPNPs, PNP816 was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in kcat/Km using inosine as the substrate at 37°C. The PNP816 or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.

Systems metabolic engineering strategies for the production of amino acids

Systems metabolic engineering is a multidisciplinary area that integrates systems biology, synthetic biology and evolutionary engineering. It is an efficient approach for strain improvement and process optimization, and has been successfully applied in the microbial production of various chemicals including amino acids. In this review, systems metabolic engineering strategies including pathway-focused approaches, systems biology-based approaches, evolutionary approaches and their applications in two major amino acid producing microorganisms: Corynebacterium glutamicum and Escherichia coli, are summarized.

Current status on metabolic engineering for the production of L-aspartate family amino acids and derivatives

The l-aspartate amino acids (AFAAs) are constituted of l-aspartate, l-lysine, l-methionine, l-threonine and l-isoleucine. Except for l-aspartate, AFAAs are essential amino acids that cannot be synthesized by humans and most farm animals, and thus possess wide applications in food, animal feed, pharmaceutical and cosmetics industries. To date, a number of amino acids, including AFAAs have been industrially produced by microbial fermentation. However, the overall metabolic and regulatory mechanisms of the synthesis of AFAAs and the recent progress on strain construction have rarely been reviewed. Aiming to promote the establishment of strains of Corynebacterium glutamicum and Escherichia coli, the two industrial amino acids producing bacteria, that are capable of producing high titers of AFAAs and derivatives, this paper systematically summarizes the current progress on metabolic engineering manipulations in both central metabolic pathways and AFAA synthesis pathways based on the category of the five-word strain breeding strategies: enter, flow, moderate, block and exit.

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5′-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.

Optimization of carbon source and glucose feeding strategy for improvement of L-isoleucine production by Escherichia coli

Fed-batch cultivations of L-isoleucine-producing Escherichia coli TRFP (SGr, α-ABAr, with a pTHR101 plasmid containing a thr operon and ilvA) were carried out on different carbon sources: glucose, sucrose, fructose, maltose and glycerol. The results indicated that sucrose was the best initial carbon source for L-isoleucine production and then sucrose concentration of 30 g·L−1 was determined in the production medium. The results of different carbon sources feeding showed that the glucose solution was the most suitable feeding media. The dissolved oxygen (DO) of L-isoleucine fermentation was maintained at 5%, 15% and 30% with DO-stat feeding, respectively. The results indicated that when the DO level was maintained at 30%, the highest biomass and L-isoleucine production were obtained. The accumulation of acetate was decreased and the production of L-isoleucine was increased markedly, when the glucose concentration was maintained at 0.15 g·L−1 by using glucose-stat feeding. Finally, the glucose concentration was maintained at 0.10 g·L−1 and the DO level was controlled at approximately 30% during the whole fermentation period, using the combined feeding strategy of glucose-stat feeding and DO feedback feeding. The acetate accumulation was decreased to 7.23 g·L−1, and biomass and production of L-isoleucine were increased to 46.8 and 11.95 g·L−1, respectively.

Expression of the Escherichia Coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw

Threonine dehydratase (EC 4.3.1.19, TDH) catalyzing the degradation of Thr to α-ketobutyrate, is a rate-limiting enzyme in L-Ile pathway. The tdcB gene encoding TDH was obtained from Escherichia coli K12 by PCR and expressed at E. coli BL21 (DE3). Then the tdcB gene was inserted into the shuttle expression vector pXMJ19 and the recombinant plasmid was electroporated into the L-isoleucine-producing strain of Corynebacterium glutamicum YILW. Crude extracts of the microbial strain containing the plasmid pXMJ19tdcB retained 60% of the original TDH activity even in the presence of 300 mM L-Ile. The recombinant strain of bacteria showed 7.5% higher enzyme activity and 11.3% higher L-Ile production compared to the original strain.

Optimization of technical conditions of producing ribavirin byBacillus subtilis

In this study, the fed-batch fermentation technique was applied to improve the yield of ribavirin produced byBacillus subtilis AG208-1. Various fermentation substrates and conditions were investigated to identify the optimal concentration of carbon source, Tween-80 and fermentation temperature in the production of ribavirin. The optimal initial glucose concentration was determined to be 120 g/L based on the results of fermentations conducted in a baffled flask. Then, different concentrations of Tween-80 and feeding time were also investigated in this work, respectively. Our results showed that the production of ribavirin byBacillus subtilis was enhanced when Tween-80 concentration was 0.5%. Furthermore, variable temperature control strategy was predominant factor for ribavirin overproduction and the optimal strategy was: temperature of fermentation at 36 °C from 0 h to 24 h, with a gradual increase in temperature of 2 °C every 6 h. Under the optimal conditions, a final ribavirin concentration of 4.21 g/L was achieved after 60 h.

Production of α-ketobutyrate using engineered Escherichia coli via temperature shift.

Alpha-ketobutyrate has been widely used in medicine and food additive industry. Because chemical and enzymatic methods are associated with many deficiencies, the recent focus shifted to fermentation for the production of α-ketobutyrate. In this study, a genetically engineered strain THRDΔrhtAΔilvIH/pWSK29-ilvA was constructed, starting from an L-threonine-producing strain, by overexpressing threonine dehydratase (TD), reducing α-ketobutyrate catabolism and L-threonine export. The shake flask cultivation of THRDΔrhtAΔilvIH/pWSK29-ilvA allowed the production of 16.2 g/L α-ketobutyrate. Accumulation of α-ketobutyrate severely inhibited the cell growth. To develop a better TD expression system and avoid the usage of the expensive inducer IPTG, a temperature-induced plasmid pBV220-ilvA was selected to transform the strain THRDΔrhtAΔilvIH for α-ketobutyrate production. The initial temperature was maintained at 35°C to guarantee normal cell growth, and then elevated to 40°C to induce the expression of TD. Under optimized conditions, the α-ketobutyrate titer reached 40.8 g/L after 28 h of fermentation, with a productivity of 1.46 g/L/h and a yield of 0.19 g/g glucose, suggesting large-scale production potential. Biotechnol. Bioeng. 2016;113: 2054-2059.