Qin Ye

The shortage of food based feedstocks has been one of the stumbling blocks in industrial biomanufacturing. The acetone bioproduction from the traditional acetone–butanol–ethanol fermentation is limited by the non-specificity of products and competitive utilization of food-based substrates. Using genetically modified Escherichia coli to produce acetone as sole product from the cost-effective non-food based substrates showed great potential to overcome these problems. A novel acetone biosynthetic pathway were constructed based on genes from Clostridium acetobutylicum (thlA encoding for thiolase, adc encoding for acetoacetate decarboxylase, ctfAB encoding for coenzyme A transferase) and Escherichia coli MG1655 (atoB encoding acetyl-CoA acetyltransferase, atoDA encoding for acetyl-CoA: acetoacetyl-CoA transferase subunit α and β). Among these constructs, one recombinant MG1655 derivative containing the hybrid pathway consisting of thlA, atoDA, and adc, produced the highest level of acetone from acetate. Reducing the gluconeogenesis pathway had little effect on acetone production, while blocking the TCA cycle by knocking out the icdA gene enhanced the yield of acetone significantly. As a result, acetone concentration increased up to 113.18 mM in 24 h by the resting cell culture coupling with gas-stripping methods. An engineered E. coli strain with optimized hybrid acetone biosynthetic pathway can utilize acetate as substrate efficiently to synthesize acetone without other non-gas byproducts. It provides a potential method for industrial biomanufacturing of acetone by engineered E. coli strains from non-food based substrate.

L-glutathione (GSH) is a non-protein thiol compound with important biological properties and is widely used in pharmaceutical, food, cosmetic and health products. The cellular GSH is determined by the activity and characteristic of GSH-synthesizing enzymes, energy and precursor supply, and degradation of formed GSH. In this study, genes encoding enzymes related to the precursor amino acid degradation and glycogen formation as well as GSH degradation were systematically manipulated in Escherichia coli strains over-expressing gshF from Actinobacillus succinogenes. The manipulation included disrupting the precursor degradation pathways (tnaA and sdaA), eliminating l-glutathione degradation (ggt and pepT), and manipulating the intracellular ATP level (disruption of glgB). However the constructed mutants showed lower levels of GshF expression. 2-D electrophoresis was performed to elucidate the reasons for this discrepancy, and the results indicated obvious changes in central metabolism and amino acid metabolism in the penta-mutant. Fed-batch culture of the penta-mutant ZJ12345 was performed where the GshF expression level was enhanced, and both the GSH production (19.10 mM) and the yield based on added l-cysteine (0.76 mmol/mmol) were significantly increased. By interrupting the degradation pathways of l-cysteine, serine and GSH and blocking glycogen formation, the GSH production efficiency was significantly improved.

We have described a metabolically engineered strain of Klebsiella pneumoniae/pUC18kan-aldHec that could convert glycerol to 3-hydroxypropionic acid (3-HP) effectively. In this study, the transcription of the genes encoding aldehyde dehydrogenase (AldH) and glycerol dehydratase (DhaB) responsible to 3-HP-biosynthesis, key enzymes in PP pathway and TCA cycle, as well as hydrogenases was observed under anaerobic, microaerobic, and aerobic conditions using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). During the fast growth and slow growth phases of cell growth, the aldH transcription levels were 7.0- and 2.1-fold enhanced under microaerobic condition, and 12.4- and 0.14-fold under fully aerobic condition, compared to the anaerobic control, while transcription of dhaB showed a downregulated manner due to aeration. Moreover, aeration increased the transcription levels of zwf, sucA, and sdhAB during the fast growth phase of the cell growth. The results also indicated that the hydrogenase 3 (hycBCDFG) and formate dehydrogenase (fdhA) mediated formate hydrogen lyase pathway was the main route to produce hydrogen and consume the excess NADH. These characteristics are useful for understanding and improving 3-HP biosynthesis in K. pneumoniae.

Gas-liquid contacting is an important unit operation in chemical and biochemical processes, but the gas utilization efficiency is low in conventional gas-liquid contactors especially for sparingly soluble gases. The gas self-inducing impeller is able to recycle gas in the headspace of a reactor to the liquid without utilization of additional equipment such as a gas compressor, and thus, the gas utilization efficiency is significantly enhanced. Gas induction is caused by the low pressure or deep vortex at a sufficiently high impeller speed, and the speed at which gas induction starts is termed the critical speed. The critical impeller speed, gas-induction flow rate, power consumption, and gas-liquid mass transfer are determined by the impeller design and operation conditions. When the reactor is operated in a dead-end mode, all the introduced gas can be completely used, and this feature is especially favorable to flammable and/or toxic gases. In this article, the principles, designs, characteristics of self-inducing reactors, and applications to biotechnology are described.

Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production.

Pseudomonas sp. F12 isolated from soil could transform dl-2-amino-Δ²-thiazoline-4-carboxylic acid (DL-ATC) to l-cysteine. It could grow in minimal medium containing DL-ATC as the sole carbon and nitrogen source, and the apparent activity of l-cysteine synthesis (CS) achieved 122 U/mL in a 5-L bioreactor. Pseudomonas sp. F12 could utilize glucose as carbon source and ammonia as nitrogen source for growth, but no CS activity was formed. To reduce the cost of DL-ATC, the cultivation process was divided into a growth stage on glucose and ammonia and a production stage induced by DL-ATC. The excessive glucose led to the production of byproduct(s) which seriously inhibited cell growth and CS production. Ammonium was accumulated when DL-ATC was consumed, and ammonium did not inhibit CS activity formation until 60 mM. Based on the above features, fed-batch cultivation of the growth stage was developed by supplying glucose restrictively. The volumetric CS activity was enhanced more than two times that obtained under the initial conditions.

We constructed a recombinant Escherichia coli strain for butanol production by cloning the cDNA sequence of the key butanol synthetic pathway genes from Clostridium acetobutylicum ATCC824. We amplified the genes of thil, adhE2 and BCS operon by PCR with C. acetobutylicum ATCC824 genome as a template. We constructed the recombinant strain E. coli pBAT (BCS operon-adhE2-thil/pTrc99a/MG1655). We used 0.1 mmol/l Isopropyl beta-D-thiogalactopyranoside (IPTG) to induce the recombinant E. coli pBAT for 5 h for recombinant protein expression. We measured acetyl-CoA acetyltransferase (THL), beta-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD) and butyraldehyde dehydrogenase (BYDH)/butanol dehydrogenase (BDH) activities in E. coli MG1655 and E. coli pBAT. The fermentation of E. coli pBAT was done in flask in aerobic, micro-aerobic and anaerobic mode separately. In the recombinant E. coli pBAT, THL activity was 0.160 U/mg protein, about 30 times higher than that of E. coli MG1655. HBD activity was 5 times higher than that of E. coli MG1655. CRT activity was 1.53 U/mg protein whereas not detectable in E. coli MG1655. BCD activity was about 32 times higher than that of E. coli MG1655. In addition, the results show that n-butanol could be produced under anaerobic and micro-aerobic conditions. The maximum n-buntanol concentration of 84 mg/l was detected in cultivation broth. The key genes of butanol synthetic pathway were expressed in E. coli and the recombinant strains would offer an alternative strategy for butanol biosynthesis.

In order to further understand the difference in metabolic regulation between Escherichia coli DH5alpha and its acetate-tolerant mutant DA19, we analyzed the effect of supplementing adenine in defined media on metabolic fluxes in the two strains. E. coli DH5alpha and DA19 were continuously cultured in nitrogen-limited defined media without or with supplemented adenine. Based on mass balance and metabolic reaction stoichiometry, the metabolic fluxes in DH5alpha and DA19 at different culture condition were calculated, and the results were compared with the activities of key enzymes. The supplementation of adenine decreased specific glucose consumption rate and specific acetate production rate, and improved growth yield coefficient on glucose of DH5alpha. Nevertheless, specific pyruvate production rate did not significantly change. Furthermore acetate split ratio decreased whereas pyruvate and TCA split ratios increased. Obvious changes were observed in the activities of phosphofructokinase, 6-phosphoglucose dehydrogenase and acetokinase. Compared with DH5alpha, the supplementation of adenine increased rate specific pyruvate production rate of DA19 nearly 57%, and other parameters did not change. In addition, it showed decreased TCA split ratio and greatly increased pyruvate split ratio, whereas no changes in the key enzyme activities were observed. The differences of enzyme activities in the two strains were reasonably consistent with those in metabolic fluxes. Because there were differences in the de novo biosynthesis capacity of purine nucleotides between DH5alpha and DA19, supplementation of adenine had completely different effect on metabolic fluxes in two strains.