Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO2 emission.

The low carbon yield from native metabolic machinery produces unfavorable process economics during the biological conversion of substrates to desirable bioproducts. To obtain higher carbon yields, we constructed a carbon conservation pathway named EP-bifido pathway in Escherichia coli by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and "bifid shunt", to generate high yield acetyl-CoA from glucose. 13C-Metabolic flux analysis confirmed the successful and appropriate employment of the EP-bifido pathway. The CO2 release during fermentation significantly reduced compared with the control strains. Then we demonstrated the in vivo effectiveness of the EP-bifido pathway using poly-ß-hydroxybutyrate (PHB), mevalonate and fatty acids as example products. The engineered EP-bifido strains showed greatly improved PHB yield (from 26.0 mol% to 63.7 mol%), fatty acid yield (from 9.17% to 14.36%), and the highest mevalonate yield yet reported (64.3 mol% without considering the substrates used for cell mass formation). The synthetic pathway can be employed in the production of chemicals that use acetyl-CoA as a precursor and can be extended to other microorganisms.

Exploring the N-terminal role of a heterologous protein in secreting out of Escherichia coli.

Escherichia coli is commonly used as a host for the extracellular production of proteins. However, its secretion capacity is often limited to a frustratingly low level compared with other expression hosts, because E. coli has a complex cell envelope with two layers. In previous report, we identified that the catalytic domain of a cellulase (Cel-CD) from Bacillus subtilis can be secreted into the medium from recombinant E. coli in large quantities without its native signal peptide. In this study, we proved the N-terminal sequence of the full length Cel-CD played a crucial role in transportation through both inner and outer membranes. By subcellular location analysis, we verified that the secretion was a two-step process via the SecB-dependent pathway through the inner membrane and an unknown pathway through the outer membrane. However, the N-terminal region of Cel-CD is polar and hydrophilic, which showed no similarities to other typical signal sequences. Random mutagenesis experiment suggested that the N-terminal sequence is a compromising result of transportation through inner and outer membranes. This is the first report that a "non-classical signal peptide" can guide recombinant proteins out of the cells from cytoplasm. Biotechnol. Bioeng. 2016;113: 2561-2567. © 2016 Wiley Periodicals, Inc.

Construction of cellulose-utilizing Escherichia coli based on a secretable cellulase.

BACKGROUND: The microbial conversion of plant biomass into value added products is an attractive option to address the impacts of petroleum dependency. The Gram-negative bacterium Escherichia coli is commonly used as host for the industrial production of various chemical products with a variety of sugars as carbon sources. However, this strain neither produces endogenous cellulose degradation enzymes nor secrets heterologous cellulases for its poor secretory capacity. Thus, a cellulolytic E. coli strain capable of growth on plant biomass would be the first step towards producing chemicals and fuels. We previously identified the catalytic domain of a cellulase (Cel-CD) and its N-terminal sequence (N20) that can serve as carriers for the efficient extracellular production of target enzymes. This finding suggested that cellulose-utilizing E. coli can be engineered with minimal heterologous enzymes. RESULTS: In this study, a ß-glucosidase (Tfu0937) was fused to Cel-CD and its N-terminal sequence respectively to obtain E. coli strains that were able to hydrolyze the cellulose. Recombinant strains were confirmed to use the amorphous cellulose as well as cellobiose as the sole carbon source for growth. Furthermore, both strains were engineered with poly (3-hydroxybutyrate) (PHB) synthesis pathway to demonstrate the production of biodegradable polyesters directly from cellulose materials without exogenously added cellulases. The yield of PHB reached 2.57-8.23 wt% content of cell dry weight directly from amorphous cellulose/cellobiose. Moreover, we found the Cel-CD and N20 secretion system can also be used for the extracellular production of other hydrolytic enzymes. CONCLUSIONS: This study suggested that a cellulose-utilizing E. coli was created based on a heterologous cellulase secretion system and can be used to produce biofuels and biochemicals directly from cellulose. This system also offers a platform for conversion of other abundant renewable biomass to biofuels and biorefinery products.

Engineering of Escherichia coli for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from glucose.

The copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has the potential to serve as a biodegradable tissue engineering material. However, the production of this kind of copolymer still suffers from high cost and uncertainty. We describe here the design of metabolic pathways to synthesize P(HB-co-HHx) directly from glucose using recombinant Escherichia coli. By combining the BktB-dependent condensation pathway with the inverted ß-oxidation cycle pathway, we were able to synthesize a P(HB-co-HHx) copolymer with a 10 mol% HHx fraction in recombinant E. coli. After optimizing the host strain and employing thioesterase mutant strains, the engineered E. coli produced 12.9 wt% P(HB-co-HHx) with a 13.2 mol% 3HHx fraction.

T cells and their products in diabetic kidney disease.

Diabetic kidney disease (DKD) is the most common cause of end-stage renal disease and has gradually become a public health problem worldwide. DKD is increasingly recognized as a comprehensive inflammatory disease that is largely regulated by T cells. Given the pivotal role of T cells and T cells-producing cytokines in DKD, we summarized recent advances concerning T cells in the progression of type 2 diabetic nephropathy and provided a novel perspective of immune-related factors in diabetes. Specific emphasis is placed on the classification of T cells, process of T cell recruitment, function of T cells in the development of diabetic kidney damage, and potential treatments and therapeutic strategies involving T cells.

Enhancing the Glucose Flux of an Engineered EP-Bifido Pathway for High Poly(Hydroxybutyrate) Yield Production.

BACKGROUND: As the greenhouse effect becomes more serious and carbon dioxide emissions continue rise, the application prospects of carbon sequestration or carbon-saving pathways increase. Previously, we constructed an EP-bifido pathway in Escherichia coli by combining Embden-Meyerhof-Parnas pathway, pentose phosphate pathway and "bifid shunt" for high acetyl-CoA production. There is much room for improvement in the EP-bifido pathway, including in production of target compounds such as poly(hydroxybutyrate) (PHB). RESULT: To optimize the EP-bifido pathway and obtain higher PHB yields, we knocked out the specific phosphoenolpyruvate phosphate transferase system (PTS) component II Cglc, encoded by ptsG. This severely inhibited the growth and sugar consumption of the bacterial cells. Subsequently, we used multiple automated genome engineering (MAGE) to optimize the ribosome binding site (RBS) sequences of galP (galactose: H (+) symporter) and glk (glucokinase gene bank: NC_017262.1), encoding galactose permease and glucokinase, respectively. Growth and glucose uptake were partially restored in the bacteria. Finally, we introduced the glf (UDP-galactopyranose) from Zymomonas mobilis mutase sugar transport vector into the host strain genome. CONCLUSION: After optimizing RBS of galP, the resulting strain L-6 obtained a PHB yield of 71.9% (mol/mol) and a 76 wt% PHB content using glucose as the carbon source. Then when glf was integrated into the genome strain L-6, the resulting strain M-6 reached a 5.81 g/L PHB titer and 85.1 wt% PHB content.