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Anaerobic fermentation is a favorable process for microbial production of bulk chemicals like ethanol and organic acids. Low productivity is the bottleneck of several anaerobic processes which has significant impact on the technique competitiveness of production strain. Improving growth rate of production strain can speed up the total production cycle and may finally increase productivity of anaerobic processes. In this work, evolutionary engineering of wild-type strain Escherichia coli W3110 was adopted to improve anaerobic growth in mineral medium. Significant increases in exponential growth rate and stationary cell density were achieved in evolved strain WE269, and a 96.5% increase in lactate productivity has also been observed in batch fermentation of this strain with M9 minimal medium. Then, an engineered strain for lactate production (BW100) was constructed by using WE269 as a platform and 98.3 g/L lactate (with an optical purity of D-lactate above 95%) was produced in a 5-L bioreactor after 48 h with a productivity of 2.05 g/(L·h). Finally, preliminary investigation demonstrated that mutation in sucD (sucD M245I) (encoding succinyl-CoA synthetase); ilvG (ilvG Δ1bp) (encoding acetolactate synthase 2 catalytic subunit), and rpoB (rpoB T1037P) (encoding RNA polymerase β subunit) significantly improved anaerobic growth of E. coli. Double-gene mutation in ilvG and sucD resumed most of the growth potential of evolved strain WE269. This work suggested that improving anaerobic growth of production host can increase productivity of organic acids like lactate, and specific mutation-enabled improved growth may also be applied to metabolic engineering for production of other bulk chemicals.

To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum. A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the − 10 and − 35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks. To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.

c-Myc is a well-characterized proto-oncogene that induces cellular transformation and modulates programmed cell death. While recent studies have demonstrated high expression of c-Myc protein in advanced and metastatic melanoma, the clinical and biological implications remain to be fully elucidated. In this study, we investigated the effect of c-Myc overexpression in melanoma tumorigenesis. Clinicopathological analysis demonstrated that c-Myc expression positively correlated with the formation of vasculogenic mimicry (VM) and linearly patterned programmed cell necrosis (LPPCN). Clinically, high c-Myc expression was significantly associated with distant metastasis and poor prognosis, while biologically, c-Myc overexpression led to significant increases in cell motility, invasiveness and metastasis. Moreover, c-Myc induced the formation of VM and promoted the expression of epithelial-mesenchymal transition (EMT)-associated protein Snail both in vivo and in vitro. High expression of c-Myc increased Bax expression in hypoxic conditions and induced cell apoptosis. Taken together, we conclude that c-Myc overexpression promotes the formation of VM by EMT and LPPCN in melanoma. Our improved understanding of the clinical and biological effects of c-Myc overexpression in melanoma highlights the incomplete understanding of this oncogene, and indicates that c-Myc is a potential therapeutic target of this disease.High c-Myc expression is associated with tumor metastasis and poor prognosis in human melanoma. c-Myc upregulates Snail expression to promote EMT via the TGF-β/Snail/Ecadherin signal pathway. c-Myc leads to cell death by upregulating Bax expression causing a lower Bcl2/Bax ratio under severe hypoxic conditions. c-Myc promotes vasculogenic mimicry and linearly patterned programmed cell necrosis.

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.

Background Succinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study. Results In this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (ΔldhAΔptaΔpqoΔcatPsod-ppcPsod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose–xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L−1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L−1 h−1 during the anaerobic stage. Conclusion This work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.

Corynebacterium glutamicum is well-established for industrial and biotechnological applications. However, its genetic manipulation has generally lagged behind traditional genetic models. In this study, a counter-selectable marker gene upp was firstly confirmed to be more efficient than traditional sacB. Furthermore, a markerless gene replacement system was developed by combining upp with double-strand break repair caused by the exogenous endonuclease I-SceI. Finally, genetic modification using a dsDNA PCR fragment was carried out with the expression of recombinase/exonuclease RecE/RecT. Our results show that the genetic modification system allows precise and markerless gene replacement without altering the chromosome, with a simplified screening procedure to generate its modification.

The regulation of metabolic flux through glycolytic versus the gluconeogenic pathway plays an important role in central carbon metabolism. In this study, we made an attempt to enhance riboflavin production by deregulating gluconeogenesis in Bacillus subtilis. To this end, gapB (code for NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase), fbp (code for fructose-1,6-bisphosphatase) and pckA (code for phosphoenolpyruvate carboxykinase) were overexpressed in parental strain B. subtilis RH33. Compared with RH33, overexpression of fbp and gapB resulted in approximately 18.0 and 14.2 % increased riboflavin production, respectively, while overexpression of pckA obtained the opposite result. Significant enhancement of riboflavin titers up to 4.89 g/l was obtained in shake flask cultures when gapB and fbp were co-overexpressed, nevertheless the specific growth rate decreased slightly and the specific glucose uptake rate remained almost unchanged. An improvement by 21.9 and 27.8 % of the riboflavin production was achieved by co-overexpression of gapB and fbp in shake flask and fed-batch fermentation, respectively. These results imply that deregulation of gluconeogenesis is an effective strategy for production of metabolites directly stemming from the pentose phosphate pathway as well as other NADPH-demanding compounds with glucose as carbon source in B. subtilis.

A dual route for anaerobic succinate production was engineered into Corynebacterium glutamicum. The glyoxylate pathway was reconstructed by overexpressing isocitrate lyase, malate synthase and citrate synthase. The engineered strain produced succinate with a yield of 1.34 mol (mol glucose)(-1). Further overexpression of succinate exporter, SucE, increased succinate yield to 1.43 mol (mol glucose)(-1). Metabolic flux analysis revealed that the glyoxylate pathway was further activated by engineering succinate export system. Using an anaerobic fed-batch fermentation process, the final strain produced 926 mM succinate (= 109 g l(-1)) with an overall volumetric productivity of 9.4 mM h(-1) and an average yield of 1.32 mol (mol glucose)(-1).

A synthetic medium for ethanol production by recombinant xylose-fermenting Zymomonas mobilis was optimized using Plackett-Burman (PB) design and response surface methodology (RSM). The effects of 19 medium components were investigated by PB design. Eight of these components were determined to have significant effects on ethanol production. The statistical model was constructed via central composite design (CCD) using five selected variables, including xylose, trisodium citrate, choline chloride, pyridoxine, and thiamine. The validity of the developed model was verified. The ethanol concentration in the optimized medium was 6.7% higher than in the RM medium. The optimized medium was then simplified to give the final synthetic medium (S2), in which the ethanol concentration was 20.7% higher than in the RM medium. Xylose and trisodium citrate were the key medium components influencing ethanol production, while calcium pantothenate was the only growth factor required by recombinant Z. mobilis. Ethanol production and growth response were directly proportional to the logarithm of the concentration of trisodium citrate in the range of 0.1–1.6 g/L.

Evolutionary engineering is a novel wholegenome wide engineering strategy inspired by natural evolution for strain improvement. Astaxanthin has been widely used in cosmetics, pharmaceutical and health care food due to its capability of quenching active oxygen. Strain improvement of Phaffia rhodozyma, one of the main sources for natural astaxanthin, is of commercial interest for astaxanthin production. In this study a selection procedure was developed for adaptive evolution of P. rhodozyma strains under endogenetic selective pressure induced by additive in environmental niches. Six agents, which can induce active oxygen in cells, were added to the culture medium respectively to produce selective pressure in process of evolution. The initial strain, P. rhodozyma AS2-1557, was mutagenized to acquire the initial strain population, which was then cultivated for 550 h at selective pressure and the culture was transferred every 48h. Finally, six evolved strains were selected after 150 generations of evolution. The evolved strains produced up to 48.2% more astaxanthin than the initial strain. Our procedure may provide a promising alternative for improvement of highproduction strain.

Improvement of acid tolerance and production of D-lactic acid by Sporolactobacillus inulinus ATCC 15538 was performed by using recursive protoplast fusion in a genome shuffling format. The starting population was generated by ultraviolet irradiation, diethyl sulfate mutagenesis, and pH-gradient filter and then, subjected for the recursive protoplast fusion. The concentration of lysozyme, time, and temperature for enzyme treatment were optimized by response surface methodology based on the central composite design. Based on contour plots and variance analysis, the model predicted a maximum Y (multiply protoplasts formation ratio by protoplasts regeneration ratio), 60.4%, and the corresponding above used values were 7.75 mg/ml lysozyme, 1.59 h, and 38 degrees C. A pH-5-resistant recombinant, F3-4, was obtained after three rounds of genome shuffling and its production of D-lactic acid reached 93.4 g/l in a 5 L bioreactor, which was increased by 39.8% and 119% in comparison with that of UV generated strain and the original strain S. inulinus ATCC 15538, respectively. The subculture experiments indicated that F3-4 was genetically stable.

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett-Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO(3), K(2)HPO(4), ZnSO(4), and MnCl(2) were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.

In the post-genome era, it is one challenge to understand the cellular metabolism at the systematic levels. Mathematical modeling of microorganisms and subsequent computer simulation are effective tools for systems biology. In this paper, based on the genome-scale Escherichia coli stoichiometric model iJR904, through the GAMS linear programming package, the in silico maximal succinate yield was estimated to be 1.714 mol/mol glucose. When another two constraints were added, the maximal succinate yield dropped to 1.60 mol/mol glucose. Further analysis substantiated the uniqueness of the flux distribution under such constraints. After comparisons with the metabolic flux analysis (MFA) results computed from the wet experimental data of the three kinds of E. coli, three potential improvement target sites, the glucose phosphotransferase transport system, the pyruvate carboxylase, and the glyoxylate shunt, were identified and selected for the genetic modifications. All the three genetic modified strains showed increased succinate yield. The final strain TUQ19/pQZ6 had a high yield of 1.29 mol succinate/mol glucose and high productivity. The success of the above experiments proved that this in silico optimal succinate production pathway is reasonable and practical. This method may also be used as a general strategy to help enhance the yields of other favorable metabolites in E. coli.

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant KAM-3, the FPS1 gene, which encodes a channel protein responsible for glycerol export, was deleted. The mutant KAM-11 had the GLT1 gene (encoding glutamate synthase) placed under the PGK1 promoter while having the FPS1 deletion. Growth rate and biomass concentration remained virtually unchanged with the mutant KAM-11, compared to that of the parent. Over-expression of GLT1 by the PGK1 promoter along with FPS1 deletion resulted in a 14% higher ethanol production and a 30% lower glycerol formation compared to the parental strain under anaerobic fermentation conditions. Furthermore, acetate and pyruvic acid formation was also reduced in order for cells to maintain redox balance.

Background Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources. Results In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source. Conclusions Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

A fed-batch fermentation of Lactobacillus lactis to produce l-lactic acid was developed in which the residual glucose concentration in the culture was used to control a continuous feeding strategy. Up to 210 g l-lactic acid l−1 (97% yield) was obtained. The maximal dry cell was 2.7 g l−1 and the average l-lactic acid productivity was 2.2 g l−1 h−1.

In Escherichia coli, disruption of ptsG, which encodes the glucose-specific permease of the phosphotransferase transport system (PTS) protein EIICB(Glc), is crucial for high succinate production. This mutation can, however, cause very slow growth and low glucose consumption rates. The ptsG mutant (TUQ2), from wild type E. coli W1485, and E. coli galP (encoding galactose permease) and glk (encoding glucose kinase) gene expression plasmids were constructed. TUQ2 increased the generation time to approximately 4 h and gave a higher final cell density of 0.5 g/l by expression of galP. However, glk expression had no effect on the mutant. After expression of pyruvate carboxylase (PYC) and galactose permease, the ptsG mutant showed higher succinate yield (1.2 mol/mol glucose) and the specific rate of glucose consumption from 0.33 to 0.6 g/1 h.