Small regulatory RNAs (sRNAs) regulate gene expression in bacteria. We designed synthetic sRNAs to identify and modulate the expression of target genes for metabolic engineering in Escherichia coli. Using synthetic sRNAs for the combinatorial knockdown of four candidate genes in 14 different strains, we isolated an engineered E. coli strain (tyrR- and csrA-repressed S17-1) capable of producing 2 g per liter of tyrosine. Using a library of 130 synthetic sRNAs, we also identified chromosomal gene targets that enabled substantial increases in cadaverine production. Repression of murE led to a 55% increase in cadaverine production compared to the reported engineered strain (XQ56 harboring the plasmid p15CadA). The design principles and the engineering strategy using synthetic sRNAs reported here are generalizable to other bacteria and applicable in developing superior producer strains. The ability to fine-tune target genes with designed sRNAs provides substantial advantages over gene-knockout strategies and other large-scale target identification strategies owing to its easy implementation, ability to modulate chromosomal gene expression without modifying those genes and because it does not require construction of strain libraries.

Butanol is an aliphatic saturated alcohol having the molecular formula of C 4 H 9 OH. Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications. Moreover, butanol has been considered as a potential fuel or fuel additive. Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century. However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes. Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production. This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes. The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed.

The L-valine production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and gene knockout simulation of the in silico genome-scale metabolic network. Feedback inhibition of acetohydroxy acid synthase isoenzyme III by L-valine was removed by site-directed mutagenesis, and the native promoter containing the transcriptional attenuator leader regions of the ilvGMEDA and ilvBN operon was replaced with the tac promoter. The ilvA, leuA, and panB genes were deleted to make more precursors available for L-valine biosynthesis. This engineered Val strain harboring a plasmid overexpressing the ilvBN genes produced 1.31 g/liter L-valine. Comparative transcriptome profiling was performed during batch fermentation of the engineered and control strains. Among the down-regulated genes, the lrp and ygaZH genes, which encode a global regulator Lrp and L-valine exporter, respectively, were overexpressed. Amplification of the lrp, ygaZH, and lrp-ygaZH genes led to the enhanced production of L-valine by 21.6%, 47.1%, and 113%, respectively. Further improvement was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and pfkA genes as knockout targets. The VAMF strain (Val ⌬aceF ⌬mdh ⌬pfkA) overexpressing the ilvBN, ilvCED, ygaZH, and lrp genes was able to produce 7.55 g/liter L-valine from 20 g/liter glucose in batch culture, resulting in a high yield of 0.378 g of L-valine per gram of glucose. These results suggest that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification, transcriptome profiling, and systems-level in silico analysis.systems biology ͉ global regulator ͉ exporter ͉ in silico prediction M ost amino acid-producing bacterial strains have been constructed by random mutagenesis. A significant disadvantage of this approach is the possibility that the random distribution of mutations in regions not directly related to amino acid biosynthesis can cause unwanted changes in physiology and growth retardation. Rational metabolic engineering by specific targeted modifications can overcome this disadvantage. The recent development of omics technology, combined with computational analysis, now provides a new avenue for strain improvement (1-4) by providing new information extracted from a large number of data, which is termed ''systems biotechnology'' (5).L-valine, an essential hydrophobic and branched-chain amino acid, is used as a component of cosmetics and pharmaceuticals as well as animal feed additives. L-valine has been produced by employing bacteria belonging to the genera Brevibacterium, Corynebacterium, and Serratia, which have been improved by random mutation and selection (6, 7). A recent report describes the production of L-valine by rationally constructed Corynebacterium glutamicum, in which a feedback inhibition-resistant small subunit of acetohydroxy acid synthase (AHAS; encoded by ilvN) was generated by site-directed mutagen...

Amino-acid producers have traditionally been developed by repeated random mutagenesis owing to the difficulty in rationally engineering the complex and highly regulated metabolic network. Here, we report the development of the genetically defined L-threonine overproducing Escherichia coli strain by systems metabolic engineering. Feedback inhibitions of aspartokinase I and III (encoded by thrA and lysC, respectively) and transcriptional attenuation regulations (located in thrL) were removed. Pathways for Thr degradation were removed by deleting tdh and mutating ilvA. The metA and lysA genes were deleted to make more precursors available for Thr biosynthesis. Further target genes to be engineered were identified by transcriptome profiling combined with in silico flux response analysis, and their expression levels were manipulated accordingly. The final engineered E. coli strain was able to produce Thr with a high yield of 0.393 g per gram of glucose, and 82.4 g/l Thr by fed-batch culture. The systems metabolic engineering strategy reported here may be broadly employed for developing genetically defined organisms for the efficient production of various bioproducts.

Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1D485G gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer.

BackgroundEscherichia coli is a model prokaryote, an important pathogen, and a key organism for industrial biotechnology. E. coli W (ATCC 9637), one of four strains designated as safe for laboratory purposes, has not been sequenced. E. coli W is a fast-growing strain and is the only safe strain that can utilize sucrose as a carbon source. Lifecycle analysis has demonstrated that sucrose from sugarcane is a preferred carbon source for industrial bioprocesses.ResultsWe have sequenced and annotated the genome of E. coli W. The chromosome is 4,900,968 bp and encodes 4,764 ORFs. Two plasmids, pRK1 (102,536 bp) and pRK2 (5,360 bp), are also present. W has unique features relative to other sequenced laboratory strains (K-12, B and Crooks): it has a larger genome and belongs to phylogroup B1 rather than A. W also grows on a much broader range of carbon sources than does K-12. A genome-scale reconstruction was developed and validated in order to interrogate metabolic properties.ConclusionsThe genome of W is more similar to commensal and pathogenic B1 strains than phylogroup A strains, and therefore has greater utility for comparative analyses with these strains. W should therefore be the strain of choice, or 'type strain' for group B1 comparative analyses. The genome annotation and tools created here are expected to allow further utilization and development of E. coli W as an industrial organism for sucrose-based bioprocesses. Refinements in our E. coli metabolic reconstruction allow it to more accurately define E. coli metabolism relative to previous models.

The second messenger nucleotide cyclic dimeric guanosine monophosphate (c-di-GMP) governs many cellular processes in the facultative human pathogen Vibrio cholerae. This organism copes with changing environmental conditions in aquatic environments and during transitions to and from human hosts. Modulation of c-di-GMP allows V. cholerae to shift between motile and sessile stages of life, thus allowing adaptation to stressors and environmental conditions during its transmission cycle. The V. cholerae genome encodes a large set of proteins predicted to degrade and produce c-di-GMP. A subset of these enzymes has been demonstrated to control cellular processes – particularly motility, biofilm formation, and virulence – through transcriptional, post-transcriptional, and translational mechanisms. Recent studies have identified and characterized enzymes that modulate or sense c-di-GMP levels and have lead towards mechanistic understanding of c-di-GMP regulatory circuits in V. cholerae.

Anion doping is one of the most widely adopted strategies to improve the electrochemical performance of cathode materials for Li-ion batteries. However, undesirable side effects are often observed together with enhanced electrochemical properties, leading to an unsatisfactory overall performance. In order to develop an anion doping strategy which enhances the positive effects and suppresses undesirable side effects, the understanding of their origin at the atomic scale is a crucial step. In this work, using density functional theory (DFT), we report a systematic study on the effects of three common anion dopants (F, S, Cl) on a wide range of properties of a model cathode material, LiNiO2, including redox potential, ionic conductivity, Li/Ni exchange, lattice distortion, and Ni migration upon delithiation. The results show that the dopants improve certain properties but worsen others, revealing some distance-dependent features. Overall, our work shows conflicting roles of anion doping on the battery voltage, rate performance, and structural stability of the cathode material. By identifying the origins of the different roles, we propose a rational anion doping strategy for the optimization of the overall electrochemical performance of the cathode material. These results for LiNiO2 can also promote anion doping studies and improved materials design in other Ni-rich layered oxide cathode materials.