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While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. In this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (

Rare-cold-inducible 2 (RCI2) genes are involved in plant response to abiotic stresses. In this study, we report the functional role of a Camelina RCI2, CsRCI2D, in plant salt stress response. The localization of CsRCI2D was observed in the plasma membrane and intracellular membranes by confocal analysis in tobacco leaf and western blot analysis in Camelina. The full length CsRCI2D cDNA clone was not able to complement the salt sensitivity of △spmp3 lacking the PMP3 gene. However, a C-terminal tail deleted CsRCI2D cDNA was able to restore the level of salt tolerance to that of WT. CsRCI2D-overexpressing Camelina showed better germination rate and seedling growth. CsRCI2D overexpression decreased Na+ accumulation in both roots and shoots but increased K+ accumulation in the shoots under salt stress. Furthermore, CsRCI2D-overexpressing Camelina displayed lower H2O2 and malondialdehyde (MDA) upon salt stress. Under normal growth condition, CsRCI2D-overexpressing Camelina showed higher transcript levels of all antioxidant genes (CsCuSODs, CsMnSOD1, CsFeSODs, CsCATs, CsAPX1, and CsGR), whereas salt stress significantly induced all antioxidant genes in WT. These results indicate that salt-upregulated CsRCI2D plays a positive role in Camelina seed germination and seedling growth under salt stress by maintenance of ion homeostasis and modulating the expression of antioxidant-related genes.

Although plant rare cold inducible 2 (RCI2), a homolog of yeast PMP3, has been considered to play important roles in responses to abiotic stress in plants, the functional role of RCI2 in salt-stress tolerance remains largely unknown for crop plants. In this study, we determined the function of CsRCI2H from Camelina under salt stress. Subcellular localization analysis of the yellow fluorescent protein (YFP)-CsRCI2H fusion protein revealed that CsRCI2H localizes to the plasma membrane in tobacco leaves. Expression of CsRCI2H was greatly increased in the presence of 150 mM NaCl. CsRCI2H compensated for the salt sensitivity of the yeast mutant ∆pmp3 lacking the PMP3 gene. CsRCI2H-overexpressing transgenic Camelina showed improved seed germination, root growth and fresh weight under high salt stress. CsRCI2H-overexpressing transgenic Camelina displayed lower accumulation of Na+ in the roots and shoots but higher K+ and Ca2+ accumulation in the shoots than wild type plants under salt stress. Furthermore, CsRCI2H-overexpressing Camelina displayed higher stomatal conductance and lower malondialdehyde under salt stress. Transcript levels of ion-homeostasis related genes, such as CsSOS1, CsSOS3, and CsHKT1, were significantly increased in the CsRCI2H-overexpressing transgenic plants upon salt treatment. These results suggest that Na+ and K+ homeostasis can be controlled by the Na+ and K+ transport systems throughout the CsRCI2H-overexpressing transgenic Camelina plant under salt-stress conditions. Collectively, these results indicate that CsRCI2H contributes to salt tolerance in Camelina during seed germination and seedling growth by reducing Na+ toxicity in the plant cells via regulation of ion homeostasis.

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizingPseudomonas putidaKT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA,gacS,oprB/PP_1446,fleQ,tktA, anduvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE,oprD, and PP_5324). We further validated the causality of the PP_5350 andemrEgenes for improved IL toleranceviareverse engineering and transcriptomic analysis. A common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that theemrEgene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bioprocesses.

Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, the analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. The inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine, inosine, or guanosine as the nitrogen source. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively., Rodionova et al. find that the putative transporter gene, ydhC and its regulator ydhB are involved in purine transportation and that the expression of the ydhC gene is activated by the YdhB in E. coli. The authors suggest renaming the regulator PunR and the transporter PunC, respectively.

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators that comprise a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed ChIP-exo assay to characterize genome-wide binding sites for these candidate TFs; 34 of them were found to be DNA-binding protein. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. These TFs were found to have various roles in regulating primary cellular processes in E. coli. Taken together, this study: (1) significantly expands the number of confirmed TFs, close to the estimated total of about 280 TFs; (2) predicts the putative functions of the newly discovered TFs, and (3) confirms the functions of representative TFs through mutant phenotypes.

Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, and biological production of 3-HP from glycerol as a carbon source using glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) has been revealed to be effective because it involves a relatively simple metabolic pathway and exhibits higher yield and productivity than other biosynthetic pathways. Despite the successful attempts of 3-HP production from glycerol, the biological process suffers from problems arising from low activity and inactivation of the two enzymes. To apply the directed evolutionary approach to engineer the 3-HP production system, we constructed a synthetic selection device using a 3-HP-responsive transcription factor and developed a selection approach for screening 3-HP-producing microorganisms. The method was applied to an ALDH library, specifically aldehyde-binding site library of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichment of strains showing increased 3-HP production, and an isolated KGSADH variant enzyme exhibited a 2.79-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. This approach will provide the simple and efficient tool to engineer the pathway enzymes in metabolic engineering.

Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of E. coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids.

Rare cold-inducible 2 (RCI2) proteins are small hydrophobic proteins that are known to be localized in cellular membranes. The function of RCI2 proteins has been reported to be associated with low-temperature, salt, and drought stress tolerances as a membrane potential regulator; however, the specific functions are still unknown. The PIP2 (plasma membrane intrinsic protein 2) aquaporins are proteins that transport water and small solutes into the cell. The expression and activity of PIP2 proteins, like RCI2, are also related to salt- and drought-stress tolerance. In this study, we identified novel protein interactions between RCI2 and PIP2; 1, including protein accumulation changes in the bioenergy crop Camelina sativa L. under various NaCl stress conditions. Accumulation of both CsRCI2E and CsRCI2F proteins increased with NaCl stress; however, to differing levels depending on the NaCl stress intensity. A co-immunoprecipitation test revealed interaction between CsRCI2E-CsPIP2 and CsRCI2F-CsPIP2. Moreover, co-expression of the four CsRCI2 proteins with CsPIP2; 1 in Xenopus laevis oocytes reduced water transport activity. Furthermore, the abundance of CsPIP2; 1 protein was decreased under CsRCI2E and CsRCI2F co-expression. These results suggest that NaCl-induced expression of CsRCI2E and CsRCI2F contributes to the regulation of CsPIP2; 1.

Summary: The production of coenzyme B12 using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B12 in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B12 using a previously engineered coenzyme B12-producing E. coli strain. To select a high producer, the coenzyme B12-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B12-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B12 validated the coenzyme B12-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B12 production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B12-producing strains. : Biological Sciences; Bioengineering; Metabolic Engineering Subject Areas: Biological Sciences, Bioengineering, Metabolic Engineering

Acetate, which is an abundant carbon source, is a potential feedstock for microbial processes that produce diverse value-added chemicals. In this study, we produced 3-hydroxypropionic acid (3-HP) from acetate with engineered Escherichia coli. For the efficient conversion of acetate to 3-HP, we initially introduced heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus. Then, the acetate assimilating pathway and glyoxylate shunt pathway were activated by overexpressing acs (encoding acetyl-CoA synthetase) and deleting iclR (encoding the glyoxylate shunt pathway repressor). Because a key precursor malonyl-CoA is also consumed for fatty acid synthesis, we decreased carbon flux to fatty acid synthesis by adding cerulenin. Subsequently, we found that inhibiting fatty acid synthesis dramatically improved 3-HP production (3.00 g/L of 3-HP from 8.98 g/L of acetate). The results indicated that acetate can be used as a promising carbon source for microbial processes and that 3-HP can be produced from acetate with a high yield (44.6% of the theoretical maximum yield).

Microbial production of 5-aminolevulinic acid (ALA) has received much attention because of its potential in clinical applications. Overexpression along with the deciphering of regulation of the related enzymes and an analogue transporter yielded remarkable achievements in ALA production. Nonetheless, there is significant room for carbon flux optimization to enhance ALA production. The aim of this study was precise carbon flux optimization for high ALA production in Escherichia coli expressing the ALA biosynthetic pathway. Initially, genes hemA and hemL were overexpressed with strong promoters and synthetic 5'-untranslated regions (5'-UTRs). Then, the tricarboxylic acid (TCA) cycle was blocked to force carbon flux toward the ALA production pathway by deletion of sucA. Although the resulting strain showed a severe metabolic imbalance and low ALA production, further precise tuning of carbon flux to the glyoxylate cycle by varying the transcriptional strength of aceA led to substantially improved cell growth and ALA production. Thus, this precise tuning of the glyoxylate cycle in a quantitative manner should also enable efficient production of other value-added products derived from the TCA cycle.

School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790-784, Republic Korea(2014년 7월 2일 접수, 2014년 7월 8일 채택)AbstractMacroalgae has been strongly touted as an alternative biomass for biofuel production due to its higher photosynthetic efficiency, carbon fixation rate, and growth rate compared to conventional cellulosic plants. However, its unique carbohydrate composition and structure limits the utilization efficiency by conventional microorganisms, resulting in reduced growth rates and lower productivity. Nevertheless, recent studies have shown that it is possible to enable micro-organisms to utilize various sugars from seaweeds and to produce some energy chemicals such as methane, ethanol, etc. This paper introduces the basic information on macroalgae and the overall conversion process from harvest to production of biofuels. Especially, we will review the successful efforts on microbial engineering through metabolic engineering and synthetic biology to utilize carbon sources from red and brown seaweed. Keywords: Macroalgae, Seaweed, Biofuel, Metabolic engineering, Synthetic biology

Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of Escherichia coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids.

Production of biochemicals by industrial fermentation using microorganisms requires maintaining cellular production capacity, because maximal productivity is economically important. High-productivity microbial strains can be developed using static engineering, but these may not maintain maximal productivity throughout the culture period as culture conditions and cell states change dynamically. Additionally, economic reasons limit heterologous protein expression using inducible promoters to prevent metabolic burden for commodity chemical and biofuel production. Recently, synthetic and systems biology has been used to design genetic circuits, precisely controlling gene expression or influencing genetic behavior toward a desired phenotype. Development of dynamic regulators can maintain cellular phenotype in a maximum production state in response to factors including cell concentration, oxygen, temperature, pH, and metabolites. Herein, we introduce dynamic regulators of industrial microorganism optimization and discuss metabolic flux fine control by dynamic regulators in response to metabolites or extracellular stimuli, robust production systems, and auto-induction systems using quorum sensing.

3-Hydroxypropionic acid (3-HP) can be biologically produced from glycerol by two consecutive enzymatic reactions, dehydration of glycerol to 3-hydroxypropionaldehyde (3-HPA) and oxidation of 3-HPA. The pathway has been proved efficient, but imbalance between the rates of the two enzymatic reactions often results in the accumulation of the toxic 3-HPA, which severely reduces cell viability and 3-HP production. In this study, we used UTR engineering to maximally increase the activities of glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) for the high conversion of glycerol to 3-HP. Thereafter, the activity of GDHt was precisely controlled to avoid the accumulation of 3-HPA by varying expression of dhaB1, a gene encoding a main subunit of GDHt. The optimally balanced E. coli HGL_DBK4 showed a substantially enhanced 3-HP titer and productivity compared with the parental strain. The yield on glycerol, 0.97 g 3-HP/g glycerol, in a fed-batch experiment, was the highest ever reported.

In this study, the Leloir pathway of galactose metabolism was rebuilt in Escherichia coli to remove CCR and amplify galactose utilization rate. All genes encoding pathway enzymes were expressed under the control of a synthetic module that included promoters, 5'-untranslated regions, and terminators as a re-organized single operon in the chromosome. The engineered strain showed both an enhanced galactose utilization rate and the capacity to simultaneously assimilate galactose and glucose. This work demonstrates the feasibility of using synthetic biology tools to re-build biological systems for engineering purpose.