Ppc Gene Research Articles

Genome-Scale Metabolic Modeling of Halomonas elongata 153B Explains Polyhydroxyalkanoate and Ectoine Biosynthesis in Hypersaline Environments.

Halomonas elongata thrives in hypersaline environments producing polyhydroxyalkanoates (PHAs) and osmoprotectants such as ectoine. Despite its biotechnological importance, several aspects of the dynamics of its metabolism remain elusive. Here, we construct and validate a genome-scale metabolic network model for H. elongata 153B. Then, we investigate the flux distribution dynamics during optimal growth, ectoine, and PHA biosynthesis using statistical methods, and a pipeline based on shadow prices. Lastly, we use optimization algorithms to uncover novel engineering targets to increase PHA production. The resulting model (iEB1239) includes 1534 metabolites, 2314 reactions, and 1239 genes. iEB1239 can reproduce growth on several carbon sources and predict growth on previously unreported ones. It also reproduces biochemical phenotypes related to Oad and Ppc gene functions in ectoine biosynthesis. A flux distribution analysis during optimal ectoine and PHA biosynthesis shows decreased energy production through oxidative phosphorylation. Furthermore, our analysis unveils a diverse spectrum of metabolic alterations that extend beyond mere flux changes to encompass heightened precursor production for ectoine and PHA synthesis. Crucially, these findings capture other metabolic changes linked to adaptation in hypersaline environments. Bottlenecks in the glycolysis and fatty acid metabolism pathways are identified, in addition to PhaC, which has been shown to increase PHA production when overexpressed. Overall, our pipeline demonstrates the potential of genome-scale metabolic models in combination with statistical approaches to obtain insights into the metabolism of H. elongata. Our platform can be exploited for researching environmental adaptation, and for designing and optimizing metabolic engineering strategies for bioproduct synthesis.

Bioelectricity facilitates carbon dioxide fixation by Alcaligenes faecalis ZS-1 in a biocathodic microbial fuel cell (MFC)

The CO2 fixation mechanism by Alcaligenes faecalis ZS-1 in a biocathode microbial fuel cell (MFC) was investigated. The closed-circuit MFC (CM) exhibited a significantly higher CO2 fixation rate (10.7%) compared to the open-circuit MFC (OC) (2.0%), indicating that bioelectricity enhances CO2 capture efficiency. During the inward extracellular electron transfer (EET) process, riboflavin concentration increased in the supernatant while cytochrome levels decreased. Genome sequencing revealed diverse metabolic pathways for CO2 fixation in strain ZS-1, with potential dominance of rTCA and C4 pathways under electrotrophic conditions as evidenced by significant upregulation of the ppc gene. Differential metabolite analysis using LC-MS demonstrated that CM promoted upregulation of various lipid metabolites. These findings collectively highlight that ZS-1 simultaneously generated electricity and fixed CO2 and that the ppc associated with bioelectricity played a critical role in CO2 capture. In conclusion, bioelectricity resulted in a significant enhancement in the efficiency of CO2 fixation and lipid production.

The Construction of the Self-Induced Sal System and Its Application in Salicylic Acid Production

The design and construction of more complex and delicate genetic control circuits suffer from poor orthogonality in quorum sensing (QS) systems. The Sal system, which relies on salicylic acid as a signaling molecule, is an artificially engineered regulatory system with a structure that differs significantly from that of natural QS signaling molecules. Salicylic acid is an important drug precursor, mainly used in the production of drugs such as aspirin and anti-HIV drugs. However, there have been no reports on the construction of a self-induced Sal system in single cells. In this study, a high-copy plasmid backbone was used to construct the regulatory proteins and a self-induced promoter of salicylic acid in E. coli by adjusting the precise regulation of key gene expression; the sensitivity and induction range of this system were improved. Subsequently, the exogenous gene pchBA was introduced in E. coli to extend the shikimate pathway and synthesize salicylic acid, resulting in the construction of the first complete self-induced Sal system. Finally, the self-induced Sal System was combined with artificial trans-encoded sRNAs (atsRNAs) to repress the growth-essential gene ppc and accumulate the precursor substance PEP, thereby increasing the titer of salicylic acid by 151%. This construction of a self-induced artificial system introduces a new tool for selecting communication tools and induction systems in synthetic biology and metabolic engineering, but also demonstrates a self-inducible pathway design strategy for salicylic acid biosynthesis.

Metabolic engineering of fast-growing Vibrio natriegens for efficient pyruvate production

BackgroundPyruvate is a widely used value-added chemical which also serves as a hub of various metabolic pathways. The fastest-growing bacterium Vibrio natriegens is a promising chassis for synthetic biology applications with high substrate uptake rates. The aim of this study was to investigate if the high substrate uptake rates of V. natriegens enable pyruvate production at high productivities.ResultsTwo prophage gene clusters and several essential genes for the biosynthesis of byproducts were first deleted. In order to promote pyruvate accumulation, the key gene aceE encoding pyruvate dehydrogenase complex E1 component was down-regulated to reduce the carbon flux into the tricarboxylic acid cycle. Afterwards, the expression of ppc gene encoding phosphoenolpyruvate carboxylase was fine-tuned to balance the cell growth and pyruvate synthesis. The resulting strain PYR32 was able to produce 54.22 g/L pyruvate from glucose within 16 h, with a yield of 1.17 mol/mol and an average productivity of 3.39 g/L/h. In addition, this strain was also able to efficiently convert sucrose or gluconate into pyruvate at high titers.ConclusionA novel strain of V. natriegens was engineered which was capable to provide higher productivity in pyruvate synthesis. This study lays the foundation for the biosynthesis of pyruvate and its derivatives in fast-growing V. natriegens.

Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase isoenzymes play an important role in the filling and quality of Arabidopsis thaliana seed

Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds. Ppc mutants exhibited moderately lower seed parameters (weight, area, yield, germination kinetics) than wild type. In contrast, ppck1-had much altered (decreased) yield. At the molecular level, ppc3-was found to be significantly deficient in global seed nitrogen (nitrate, amino-acids, and soluble protein pools). Also, N-deficiency was much more marked in ppck1-, which exhibited a tremendous loss of 95% and 90% in nitrate and proteins, respectively. The line ppck2-had accumulated amino-acids but lower levels of soluble proteins. Regarding carboxylic acid pools, Krebs cycle intermediates were found to be diminished in all mutants; this was accompanied by a consistent decrease in ATP. Lipids were stable in ppc mutants, however ppck1-seeds accumulated more lipids while ppck2-seeds showed high level of polyunsaturated fatty acid oleic and linolenic (omega 3). Altogether, the results indicate that the complete PEPC and PPCK family are needed for normal C/N metabolism ratio, growth, development, yield and quality of the seed.

A well-supported nuclear phylogeny of Poaceae and implications for the evolution of C4 photosynthesis

Poaceae (the grasses) includes rice, maize, wheat, and other crops, and is the most economically important angiosperm family. Poaceae is also one of the largest plant families, consisting of over 11 000 species with a global distribution that contributes to diverse ecosystems. Poaceae species are classified into 12 subfamilies, with generally strong phylogenetic support for their monophyly. However, many relationships within subfamilies, among tribes and/or subtribes, remain uncertain. To better resolve the Poaceae phylogeny, we generated 342 transcriptomic and seven genomic datasets; these were combined with other genomic and transcriptomic datasets to provide sequences for 357 Poaceae species in 231 genera, representing 45 tribes and all 12 subfamilies. Over 1200 low-copy nuclear genes were retrieved from these datasets, with several subsets obtained using additional criteria, and used for coalescent analyses to reconstruct a Poaceae phylogeny. Our results strongly support the monophyly of 11 subfamilies; however, the subfamily Puelioideae was separated into two non-sister clades, one for each of the two previously defined tribes, supporting a hypothesis that places each tribe in a separate subfamily. Molecular clock analyses estimated the crown age of Poaceae to be ∼101 million years old. Ancestral character reconstruction of C3/C4 photosynthesis supports the hypothesis of multiple independent origins of C4 photosynthesis. These origins are further supported by phylogenetic analysis of the ppc gene family that encodes the phosphoenolpyruvate carboxylase, which suggests that members of three paralogous subclades (ppc-aL1a, ppc-aL1b, and ppc-B2) were recruited as functional C4ppc genes. This study provides valuable resources and a robust phylogenetic framework for evolutionary analyses of the grass family.

Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli.

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.

The Physiological Responses of Escherichia coli Triggered by Phosphoribulokinase (PrkA) and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco).

Phosphoribulokinase (PrkA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to create a heterologous Rubisco-based engineered pathway in Escherichia coli for in situ CO2 recycling. While the feasibility of a Rubisco-based engineered pathway has been shown, heterologous expressions of PrkA and Rubisco also induced physiological responses in E. coli that may compete with CO2 recycling. In this study, the metabolic shifts caused by PrkA and Rubisco were investigated in recombinant strains where ppc and pta genes (encodes phosphoenolpyruvate carboxylase and phosphate acetyltransferase, respectively) were deleted from E. coli MZLF (E. coli BL21(DE3) Δzwf, ΔldhA, Δfrd). It has been shown that the demand for ATP created by the expression of PrkA significantly enhanced the glucose consumptions of E. coli CC (MZLF Δppc) and E. coli CA (MZLF Δppc, Δpta). The accompanying metabolic shift is suggested to be the mgsA route (the methylglyoxal pathway) which results in the lactate production for reaching the redox balance. The overexpression of Rubisco not only enhanced glucose consumption but also bacterial growth. Instead of the mgsA route, the overproduction of the reducing power was balanced by the ethanol production. It is suggested that Rubisco induces a high demand for acetyl-CoA which is subsequently used by the glyoxylate shunt. Therefore, Rubisco can enhance bacterial growth. This study suggests that responses induced by the expression of PrkA and Rubisco will reach a new energy balance profile inside the cell. The new profile results in a new distribution of the carbon flow and thus carbons cannot be majorly directed to the Rubisco-based engineered pathway.

Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV)

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength Pporin-194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (ΔsdhEΔicl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (ΔsdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3–18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (ΔsdhE, G7::Pporin-ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mol% 3HV in PHBV when grown in glucose and gluconate. 25mol% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0∼25mol% 3HV) in a convenient way with reduced production complexity and cost.

Betaine supplementation improved l-threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression

Abstract Background The supplementation of betaine, an osmoprotective compatible solute, in the cultivation media has been widely used to protect bacterial cells. To explore the effects of betaine addition on industrial fermentation, Escherichia coli THRD, an l -threonine producer, was used to examine the production of l -threonine with betaine supplementation and the underlying mechanism through which betaine functions was investigated. Results Betaine supplementation in the medium of E. coli THRD significantly improved l -threonine fermentation parameters. The transcription of zwf and corresponding enzyme activity of glucose-6-phosphate dehydrogenase were significantly promoted by betaine addition, which contributed to an enhanced expression of zwf that provided more nicotinamide adenine dinucleotide phosphate (NADPH) for l -threonine synthesis. In addition, as a result of the betaine addition, the betaine-stimulated expression of enhanced green fluorescent protein (eGFP) under the zwf promoter within a plasmid-based cassette proved to be a transcription-level response of zwf. Finally, the promoter of the phosphoenolpyruvate carboxylase gene ppc in THRD was replaced with that of zwf, while l -threonine fermentation of the new strain was promoted by betaine addition. Conclusions We reveal a novel mode of betaine that facilitates the microbial production of useful compounds. Betaine supplementation upregulates the expression of zwf and increases the NADPH synthesis, which may be beneficial for the cell growth and thereby promote the production of l -threonine. This finding might be useful for the production of NADPH-dependent amino acids and derivatives in E. coli THRD or other E. coli strains. How to cite: Li Y, Zhang D, Cai N, et al. Betaine supplementation improved l -threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression. Electron J Biotechnol 2019;39. https://doi.org/10.1016/j.ejbt.2019.03.004 .

EngineeringCorynebacterium crenatumfor enhancing succinic acid production

Succinic acid is valuable due to its historical use in food industry. In this paper, Corynebacterium crenatum was engineered to enhance succinic acid production and the fermentation properties of engineered strains were investigated. The lactate dehydrogenase gene (ldhA) inactivation mutant was first constructed, and then the pyruvate carboxylase gene (pyc) or phosphoenolpyruvate carboxylase gene (ppc) was expressed in the ldhA mutant. The ldhA inactivation resulted in a 52% decrease on glucose consumption, and the succinic acid yield produced by the ldhA mutant was 0.62 g/g, representing a 2-fold increase compared with the yield obtained from the original strain. Overexpression of pyc gene could improve the glucose consumption rate, resulting in a 37% increase compared with the ldhA mutant strain. Meanwhile, the final succinic acid concentration represented a 50% increase than the ldhA mutant strain, and a 106% increase than the original strain. Overexpression of ppc gene had no significant effect. Practical applications The biobased succinic acid and the derivative can be used as food additives. C. crenatum is used for producing amino acid for many years, and it was found that succinic acid and lactic acid are the major products when fermented under anaerobic conditions. This study suggests that C. crenatum can be utilized as a potential cell factory for producing succinic acid after genetic modification. The results obtained in this paper could contribute to the depth development of C. crenatum for producing succinic acid or other products in food industry, not only for amino acid production.

Enzymes involved in the anaerobic degradation of phenol by the sulfate-reducing bacterium Desulfatiglans anilini

BackgroundThe sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet.ResultsThe pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsβ (phenylphosphate synthase beta subunit), ppcβ (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors.ConclusionsThe genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

Rational modification of tricarboxylic acid cycle for improving l-lysine production in Corynebacterium glutamicum

BackgroundOxaloacetate (OAA) and l-glutamate are essential precursors for the biosynthesis of l-lysine. Reasonable control of all potentially rate-limiting steps, including the precursors supply rate, is of vital importance to maximize the efficiency of l-lysine fermentation process.ResultsIn this paper, we have rationally engineered the tricarboxylic acid (TCA) cycle that increased the carbon yield (from 36.18 to 59.65%), final titer (from 14.47 ± 0.41 to 23.86 ± 2.16 g L−1) and productivity (from 0.30 to 0.50 g L−1 h−1) of l-lysine by Corynebacterium glutamicum in shake-flask fermentation because of improving the OAA and l-glutamate availability. To do this, the phosphoenolpyruvate–pyruvate–oxaloacetate (PEP–pyruvate–OAA) node’s genes ppc and pyc were inserted in the genes pck and odx loci, the P1 promoter of the TCA cycle’s gene gltA was deleted, and the nature promoter of glutamate dehydrogenase-coding gene gdh was replaced by Ptac-M promoter that resulted in the final engineered strain C. glutamicum JL-69Ptac-Mgdh. Furthermore, the suitable addition of biotin accelerates the l-lysine production in strain JL-69Ptac-Mgdh because it elastically adjusts the carbon flux for cell growth and precursor supply. The final strain JL-69Ptac-Mgdh could produce 181.5 ± 11.74 g L−1 of l-lysine with a productivity of 3.78 g L−1 h−1 and maximal specific production rate (qLys, max.) of 0.73 ± 0.16 g g−1 h−1 in fed-batch culture during adding 2.4 mg L−1 biotin with four times.ConclusionsOur results reveal that sufficient biomass, OAA and l-glutamate are equally important in the development of l-lysine high-yielding strain, and it is the first time to verify that fed-batch biotin plays a positive role in improving l-lysine production.

Overexpression of ppc or deletion of mdh for improving production of γ-aminobutyric acid in recombinant Corynebacterium glutamicum.

L-Glutamate decarboxylase (GAD) transforms L-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced L-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, L-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72h, GABA production increased to 26.3 ± 3.4, 24.8 ± 0.7, and 25.5 ± 3.3g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7 ± 0.5g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0 ± 0.6g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.

Enzymatic activity, gene expression and posttranslational modifications of photosynthetic and non-photosynthetic phosphoenolpyruvate carboxylase in ammonium-stressed sorghum plants

Sorghum plants grown with 5mM (NH4)2SO4 showed symptoms of stress, such as reduced growth and photosynthesis, leaf chlorosis, and reddish roots. Phosphoenolpyruvate carboxylase (PEPC) activity, by supplying carbon skeletons for ammonium assimilation, plays a pivotal role in tolerance to ammonium stress. This work investigated the effect of ammonium nutrition on PPC and PPCK gene expression, on PEPC activity, and on post-translational modifications (PTMs) of PEPC in leaves and roots of sorghum plants. Ammonium increased PEPC kinase (PEPCk) activity and the phosphorylation state of PEPC in leaves, both in light and in the dark, due to increased PPCK1 expression in leaves. This result resembled the effect of salinity on sorghum leaf PEPC and PEPCk, which is thought to allow a better functioning of PEPC in conditions that limit the income of reduced C. In roots, ammonium increased PEPC activity and the amount of monoubiquitinated PEPC. The first effect was related to increased PPC3 expression in roots. These results highlight the relevance of this specific isoenzyme (PPC3) in sorghum responses to ammonium stress. Although the role of monoubiquitination is not fully understood, it also increased in germinating seeds along with massive mobilization of reserves, a process in which the anaplerotic function of PEPC is of major importance.

Construction of pyruvate producing strain with intact pyruvate dehydrogenase and genome-wide transcription analysis.

To obtain strain YP211 with a high tendency for accumulating pyruvate, central metabolic pathways were modified in Escherichia coli MG1655. Specifically, seven genes (ldhA, pflB, pta-ackA, poxB, ppc, frdBC) were knocked out sequentially and full pyruvate dehydrogenase was retained. In batch fermentation with M9 medium, pyruvate yield and production rate reached 0.63g/g glucose and 1.89g/(1h), respectively. Meanwhile, the production of acetate, succinate, and other carboxylates was effectively controlled. To understand the physiological observations, we further completed genome-wide transcription analysis of wild-type and YP211. As the acetic acid pathways were blocked, the pathways of convertion of pyruvate to phosphoenol pyruvate and acetyl CoA were enhanced. The transcription of pck, as an alternative gene for ppc, was increased by 2.6 times. So even if gene ppc was inactivated, the tricarboxylic acid pathway was still enhanced in YP211. In order to balance intracellular NADH/NAD+, oxidative phosphorylation and flagellar assembly system were also up-regulated significantly. Biochemical pathways involved in pyruvate accumulation in YP211 (a). Transcriptional differences of genes related to pyruvate metabolism between strain YP211 and E. coli wild-type (b).

Metabolic engineering of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid.

5-Aminolevulinic acid (5-ALA) has recently attracted attention for its potential applications in the fields of medicine and agriculture. In this study, Corynebacterium glutamicum was firstly engineered for 5-ALA production via the C4 pathway. HemA encoding 5-aminolevulinic acid synthase from Rhodobacter sphaeroides was codon optimized and expressed in C. glutamicum ATCC13032, resulting in accumulation of 5-ALA. Deletion of all known genes responsible for the formation of acetate and lactate further enhanced production of 5-ALA. Overexpression of ppc gene encoding phoenolpyruvate carboxylase resulted in an accumulation of 5-ALA up to 2.06 ± 0.05 g/L. Furthermore, deletion of high-molecular-weight penicillin-binding proteins (HMW-PBPs) genes pbp1a, pbp1b, and pbp2b led to an increase in 5-ALA production of 13.53%, 29.47%, and 22.22%, respectively. Finally, 5-ALA production was enhanced to 3.14 ± 0.02 g/L in shake flask by heterologously expressing rhtA encoding threonine/homoserine exporter, and 86.77% of supplemented glycine was channeled toward 5-ALA production in shake flask. The engineered C. glutamicum ALA7 strain produced 7.53 g/L 5-ALA in a 5 L bioreactor. This study demonstrated the potential utility of C. glutamicum as a platform for metabolic production of 5-ALA. Change of cell permeability by metabolic engineering HMW-PBPs may provide a new strategy for biochemicals production in Corynebacterium glutamicum. Biotechnol. Bioeng. 2016;113: 1284-1293. © 2015 Wiley Periodicals, Inc.

Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid

A novel metabolic pathway was designed for the production of 3-aminopropionic acid (3-AP), an important platform chemical for manufacturing acrylamide and acrylonitrile. Using a fumaric acid producing Escherichia coli strain as a host, the Corynebacterium glutamicum panD gene (encoding l-aspartate-α-decarboxylase) was overexpressed and the native promoter of the aspA gene was replaced with the strong trc promoter, which allowed aspartic acid production through the aspartase-catalyzed reaction. Additional overexpression of aspA and ppc genes, and supplementation of ammonium sulfate in the medium allowed production of 3.49g/L 3-AP. The 3-AP titer was further increased to 3.94g/L by optimizing the expression level of PPC using synthetic promoters and RBS sequences. Finally, native promoter of the acs gene was replaced with strong trc promoter to reduce acetic acid accumulation. Fed-batch culture of the final strain allowed production of 32.3g/L 3-AP in 39h.

Transformation of Barnyardgrass ( Echinochloa crusgalli ) Root Type Phosphoenolpyruvate Carboxylase Gene into Rice ( Oryza sativa ) Plants and Their Effects on Photosynthesitic Gas Exchange

Barnyardgrass(Echinochloa crusgalli) is a C4 weed commonly found in rice field. To fully utilize the photosynthestic potential of Barnyardgrass C4 gene, we transformed Barnyardgrass root Phosphoenolpyruvate Carboxylase gene into rice plant with vectors contained promoters of Ubiqitin gene and Rubisco small unit gene by Agrobactirium- mediated transformation. Both marker genes Hygr and ppc were detected by PCR in regenerated plants. RT-PCR and Western blot analysis confirmed that the ppc gene was incorporated into rice plant and expressed with stable transcripts and proteins. PEPC activity as measured in most of the transgenic rice plants was higher than that in control, being up to 5.85-fold of that in untransformed rice. At T0 generation, net photosynthetic rate(Pn) in most of transgenic rice plants was 20.00% higher than that in untransformed rice, with the highest increase of 47.16%. Water utilization efficiency(WUE) in transgenic rice was also improved. At T6 generation, PEPC activity and Pn of transgenic lines remained higher than those of the wild type. These indicate that over-expressing C3 Eppc gene also can improve rice photosynthesis.

Engineering Escherichia coli for fumaric acid production from glycerol

The evolved mutant Escherichia coli E2 previously developed for succinate production from glycerol was engineered in this study for fumaric acid production under aerobic conditions. Through deletion of three fumarases, 3.65g/L fumaric acid was produced with the yield of 0.25mol/mol glycerol and a large amount of acetate was accumulated as the main byproduct. In order to reduce acetate production several strategies were attempted, among which increasing the flux of the anaplerotic pathways through overexpression of phosphoenolpyruvate carboxylase gene ppc or the glyoxylate shunt operon aceBA effectively reduced acetate and improved fumaric acid production. In fed-batch culture, the resulting strain EF02(pSCppc) produced 41.5g/L fumaric acid from glycerol with 70% of the maximum theoretical yield and an overall productivity of 0.51g/L/h.