Pyridine Nucleotide Transhydrogenase Research Articles

Efficient production of ginsenoside Rh2 from xylose by remodeling metabolism in Saccharomyces cerevisiae

Ginsenoside Rh2, a promising candidate for cancer prevention and therapy, has applications in the fields of health products and pharmaceuticals. However, the current production of Rh2 through the extraction of Panax ginseng is not sustainable and cannot meet the increasing market demand. Here, we increased the Rh2 production in Saccharomyces cerevisiae by engineering the heterologous xylose metabolic pathway. The Candida tropicalis derived xylose reductase-xylitol dehydrogenase pathway was successfully constructed in in the previously engineered protopanaxadiol (PPD)-producing strain. A PPD high-yield strain with strong xylose utilization capacity was optimized to reduce the generation of byproduct xylitol with the expression of a soluble pyridine nucleotide transhydrogenase. The new yeast chassis produced 494.0 mg/L PPD in xylose medium, which could provide sufficient precursors for Rh2. The Rh2 production strain was then constructed by integrating the glycosyltransferases UGTPg45 overexpression module and further optimized through systematic enhancement of its mevalonic acid (MVA) pathway, resulting in a Rh2 titer of 209.5 mg/L in xylose medium. In addition, we confirmed that the engineered strain also exhibited good fermentation performance in the lignocellulose hydrolysate mimic glucose-xylose mixed medium, and we further elucidated the reasons of higher Rh2 production in xylose fermentation stage through transcriptomic and metabolic analysis. Taken all strategies together, the resulting strain produced 1.47 g/L Rh2 in a 5 L bioreactor with mixed sugar medium, making it possible for a green and sustainable biotechnology to produce ginsenoside Rh2 using xylose efficiently. This is the first report on the use of xylose for high-value chemical ginsenoside Rh2 production, which will pave the way for the future industrial production of ginsenoside Rh2 from a renewable carbon source such as lignocellulose.

Dynamic regulation and cofactor engineering of escherichia coli to enhance production of glycolate from corn stover hydrolysate

Glycolic acid is widely employed in chemical cleaning, the production of polyglycolic acid-lactic acid, and polyglycolic acid. Currently, the bottleneck of glycolate biosynthesis lies on the imbalance of metabolic flux and the deficiency of NADPH. In this study, a dynamic regulation system was developed and optimized to enhance the metabolic flux from glucose to glycolate. Additionally, the knockout of transhydrogenase (sthA), along with the overexpression of pyridine nucleotide transhydrogenase (pntAB) and the implementation of the Entner-Doudoroff pathway, were performed to further increase the production of the NADPH, thereby increasing the titer of glycolate to 5.6 g/L. To produce glycolate from corn stover hydrolysate, carbon catabolite repression was alleviated and glucose utilization was accelerated. The final strain, E. coli Mgly10-245, is inducer-free, achieving a glycolate titer of 46.1 g/L using corn stover hydrolysate (77.1 % of theoretical yield). These findings will contribute to the advancement of industrial glycolate production.

Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date.

Identification of purine biosynthesis as an NADH-sensing pathway to mediate energy stress

An enhanced NADH/NAD+ ratio, termed reductive stress, is associated with many diseases. However, whether a downstream sensing pathway exists to mediate pathogenic outcomes remains unclear. Here, we generate a soluble pyridine nucleotide transhydrogenase from Escherichia coli (EcSTH), which can elevate the NADH/NAD+ ratio and meantime reduce the NADPH/NADP+ ratio. Additionally, we fuse EcSTH with previously described LbNOX (a water-forming NADH oxidase from Lactobacillus brevis) to resume the NADH/NAD+ ratio. With these tools and by using genome-wide CRISPR/Cas9 library screens and metabolic profiling in mammalian cells, we find that accumulated NADH deregulates PRPS2 (Ribose-phosphate pyrophosphokinase 2)-mediated downstream purine biosynthesis to provoke massive energy consumption, and therefore, the induction of energy stress. Blocking purine biosynthesis prevents NADH accumulation-associated cell death in vitro and tissue injury in vivo. These results underscore the pathophysiological role of deregulated purine biosynthesis in NADH accumulation-associated disorders and demonstrate the utility of EcSTH in manipulating NADH/NAD+ and NADPH/NADP+.

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

Identification and Characterization of a Novel Soluble Pyridine Nucleotide Transhydrogenase from Streptomyces avermitilis.

Soluble pyridine nucleotide transhydrogenase (STH) transfers hydride between NADH and NADPH to maintain redox balance. In the present study, the sth gene from Gram-positive bacterium Streptomyces avermitilis (SaSTH) was expressed in Escherichia coli, and the recombinant STH protein was purified to homogeneity. Activity assays indicated that SaSTH was able to catalyze transhydrogenase reactions by using NADH or NADPH as reductants and thio-NAD+ as an oxidant. The apparent Km value for NADPH (74.5μM) was lower than that for NADH (104.0μM) and the apparent kcat/Km for NADPH (2704.7mM-1s-1) was higher than that for NADH (1129.8mM-1s-1). SaSTH showed optimal activity at 25°C and at a pH of 6.2. Heat-inactivation studies revealed that SaSTH remained stable below 55°C and that approximately 50% activity was preserved at 57°C for 20min. Analyses also showed that SaSTH activity was inhibited by divalent ions, particularly Co2+, Ni2+, and Zn2+. In addition, the transhydrogenase activity of SaSTH was inhibited by ATP and strongly stimulated by ADP and AMP. In summary, we characterized a recombinant enzyme exhibiting STH activity from Gram-positive bacteria for the first time. Our findings provide new options for cofactor engineering and industrial biocatalytic processes.

Ferulic acid production by metabolically engineered Escherichia coli

Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics. Production of FA by eco‐friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p-coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as caffeic acid O-methytransferase. Fermentation result revealed JM109(DE3) as a more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 or p15A), and the combination p15a-T5 works best. To further improve FA production, E. coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also overexpressed. Based on the flask fermentation data which show that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E. coli K-12 strains reported to the best of our knowledge.

Heterologous expression and enzymatic identification of two novel soluble pyridine nucleotide transhydrogenases from Acidobacteria bacterium KBS 146 and Nocardia jiangxiensis

Soluble pyridine nucleotide transhydrogenase (STH), catalyzes a reversible hydrogen transfer between NADH and NADPH, is widely utilized in cofactor engineering. In the present study, we expanded the distribution range of STH to more unreported bacteria. Meanwhile, STH was mainly found in α, γ, δ-proteobacteria, acidobacteria, actinobacteria and planctomycetes. The enzymological properties of two novel STHs from Acidobacteria bacterium KBS 146 (AbSTH) and Nocardia jiangxiensis (NjSTH) were characterized. The optimum temperature and pH of AbSTH and NjSTH were pH 6.2 and 35 °C, pH 5.7 and 50 °C, respectively. When using thio-NAD+ as a hydrogen accepter, the 1/K m and k cat/K m for NADH of AbSTH were 1.8 and 1.7-fold greater than NADPH, whereas the 1/K m and k cat/K m of NjSTH for NADPH were 2.0 and 2.2-fold greater than NADH. The physiological hydrogen donor substrate of AbSTH and NjSTH may be NADH and NADPH, respectively. AbSTH activity was inhibited by ATP and strongly stimulated by ADP and AMP. These results may provide new insights into the physiological roles and cofactor engineering application of STH. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1988708 .

Improving squalene production by enhancing the NADPH/NADP+ ratio, modifying the isoprenoid-feeding module and blocking the menaquinone pathway in Escherichia coli

BackgroundSqualene is currently used widely in the food, cosmetics, and medicine industries. It could also replace petroleum as a raw material for fuels. Microbial fermentation processes for squalene production have been emerging over recent years. In this study, to study the squalene-producing potential of Escherichia coli (E. coli), we employed several increasing strategies for systematic metabolic engineering. These include the expression of human truncated squalene synthase, the overexpression of rate-limiting enzymes in isoprenoid pathway, the modification of isoprenoid-feeding module and the blocking of menaquinone pathway.ResultsHerein, human truncated squalene synthase was engineered in Escherichia coli to create a squalene-producing bacterial strain. To increase squalene yield, we employed several metabolic engineering strategies. A fivefold squalene titer increase was achieved by expressing rate-limiting enzymes (IDI, DXS, and FPS) involved in the isoprenoid pathway. Pyridine nucleotide transhydrogenase (UdhA) was then expressed to improve the cellular NADPH/NADP+ ratio, resulting in a 59% increase in squalene titer. The Embden–Meyerhof pathway (EMP) was replaced with the Entner–Doudoroff pathway (EDP) and pentose phosphate pathway (PPP) to feed the isoprenoid pathway, along with the overexpression of zwf and pgl genes which encode rate-limiting enzymes in the EDP and PPP, leading to a 104% squalene content increase. Based on the blocking of menaquinone pathway, a further 17.7% increase in squalene content was achieved. Squalene content reached a final 28.5 mg/g DCW and 52.1 mg/L.ConclusionsThis study provided novel strategies for improving squalene yield and demonstrated the potential of producing squalene by E. coli.

Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane-bound pyridine nucleotide transhydrogenase.

The non-conventional D-xylose metabolism called the Dahms pathway which only requires the expression of at least three enzymes to produce pyruvate and glycolaldehyde has been previously engineered in Escherichia coli. Strains that rely on this pathway exhibit lower growth rates which were initially attributed to the perturbed redox homeostasis as evidenced by the lower intracellular NADPH concentrations during exponential growth phase. NADPH-regenerating systems were then tested to restore the redox homeostasis. The membrane-bound pyridine nucleotide transhydrogenase, PntAB, was overexpressed and resulted to a significant increase in biomass and glycolic acid titer and yield. Furthermore, expression of PntAB in an optimized glycolic acid-producing strain improved the growth and product titer significantly. This work demonstrated that compensating for the NADPH demand can be achieved by overexpression of PntAB in E. coli strains assimilating D-xylose through the Dahms pathway. Consequently, increase in biomass accumulation and product concentration was also observed.

Two‐stage carbon distribution and cofactor generation for improving l‐threonine production of Escherichia coli

L-Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l-threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l-threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two-stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l-threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l-threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l-threonine production, we developed an l-glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02- and 1.21-fold increase in l-threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions.

Metabolic Engineering of Escherichia coli for Efficient Production of 2-Pyrone-4,6-dicarboxylic Acid from Glucose.

2-Pyrone-4,6-dicarboxylic acid (PDC) is a pseudoaromatic dicarboxylic acid and is a promising biobased building block chemical that can be used to make diverse polyesters with novel functionalities. In this study, Escherichia coli was metabolically engineered to produce PDC from glucose. First, an efficient biosynthetic pathway for PDC production from glucose was suggested by in silico metabolic flux simulation. This best pathway employs a single-step biosynthetic route to protocatechuic acid (PCA), a metabolic precursor for PDC biosynthesis. On the basis of the selected PDC biosynthetic pathway, a shikimate dehydrogenase (encoded by aroE)-deficient E.coli strain was engineered by introducing heterologous genes of different microbial origin encoding enzymes responsible for converting 3-dehydroshikimate (DHS) to PDC, which allowed de novo biosynthesis of PDC from glucose. Next, production of PDC was further improved by applying stepwise rational metabolic engineering strategies. These include elimination of feedback inhibition on 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (encoded by aroG) by overexpressing a feedback-resistant variant, enhancement of the precursor phosphoenolpyruvate supply by changing the native promoter of the ppsA gene with the strong trc promoter, and reducing accumulation of the major byproduct DHS by overexpression of a DHS importer (encoded by shiA). Furthermore, cofactor (NADP+/NADPH) utilization was manipulated through genetic modifications of the E.coli soluble pyridine nucleotide transhydrogenase (encoded by sthA), and the resultant impact on PDC production was investigated. Fed-batch fermentation of the final engineered E.coli strain allowed production of 16.72 g/L of PDC from glucose with the yield and productivity of 0.201 g/g and 0.172 g/L/h, respectively, representing the highest PDC production performance indices reported to date.

Transcriptome analysis of the cyanobacterium Synechocystis sp. PCC 6803 and mechanisms of photoinhibition tolerance under extreme high light conditions

Photoinhibition, or cell damage caused by excessively intense light is a major issue for the industrial use of cyanobacteria. To investigate the mechanism of responses to extreme high light intensity, gene expression analysis was performed using the model cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803) cultured under various light intensities. The culture profile data demonstrated that, in contrast to the slow cell growth observed under low light intensities (30 and 50μmolm-2s-1), maximal cell growth was observed under mid light conditions (300 and 1000μmolm-2s-1). PCC 6803cells exhibited photoinhibition when cultured under excessive high light intensities of 1100 and 1300μmolm-2s-1. From the low to the mid light conditions, the expression of genes related to light harvesting systems was repressed, whereas that of CO2 fixation and of D1 protein turnover-related genes was induced. Gene expression data also revealed that the down-regulation of genes related to flagellum synthesis (pilA2), pyridine nucleotide transhydrogenase (pntA and pntB), and sigma factor (sigA and sigF) represents the key responses of PCC 6803 under excessive high light conditions. The results obtained in this study provide further understanding of high light tolerance mechanisms and should help to improve the productivity of bioprocess using cyanobacteria.

Identification of Genes Involved in Low Temperature Growth of Cronobacter sakazakii ATCC 29544

Cronobacter sakazakii ATCC 29544 is a gram‐negative opportunistic pathogen, which can cause local necrotizing enterocolitis, bacteremia, and meningitis. Powdered infant formula is considered to be the major transmission route for newborn infections. Even though powdered infant formula is stored at a refrigerator temperature (5~10°C), Cronobacter sakazakii can still cause infection. In order to understand its molecular mechanism for low temperature growth, we first constructed random mutant library and screened for no growth at 8 °C. By using the plasmid recovery technique, soluble pyridine nucleotide transhydrogenase (sth), nusB and two ribosomal RNA genes were identified to be responsible for the reduced growth at low temperature. Overall, we report 11 genes that play an important role in the growth of C. sakazakii ATCC 29544 at low temperature.Support or Funding InformationThis research was financially supported by the “Global accompanied growth R&BD program (N042600010)” through the Ministry of Trade, Industry and Energy (MOTIE, Korea).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pyridine nucleotide transhydrogenase PntAB is essential for optimal growth and photosynthetic integrity under low‐light mixotrophic conditions in Synechocystis sp. PCC 6803

Pyridine nucleotide transhydrogenase (PntAB) is an integral membrane protein complex participating in the regulation of NAD(P)+ :NAD(P)H redox homeostasis in various prokaryotic and eukaryotic organisms. In the present study we addressed the function and biological role of PntAB in oxygenic photosynthetic cyanobacteria capable of both autotrophic and heterotrophic growth, with support from structural three-dimensional (3D)-modeling. The pntA gene encoding the α subunit of heteromultimeric PntAB in Synechocystis sp. PCC 6803 was inactivated, followed by phenotypic and biophysical characterization of the ΔpntA mutant under autotrophic and mixotrophic conditions. Disruption of pntA resulted in phenotypic growth defects observed under low light intensities in the presence of glucose, whereas under autotrophic conditions the mutant did not differ from the wild-type strain. Biophysical characterization and protein-level analysis of the ΔpntA mutant revealed that the phenotypic defects were accompanied by significant malfunction and damage of the photosynthetic machinery. Our observations link the activity of PntAB in Synechocystis directly to mixotrophic growth, implicating that under these conditions PntAB functions to balance the NADH: NADPH equilibrium specifically in the direction of NADPH. The results also emphasize the importance of NAD(P)+ :NAD(P)H redox homeostasis and associated ATP:ADP equilibrium for maintaining the integrity of the photosynthetic apparatus under low-light glycolytic metabolism.

Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegradation of aromatic compounds

The metabolic versatility of the soil bacterium Pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. Biodegradation of aromatics occurs via the pathway encoded in the archetypal TOL plasmid pWW0, yet the effect of running such oxidative route on redox balance against the background metabolism of P. putida remains unexplored. To answer this question, the activity of pyridine nucleotide transhydrogenases (that catalyze the reversible interconversion of NADH and NADPH) was inspected under various physiological and oxidative stress regimes. The genome of P. putida KT2440 encodes a soluble transhydrogenase (SthA) and a membrane-bound, proton-pumping counterpart (PntAB). Mutant strains, lacking sthA and/or pntAB, were subjected to a panoply of genetic, biochemical, phenomic and functional assays in cells grown on customary carbon sources (e.g., citrate) versus difficult-to-degrade aromatic substrates. The results consistently indicated that redox homeostasis is compromised in the transhydrogenases-defective variant, rendering the mutant sensitive to oxidants. This metabolic deficiency was, however, counteracted by an increase in the activity of NADP+ -dependent dehydrogenases in central carbon metabolism. Taken together, these observations demonstrate that transhydrogenases enable a redox-adjusting mechanism that comes into play when biodegradation reactions are executed to metabolize unusual carbon compounds.

Effects of the protonophore carbonyl-cyanide m-chlorophenylhydrazone on intracytoplasmic membrane assembly in Rhodobacter sphaeroides

The effect of carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP) on intracytoplasmic membrane (ICM) assembly was examined in the purple bacterium Rhodobacter sphaeroides. CCCP blocks generation of the electrochemical proton gradient required for integral membrane protein insertion. ICM formation was induced for 8h, followed by a 4-h exposure to CCCP. Measurements of fluorescence induction/relaxation kinetics showed that CCCP caused a diminished quantum yield, a cessation in expansion of the functional absorption cross-section and a 4- to 10-fold slowing in the electron transfer turnover rate. ICM vesicles (chromatophores) and an upper-pigmented band (UPB) containing ICM growth initiation sites, were isolated and subjected to clear-native electrophoresis. Proteomic analysis of the chromatophore gel bands indicated that CCCP produced a 2.7-fold reduction in spectral counts in the preferentially assembled light-harvesting 2 (LH2) antenna, while the RC-LH1 complex, F1FO-ATPase and pyridine nucleotide transhydrogenase decreased by 1.7–1.9-fold. For 35 soluble enzymes, the ratio of 0.99 for treated/control proteins demonstrated that protein synthesis was unaffected by CCCP, suggesting that the membrane complex decline arose from the turnover of unassembled apoproteins. In the UPB fraction, an ~2-fold accumulation was observed for the preprotein translocase SecY, the SecA translocation ATPase, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, consistent with the possibility that these factors, which act early in the assembly process, have accumulated in association with nascent polypeptides as stabilized assembly intermediates.

Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from unrelated carbon sources in engineered Rhodospirillum rubrum.

Different genes encoding pyridine nucleotide transhydrogenases (pntAB, udhA) and acetoacetyl-CoA reductases (phaB) were heterologously overexpressed in Rhodospirillum rubrum S1. A recombinant strain, which harbored the gene encoding the membrane-bound transhydrogenase PntAB from Escherichia coli MG1655 and the phaB1 gene coding for an NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha H16, accumulated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [Poly(3HB-co-3HV)] with a 3HV fraction of up to 13 mol% from fructose. This was a 13-fold increase of the 3HV content when compared to the wild-type strain. Higher contents of 3HV are known to reduce the brittleness of this polymer, which is advantageous for most applications. The engineered R. rubrum strain was also able to synthesize this industrially relevant copolymer from CO2 and CO from artificial synthesis gas (syngas) with a 3HV content of 56 mol%. The increased incorporation of 3HV was attributed to an excess of propionyl-CoA, which was generated from threonine and related amino acids to compensate for the intracellular redox imbalance resulting from the transhydrogenase reaction. Thereby, our study presents a novel, molecular approach to alter the composition of bacterial PHAs independently from external precursor supply. Moreover, this study also provides a promising production strain for syngas-derived second-generation biopolymers.

Enhanced 1-Butanol Production in Engineered Klebsiella pneumoniae by NADH Regeneration

1-Butanol, as a next-generation biofuel, is an important target product of biorefinery research. With the introduction of the CoA-dependent pathway or Ehrlich pathway, many engineered strains have been developed to produce 1-butanol, although cofactor imbalance occurred in engineered strains by the introduction of the 1-butanol synthesis pathway. Several studies have been performed to regenerate NADH by overexpressing NAD+-dependent enzymes. However, the significant role of cofactor regeneration in 1-butanol production and the transcription level of the target genes have seldom been studied. The 1-butanol producer, recombinant Klebsiella pneumoniae (KLA), was constructed by overexpressing the genes kivd, leuABCD, and adhE1 under the control of tac promoter in this study, and several NADH regeneration strategies were adopted to solve the problem of NADH imbalance, including the introduction of NAD+-dependent enzymes (formate dehydrogenase, pyridine nucleotide transhydrogenase, and glucose dehydrogenase) or elimination of the NADH competition pathway (1,3-propanediol synthesis). The resultant NADH/NAD+ ratio, 1-butanol production, and transcription levels have been significantly affected. In comparison to the wild-type strain, the NADH/NAD+ ratio in the reengineered strains was increased by 78–135%, and the transcript levels of target genes have been obviously interfered. Moreover, the resultant 1-butanol titer was increased by 83–114% in comparison to KLA.

Development of Halomonas TD01 as a host for open production of chemicals

Genetic engineering of Halomonas spp. was seldom reported due to the difficulty of genetic manipulation and lack of molecular biology tools. Halomonas TD01 can grow in a continuous and unsterile process without other microbial contaminations. It can be therefore exploited for economic production of chemicals. Here, Halomonas TD01 was metabolically engineered using the gene knockout procedure based on markerless gene replacement stimulated by double-strand breaks in the chromosome. When gene encoding 2-methylcitrate synthase in Halomonas TD01 was deleted, the conversion efficiency of propionic acid to 3-hydroxyvalerate (3HV) monomer fraction in random PHBV copolymers of 3-hydroxybutyrate (3HB) and 3HV was increased from around 10% to almost 100%, as a result, cells were grown to accumulate 70% PHBV in dry weight (CDW) consisting of 12mol% 3HV from 0.5g/L propionic acid in glucose mineral medium. Furthermore, successful deletions on three PHA depolymerases eliminate the possible influence of PHA depolymerases on PHA degradation in the complicated industrial fermentation process even though significant enhanced PHA content was not observed. In two 500L pilot-scale fermentor studies lasting 70h, the above engineered Halomonas TD01 grew to 112g/L CDW containing 70wt% P3HB, and to 80g/L CDW with 70wt% P(3HB-co-8mol% 3HV) in the presence of propionic acid. The cells grown in shake flasks even accumulated close to 92% PHB in CDW with a significant increase of glucose to PHB conversion efficiency from around 30% to 42% after 48h cultivation when pyridine nucleotide transhydrogenase was overexpressed. Halomonas TD01 was also engineered for producing a PHA regulatory protein PhaR which is a robust biosurfactant.