Phenotype-genotype mapping reveals the betaine-triggered L-arginine overproduction mechanism in Escherichia coli.

The production phenotype improvement of industrial microbes is extremely needed and challenging. Environmental factors optimization provides insightful ideas to trigger the superior production phenotype by activating potential genetic determiners. Here, phenotype-genotype mapping was used to dissect the betaine-triggered L-arginine overproduction mechanism and mine beneficial genes for further improving production phenotype. The comparative transcriptomic analysis revealed a novel role for betaine in modulating global gene transcription. Guided by this finding, 4 novel genes (cynX, cynT, pyrB, and rhaB) for L-arginine biosynthesis were identified via reverse engineering. Moreover, the rhaB deletion was demonstrated as a common metabolic engineering strategy to improve ATP pool in E. coli. By combinatorial genes manipulation, the L-arginine titer and yield increased by 17.9% and 28.9% in a 5-L bioreactor without betaine addition. This study revealed the molecular mechanism of gene transcription regulation by betaine and developed a superior L-arginine overproducer that does not require betaine.

Efficient fermentative production of L-theanine by Corynebacterium glutamicum.

L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.

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

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.

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.

Strategy for enhancing adenosine production under the guidance of transcriptional and metabolite pool analysis.

OBJECTIVES: To rationally identify targets for enhancing adenosine production, transcription level of genes involved in adenosine synthesis of Bacillus subtilis XGL was detected during the fermentation process, complemented with metabolite pool analysis. RESULTS: PurR-regulated genes (pur operon and purA) and prs were down-regulated and 5-phosphoribosyl 1-pyrophosphate (PRPP) decreased considerably after 24 h when adenosine significantly accumulated. Since PRPP could strongly antagonize the binding of PurR to its targets, it was inferred that down-regulation of pur operon and purA might be due to a low PRPP pool, which was confirmed by metabolite analysis. So desensitized prs responsible for PRPP synthesis was overexpressed, resulting in increased PRPP concentration and pur operon transcription. To further enhance the adenosine production, desensitized purF and prs were co-overexpressed with integrating additional copy of purA to B. subtilis XGL genome, resulting in 24.3 % (1.29 g/g DCW) higher adenosine production than that by B. subtilis XG. CONCLUSIONS: Overexpression of prs, purF and purA under the guidance of transcriptional and metabolite pool analysis significantly increased adenosine production. Strategies used in this study have potential applications for rational modification of industrial microorganisms.

Reducing lactate secretion by ldhA Deletion in L-glutamate- producing strain Corynebacterium glutamicum GDK-9

L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.

[Modification in de novo purine pathway for adenosine accumulation by Bacillus subtilis].

OBJECTIVE: To study the effects of co-overexpression of purF, purM, purN, purH and purD genes on adenosine production in Bacillus subtilis. METHODS: First, an extra purF gene under control of the P43 promoter was integrated into the B. subtilis chromosome at the native purF locus by single crossover, resulting in simultaneous expression of purF, purM, purN, purH and purD. Then the transcriptional levels of the five genes in the engineering strain were tested by Realtime Quantitative PCR. The activity of PRPP amidotransferase was also detected. Finally, cell growth, glucose consumption and adenosine production in engineering strain along with original strain were examined. RESULTS: The transcriptional analysis showed that purF and its downstream genes purM, purN, purH and purD were simultaneously upregulated at different level. The PRPP amidotransferase activity of engineering strain was about 2.4-fold that of original strain. Shake flask fermentation showed the improvement in adenosine yield and conversion ratio from glucose to adenosine (17.5% and 26.1%, respectively). Fed-batch fermentation by the engineering strain was conducted. Compared with the original strain, adenosine accumulation of engineering strain increased within the same fermentation time. However, the cell growth of engineering strain was retarded. CONCLUSION: The co-overexpression of purF and its downstream genes purM, purN, purH and purD could enhance the adenosine yield in the culture broth. This paper could facilitate future research by providing theoretical evidence and method of metabolic engineering.

[Molecular cloning, gene expression and characterization of purine nucleoside phosphorylase from Pseudoalteromonas sp. XM2107].

OBJECTIVE: Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is an important enzyme which is applied in nucleoside medication and intermediate biosynthesis. In this paper, we aimed to obtain the PNP gene from cold-adapted marine bacterium Pseudoalteromonas sp. XM2107 and study the characteristics of enzyme for applying in nucleoside medication and intermediate biosynthesis. METHODS: Purine nucleoside phosphorylas gene which amplified from the cold-adapted marine bacterium Pseudoalteromonas sp. XM2107 genome by homology-based PCR cloning was cloned, sequenced and expressed at E. coli BL21 (DE3) by using expression vector pET-His. The recombinant purine nucleoside phosphorylas enzyme (XmPNP) was purified by metal chelate chromatography and its several characteristics were determined completely. RESULTS: Analysis of entire sequences of XmPNP revealed that the whole sequence is 702 bp and coded a peptide of 233 amino acids with a calculated molecular mass of 25 kDa. Compared with mesophilic counterparts, XmPNP showed a lower temperature optimum (50 degrees C). The optimal pH for inosine phosphorolysis catalyzed by XmPNP was around 7.6 at sodium phosphate buffer. XmPNP showed the highest activity toward inosine (K(m) value, 0.382 mmol/L, at 37 degrees C) and the activity decreased in the order of guanosine and adenosine. Furthermore, XmPNP still expressed high catalytic activity and excellent thermalstability at ordinary temperature. CONCLUSION: Both high catalytic activity and good thermalstability at ordinary temperature indicated that it will provide attractive candidate for prodrug activation and nucleoside medication biotransformation.

Proteomic analysis of the major envelope and nucleocapsid proteins of white spot syndrome virus.

White spot syndrome virus (WSSV) virions were purified from the tissues of infected Procambarus clarkii (crayfish) isolates. Pure WSSV preparations were subjected to Triton X-100 treatment to separate into the envelope and nucleocapsid fractions, which were subsequently separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major envelope and nucleocapsid proteins were identified by either matrix-assisted laser desorption ionization-time of flight mass spectrometry or defined antibody. A total of 30 structural proteins of WSSV were identified in this study; 22 of these were detected in the envelope fraction, 7 in the nucleocapsid fraction, and 1 in both the envelope and the nucleocapsid fractions. With the aid of specific antibodies, the localizations of eight proteins were further studied. The analysis of posttranslational modifications revealed that none of the WSSV structural proteins was glycosylated and that VP28 and VP19 were threonine phosphorylated. In addition, far-Western and coimmunoprecipitation experiments showed that VP28 interacted with both VP26 and VP24. In summary, the data obtained in this study should provide an important reference for future molecular studies of WSSV morphogenesis.

White spot syndrome virus VP24 interacts with VP28 and is involved in virus infection.

White spot syndrome virus (WSSV) is one of the most virulent pathogens causing high mortality in shrimp. Herein, the characterization of VP24, a major structural protein of WSSV, is described. When purified virions were subjected to Nonidet P-40 treatment to separate the envelopes from the nucleocapsids, VP24 was found to be present exclusively in the envelope fraction. Triton X-114 extraction also indicated that VP24 behaves as an envelope protein. Immunoelectron microscopy further confirmed that VP24 is located in the virion envelope. Far-Western experiments showed that VP24 interacts with VP28, another major envelope protein of the WSSV virion. To investigate the function of VP24, WSSV was neutralized with various amounts of anti-VP24 IgG and injected into crayfish. The results showed that anti-VP24 IgG could partially attenuate infection with WSSV. It is concluded that VP24 is an envelope protein and functions at an early stage in virus infection.

Identification of a novel envelope protein (VP187) gene from shrimp white spot syndrome virus.

A novel protein from white spot syndrome virus (WSSV) was identified to match an open reading frame (wsv209) of WSSV genome by combining SDS-PAGE with mass spectrometry. This ORF contained 4,818 bp, encoding 1,606 aa. The apparent molecular mass of the protein from WSSV virions on SDS-PAGE gel is 187 kDa, so it was named VP187 and its gene was termed vp187. Temporal transcription analysis revealed that vp187 was a late gene. To characterize VP187, a segment of the vp187 gene (vp187p) was cloned into pET-GST vector and expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli strain BL21 (DE3). Specific antibody was raised using the purified fusion protein (GST-VP187P). Western blot analysis showed that the mouse anti-GST-VP187P serum reacted specifically with VP187 present either in the WSSV virions or in the viral envelopes, and did not react with fractions of the viral nucleocapsids. VP187 was proved to locate in the WSSV virions as an envelope protein using immunoelectron microscopy.

Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31.

Based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 kDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag, and then specific antibody was raised. Western blot analysis and the immunoelectron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity.

Transcription and identification of a novel envelope protein (VP124) gene of shrimp white spot syndrome virus.

White spot syndrome virus (WSSV) is one of the most virulent pathogens in shrimp culture worldwide. Combining SDS-PAGE with mass spectrometry, a novel envelope protein from WSSV was identified to match an open reading frame (ORF) of WSSV genome. This ORF contained 3582nt, encoding 1194 aa, and was termed the vp124 gene. One part of the whole gene (named vp124p) was cloned into pET-GST vector and expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli strain BL21 (DE3). Specific antibodies were raised using the purified fusion protein (GST-VP124P). Temporal transcription analysis revealed that the vp124 gene was a late gene. Western blot analysis showed that the mouse anti-GST-VP124P antibodies reacted specifically with VP124 present either in the WSSV virions or in the viral envelopes, and did not react with the proteins of the viral nucleocapsids. VP124 was located in the WSSV virions as an envelope protein using immunoelectron microscopy.

Interaction of white spot syndrome virus VP26 protein with actin.

VP26 protein, the product of the WSV311 gene of white spot syndrome virus (WSSV), is one of major structural proteins of virus. In this study, when purified virions were treated with Triton X-100 detergent, VP26 protein was present in both the envelope and the nucleocapsid fraction. We have rationalized this finding by suggesting that VP26 protein might be located in the space between the envelope and the nucleocapsid. By using a fluorescent probe method, we have investigated the interaction between VP26 protein and some proteins of host cells. Three major VP26-binding proteins were purified from crayfish hemocytes by affinity-chromatography, in which the protein with an apparent molecular mass of 42 kDa was identified as actin by mass spectrometry (MS). Moreover, the association of VP26 protein with actin microfilaments was confirmed by coimmunoprecipitation.