Genome shuffling and high-throughput screening of <Emphasis Type="Italic">Brevibacterium flavum</Emphasis> MDV1 for enhanced <Emphasis Type="SmallCaps">l</Emphasis>-valine production

l-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for l-valine grows rapidly, there is an increasing interest to develop an efficient l-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield l-valine strains to replace the traditional l-valine assay. l-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six l-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an l-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of l-valine (0.598 mol l-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m³ fermentor, this strain produced?>?66.8 g/L l-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of l-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.     

Effect of Active Site Pocket Structure Modification of <Emphasis Type="SmallCaps">d</Emphasis>-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

d-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of d/l configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound d-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with d-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of d/l-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of d/l-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-alanine from <Emphasis Type="SmallCaps">dl</Emphasis>-alanine using immobilized cells of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> HLZ-68

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce d-alanine from dl-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher l-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on l-alanine consumption were examined. Maximum l-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of dl-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete l-alanine degradation within 60 h, leaving 185 g of d-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. d-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted d-alanine was 99.1 and 99.6%, respectively.     

The occurrence and accumulation of <Emphasis Type="SmallCaps">d</Emphasis>-pinitol in fenugreek (<Emphasis Type="Italic">Trigonella foenum graecum</Emphasis> L.)

This article presents changes in concentrations of d-pinitol (and other cyclitols as well as low molecular weight carbohydrates) in vegetative and reproductive organs of fenugreek (Trigonella foenumgraecum L.) during an entire plant growing period. d-Pinitol was the major cyclitol in all tested organs, representing 43–94% of total cyclitols and 2–77% of total soluble carbohydrates. The highest concentration of d-pinitol was found in pods (14–23 mg g^?1 of dry weight, DW), lower in leaves and stems (5–20 and 9–10 mg g^?1 DW, respectively), and the lowest in maturing seeds (2–5 mg g^?1 DW). Although maturing seeds accumulate α-d-galactosides of d-pinitol (galactosyl pinitols, up to 6.6 mg g^?1 DW), the major storage sugars were raffinose family oligosaccharides (RFOs, 65.37 mg g^?1 DW). Both RFOs and galactosyl pinitols are hydrolyzed during seed germination, releasing sucrose and d-pinitol, respectively. Accumulation of free galactose was not detected. Owing to the high concentration of d-pinitol (up to 23.70 mg g^?1 DW) and low concentration of soluble sugars, developing pods seem to be the best source of d-pinitol.     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

Synthesis of a tetra- and a trisaccharide related to an anti-tumor saponin “Julibroside J<Subscript>28</Subscript>” from <Emphasis Type="Italic">Albizia julibrissin</Emphasis>

Simple and convergent synthesis of a tetra- and a trisaccharide portions of an antitumor compound Julibroside J₂₈, isolated from Albizia julibrissin, that showed significant in vitro antitumor activity against HeLa, Bel-7402 and PC-3M-1E8 cancer cell lines is reported. The tetrasaccharide has been synthesized as its p-methoxyphenyl glycoside starting from commercially available d-glucose, l-rhamnose and l-arabinose. The trisaccharide part has been synthesized from commercially available N-acetyl d-glucosamine, d-fucose and d-xylose using simple protecting group manipulations. Sulfuric acid immobilized on silica has been used successfully as a Brönsted acid catalyst for the crucial glycosylation steps.     

Application and microbial preparation of <Emphasis Type="SmallCaps">d</Emphasis>-valine

d-Valine is an important organic chiral source and has extensive industrial application, which is used as intermediate for the synthesis of agricultural pesticides, semi-synthetic veterinary antibiotics and pharmaceutical drugs. Its derivatives have shown great activity in clinical use, such as penicillamine for the treatment of immune-deficiency diseases, and actinomycin D for antitumor therapy. Fluvalinate, a pyrethroid pesticide made from d-valine, is a broad-spectrum insecticide with low mammalian toxicity. Valnemulin, a semi-synthetic pleuromutilin derivative synthesized from d-valine, is an antibiotic for animals. Moreover, d-valine is also used in cell culture for selectively inhibiting fibroblasts proliferation. Due to its widespread application, d-valine is gaining more and more attention and some approaches for d-valine preparation have been investigated. In comparison with other approaches, microbial preparation of d-valine is more competitive and promising because of its high stereo selectivity, mild reaction conditions and environmental friendly process. So far, microbial preparation of d-valine can be mainly classified into three categories: microbial asymmetric degradation of dl-valine, microbial stereoselective hydrolysis of N-acyl-dl-valine by d-aminoacylase, and microbial specific hydrolysis of dl-5-isopropylhydantoin by d-hydantoinase coupled with d-carbamoylase. In this paper, the industrial application of d-valine and its microbial preparation are reviewed.     

An <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine Transporter Isoform for Neurogenesis Facilitated by <Emphasis Type="SmallCaps">l</Emphasis>-Theanine

l-Theanine (=γ-glutamylethylamide) is an amino acid ingredient in green tea with a structural analogy to l-glutamine (l-GLN) rather than l-glutamic acid (l-GLU), with regards to the absence of a free carboxylic acid moiety from the gamma carbon position. l-theanine markedly inhibits [³H]l-GLN uptake without affecting [³H]l-GLU uptake in cultured neurons and astroglia. In neural progenitor cells with sustained exposure to l-theanine, upregulation of the l-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neuronal commitment are seen along with marked acceleration of the phosphorylation of mammalian target of rapamycin (mTOR) and relevant downstream proteins. Stable overexpression of Slc38a1 leads to promotion of cellular growth with facilitated neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells stably overexpressing Slc38a1, marked phosphorylation is seen with mTOR and downstream proteins in a fashion insensitive to the additional stimulation by l-theanine. The green tea amino acid l-theanine could thus elicit pharmacological actions to up-regulate Slc38a1 expression for activation of the mTOR signaling pathway required for cell growth together with accelerated neurogenesis after sustained exposure in undifferentiated neural progenitor cells. In this review, I summarize a novel pharmacological property of the green tea amino acid l-theanine for embryonic and adult neurogenesis with a focus on the endogenous amino acid analog l-GLN. A possible translational strategy is also discussed on the development of dietary supplements and nutraceuticals enriched of l-theanine for the prophylaxis of a variety of untoward impairments and malfunctions seen in patients with different neurodegenerative and/or neuropsychiatric disorders.     

Safety of long-term dietary supplementation with <Emphasis Type="SmallCaps">l</Emphasis>-arginine in rats

This study was conducted with rats to determine the safety of long-term dietary supplementation with l-arginine. Beginning at 6 weeks of age, male and female rats were fed a casein-based semi-purified diet containing 0.61 % l-arginine and received drinking water containing l-arginine-HCl (0, 1.8, or 3.6 g l-arginine/kg body-weight/day; n = 10/group). These supplemental doses of l-arginine were equivalent to 0, 286, and 573 mg l-arginine/kg body-weight/day, respectively, in humans. After a 13-week supplementation period, blood samples were obtained from rats for biochemical analyses. Supplementation with l-arginine increased plasma concentrations of arginine, ornithine, proline, homoarginine, urea, and nitric oxide metabolites without affecting those for lysine, histidine, or methylarginines, while reducing plasma concentrations of ammonia, glutamine, free fatty acids, and triglycerides. l-Arginine supplementation enhanced protein gain and reduced white-fat deposition in the body. Based on general appearance, feeding behavior, and physiological parameters, all animals showed good health during the entire experimental period; Plasma concentrations of all measured hormones (except leptin) did not differ between control and arginine-supplemented rats. l-Arginine supplementation reduced plasma levels of leptin. Additionally, l-arginine supplementation increased l-arginine:glycine amidinotransferase activity in kidneys but not in the liver or small intestine, suggesting tissue-specific regulation of enzyme expression by l-arginine. Collectively, these results indicate that dietary supplementation with l-arginine (e.g., 3.6 g/kg body-weight/day) is safe in rats for at least 91 days. This dose is equivalent to 40 g l-arginine/kg body-weight/day for a 70-kg person. Our findings help guide clinical studies to determine the safety of long-term oral administration of l-arginine to humans.     

Highly efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from chicory-derived inulin by <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of d-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for d-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of d-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest d-lactic acid concentration (123.6 ± 0.9 g/L) with a yield of 97.9 % was obtained from 120 g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of d-lactic acid (>99.9 %) and reduced number of residual sugars. The excellent performance of d-lactic acid production from inulin by SSF represents a high-yield method for d-lactic acid production from non-food grains.     

Engineering an ATP-dependent <Emphasis Type="SmallCaps">d</Emphasis>-Ala:<Emphasis Type="SmallCaps">d</Emphasis>-Ala ligase for synthesizing amino acid amides from amino acids

We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH₂) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH₂ production. The S293E variant, which was selected as the best enzyme for d-AlaNH₂ production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH₂ production. The apparent K _m values of this variant for d-Ala and NH₃ were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH₂ from 10 and 50 mM d-Ala and 3 M NH₄Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.     

Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

Increased productivity of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid and total turnover number of NAD<Superscript>+</Superscript>/NADH in a one-pot system through enhanced thermostability of <Emphasis Type="SmallCaps">l</Emphasis>-threonine deaminase

Objective

To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).

Results

l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD⁺/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.

Conclusions

By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.

     

<Emphasis Type="SmallCaps">l</Emphasis>-Serine overproduction with minimization of by-product synthesis by engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The direct fermentative production of l-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low l-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing l-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both l-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products l-alanine and l-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards l-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as l-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the l-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of l-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of l-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve l-serine productivity.     

Synthesis and preliminary biological evaluation of <Emphasis Type="Italic">S</Emphasis>-<Superscript>11</Superscript>C-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-cysteine as a new amino acid PET tracer for cancer imaging

S-¹¹C-methyl-l-cysteine (LMCYS) is an attractive amino acid tracer for clinical tumor positron emission tomography (PET) imaging. d-isomers of some radiolabeled amino acids are potential PET tracers for tumor imaging. In this work, S-¹¹C-methyl-d-cysteine (DMCYS), a d-amino acid isomer of S-¹¹C-methyl-cysteine for tumor imaging was developed and evaluated. DMCYS was prepared by ¹¹C-methylation of the precursor d-cysteine, with an uncorrected radiochemical yield over 50 % from ¹¹CH₃I within a total synthesis time from ¹¹CO₂ about 12 min. In vitro competitive inhibition studies showed that DMCYS uptake was primarily transported through the Na⁺-independent system L, and also the Na⁺-dependent system B^0,+ and system ASC, with almost no system A. In vitro incorporation experiments indicated that almost no protein incorporation was found in Hepa 1–6 hepatoma cell lines. Biodistribution studies demonstrated higher uptake of DMCYS in pancreas and liver at 5 min post-injection, relatively lower uptake in brain and muscle, and faster radioactivity clearance from most tissues than those of l-isomer during the entire observation time. In the PET imaging of S180 fibrosarcoma–bearing mice and turpentine-induced inflammatory model mice, 2-¹⁸F-fluoro-2-deoxy-d-glucose (FDG) exhibited significantly high accumulation in both tumor and inflammatory lesion with low tumor-to-inflammation ratio of 1.40, and LMCYS showed low tumor-to-inflammation ratio of 1.64 at 60 min post-injection. By contrast, DMCYS showed moderate accumulation in tumor and very low uptake in inflammatory lesion, leading to relatively higher tumor-to-inflammation ratio of 2.25 than ¹¹C-methyl-l-methionine (MET) (1.85) at 60 min post-injection. Also, PET images of orthotopic transplanted glioma models demonstrated that low uptake of DMCYS in normal brain tissue and high uptake in brain glioma tissue were observed. The results suggest that DMCYS is a little better than the corresponding l-isomers as a potential PET tumor-detecting agent and is superior to MET and FDG in the differentiation of tumor from inflammation.     

The decreased expression of Stat3 and p-Stat3 in preeclampsia-like rat placenta

This study aims to investigate the expression of Stat3 and p-Stat3 in the placenta of a preeclampsia-like rat model induced by Nωnitro-l-arginine methyl ester (l-NAME). Two-to three-month-old (20 males, 40 females) Sprague–Dawley rats were used in this study. After conception was confirmed by vaginal smears, on the thirteenth day of pregnancy, the animals were allocated into two groups: control (0.9% NaCl administered) group and l-NAME (75 mg/kg) group. After the treatment of l-NAME, there was a significant increase in systolic blood pressure (SBP) levels in the l-NAME group (148.5?±?5.71 mmHg) on day 21 compared to the SBPs in the control group (117.5?±?4.57 mmHg) (P?<?0.001). There was also an increase in total proteinuria on day 21 in the l-NAME group (766.57?±?17.7 mg/L), when compared to the control group (459.89?±?20.1 mg/L) (P?<?0.001). Moreover, we also found a decrease in fetal numbers and fetal weight in the PE group in comparison to the control group. After the rats were sacrificed, the placentas were obtained from both groups. We found that the l-NAME group exhibited fewer placentas compared with the control group. Furthermore, the immunohistochemistry (IHC) and Western blot results showed that decreased expression of Stat3 and p-Stat3 were detected in the placenta of the preeclampsia-like rat model compared to Stat3 and p-Stat3 in the control group. We found the expression and activation of Stat3 and p-Stat3 were decreased in the placenta of the l-NAME-induced preeclampsia rat model.     

Ectopic expression of sorbitol-6-phosphate 2-dehydrogenase gene from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

d-Sorbitol-6-phosphate 2-dehydrogenase (S6PDH, E.C. 1.1.1.140) catalyzes the NADH-dependent conversion of d-fructose 6-phosphate (F6P) to d-sorbitol 6-phosphate (S6P). In this work, recombination and characterization of Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase are reported. Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase was expressed in P. pastoris and Arabidopsis thaliana. Enzyme assay indicated that HmS6PDH catalyzes the reduction of d-fructose 6-phosphate to d-sorbitol 6-phosphate and HmS6PDH activity was enhanced by NaCl. Furthermore, transgenic A. thaliana ectopic expressing HmS6PDH accumulate more sorbitol under salt stress. These results suggest that the ectopic expression of HmS6PDH in plants can facilitate future studies regarding the engineering and breeding of salt-tolerant crops.     

Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.     

Characterization of the cell surface glycolipid from <Emphasis Type="Italic">Spirochaeta aurantia</Emphasis>

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, l-rhamnose, l-fucose, d-xylose, d-mannose, d-glucosamine, d-glycero-d-gluco-heptose (DDglcHep), d-glycero-d-manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as:

$\matrix {{\quad \quad \quad \quad \quad {\text{ - [ - 3 - }}\beta {\text{ - DDglcHep - }}3{\text{ - }}\beta {\text{ - D - GlcNAc - 2 - }}\beta {\text{ - D - Man - ] - }}}} \\ {{\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad |}} \\ {{\alpha {\text{ - Aur - 3 - }}\beta {\text{ - L - Rha - 4 - }}\beta {\text{ - D - Xyl - 4 - }}\alpha {\text{ - L - Fuc - 3 - }}\beta {\text{ - DDHep - 4}}}} \\ {{\quad \quad \quad \quad \quad \quad \quad \quad \quad |}} \\ {{\alpha {\text{ - L - Rha - 3}}}} \ $

which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

     

An optimized fed-batch culture strategy integrated with a one-step fermentation improves <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate l-lactic acid (l-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of l-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing l-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of l-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in l-LA production or yield; however, the productivity of l-LA increased by 14.3%.