Sweet Sorghum Juice and Bagasse as Feedstocks for the Production of Optically Pure Lactic Acid by Native and Engineered <Emphasis Type="Italic">Bacillus coagulans</Emphasis> Strains

Sweet sorghum is a bioenergy crop that produces large amounts of soluble sugars in its stems (3–7 Mg ha^?1) and generates significant amounts of bagasse (15–20 Mg ha^?1) as a lignocellulosic feedstock. These sugars can be fermented not only to biofuels but also to bio-based chemicals. The market potential of the latter may be higher given the current prices of petroleum and natural gas. The yield and rate of production of optically pure d-(?)- and l-(+)-lactic acid as precursors for the biodegradable plastic polylactide was optimized for two thermotolerant Bacillus coagulans strains. Strain 36D1 fermented the sugars in unsterilized sweet sorghum juice at 50 °C to l-(+)-lactic acid (～150 g L^?1; productivity, 7.2 g L^?1 h^?1). B. coagulans strain QZ19-2 was used to ferment sorghum juice to d-(?)-lactic acid (～125 g L^?1; productivity, 5 g L^?1 h^?1). Carbohydrates in the sorghum bagasse were also fermented after pretreatment with 0.5 % phosphoric acid at 190 °C for 5 min. Simultaneous saccharification and co-fermentation of all the sugars (SScF) by B. coagulans resulted in a conversion of 80 % of available carbohydrates to optically pure lactic acid depending on the B. coagulans strain used as the microbial biocatalyst. Liquefaction of pretreated bagasse with cellulases before SScF (L + SScF) increased the productivity of lactic acid. These results show that B. coagulans is an effective biocatalyst for fermentation of all the sugars present in sweet sorghum juice and bagasse to optically pure lactic acid at high titer and productivity as feedstock for bio-based plastics.     

Co-generation of ethanol and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from corn stalk under a hybrid process

Background

Corn stover, as one important lignocellulosic material, has characteristics of low price, abundant output and easy availability. Using corn stover as carbon source in the fermentation of valuable organic chemicals contributes to reducing the negative environmental problems and the cost of production. In ethanol fermentation based on the hydrolysate of corn stover, the conversion rate of fermentable sugars is at a low level because the native S. cerevisiae does not utilize xylose. In order to increase the conversion rate of fermentable sugars deriving from corn stover, an effective and energy saving biochemical process was developed in this study and the residual xylose after ethanol fermentation was further converted to l-lactic acid.

Results

In the hybrid process based on the hydrolysate of corn stover, the ethanol concentration and productivity reached 50.50 g L^?1 and 1.84 g L^?1 h^?1, respectively, and the yield of ethanol was 0.46 g g^?1. The following fermentation of l-lactic acid provided a product titer of 21.50 g L^?1 with a productivity of 2.08 g L^?1 h^?1, and the yield of l-lactic acid was 0.76 g g^?1. By adopting a blank aeration before the inoculation of B. coagulans LA1507 and reducing the final cell density, the l-lactic acid titer and yield reached 24.25 g L^?1 and 0.86 g g^?1, respectively, with a productivity of 1.96 g L^?1 h^?1.

Conclusions

In this work, the air pumped into the fermentor was used as both the carrier gas for single-pass gas stripping of ethanol and the oxygen provider for the aerobic growth of B. coagulans LA1507. Ethanol was effectively separated from the fermentation broth, while the residual medium containing xylose was reused for l-lactic acid production. As an energy-saving and environmental-friendly process, it introduced a potential way to produce bioproducts under the concept of biorefinery, while making full use of the hydrolysate of corn stover.

     

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.     

Semi-industrial scale (30 m<Superscript>3</Superscript>) fed-batch fermentation for the production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Escherichia coli</Emphasis> strain HBUT-D15

d(?)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale d-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L^?1 of glucose, producing 184–191 g L^?1 of d-lactic acid, with a volumetric productivity of 4.38 g L^?1 h^?1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m³) via fed-batch fermentation with up to 160 g L^?1 of glucose, producing 146–150 g L^?1 of d-lactic acid, with a volumetric productivity of 3.95–4.29 g L^?1 h^?1, a yield of 91–94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale l(+)-lactic acid fermentation.     

Production of optically pure <Emphasis Type="SmallCaps">l</Emphasis>(+)-lactic acid from waste plywood chips using an isolated thermotolerant <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> SI at a pilot scale

Utilization of renewable and low-cost lignocellulosic wastes has received major focus in industrial lactic acid production. The use of high solid loadings in biomass pretreatment potentially offers advantages over low solid loadings including higher lactic acid concentration with decreased production and capital costs. In this study, an isolated Enterococcus faecalis SI with optimal temperature 42 °C was used to produce optically pure l-lactic acid (>?99%) from enzyme-saccharified hydrolysates of acid-impregnated steam explosion (AISE)-treated plywood chips. The l-lactic acid production increased by 10% at 5 L scale compared to the similar fermentation scheme reported by Wee et al. The fermentation with a high solid loading of 20% and 35% (w/v) AISE-pretreated plywood chips had been successfully scaled up to process development unit scale (100 L) and pilot scale (9 m³), respectively. This is the first report of pilot-scale lignocellulosic lactic acid fermentation by E. faecalis with high lactic acid titer (nearly 92 g L^?1) and yield (0.97 kg kg^?1). Therefore, large-scale l-lactic acid production by E. faecalis SI shows the potential application for industries.     

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.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

     

Enhanced cadaverine production from <Emphasis Type="SmallCaps">l</Emphasis>-lysine using recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> co-overexpressing CadA and CadB

The effect of fusing the PelB signal sequence to lysine/cadaverine antiporter (CadB) on the bioconversion of l-lysine to cadaverine was investigated. To construct a whole-cell biocatalyst for cadaverine production, four expression plasmids were constructed for the co-expression of lysine decarboxylase (CadA) and lysine/cadaverine antiporter (CadB) in Escherichia coli. Expressing CadB with the PelB signal sequence increased cadaverine production by 12 %, and the optimal expression plasmid, pETDuet-pelB-CadB-CadA, contained two T7 promoter-controlled genes, CadA and the PelB-CadB fusion protein. Based on pETDuet-pelB-CadB-CadA, a whole-cell system for the bioconversion of l-lysine to cadaverine was constructed, and three strategies for l-lysine feeding were evaluated to eliminate the substrate inhibition problem. A cadaverine titer of 221 g l^?1 with a molar yield of 92 % from lysine was obtained.     

A saponin isolated from <Emphasis Type="Italic">Agapanthus africanus</Emphasis> differentially induces apoplastic peroxidase activity in wheat and displays fungicidal properties

A spirostane with an attached trisaccharide, (25R)-5α-spirostane-2α,3β,5α-triol 3-O-(O-α-l-rhamnopyranosyl-(1 → 2)-O-(β-d-galactopyranosyl-(1 → 3))-β-d-glucopyranoside), was isolated and identified from the aerial parts of Agapanthus africanus by activity-guided fractionation. Fungicidal properties of the crude extract, semi-purified fractions as well as the purified active saponin from A. africanus were screened in vitro against Fusarium oxysporum. At a concentration of 1 mg mL^?1, the crude extract and semi-purified ethyl acetate and dichloromethane fractions showed significant antifungal activity. The purified saponin inhibited the in vitro mycelial growth of F. oxysporum completely (100 %) at a concentration of 125 µg mL^?1. Furthermore, to verify previously observed induced resistance by crude extracts of A. africanus towards leaf rust, intercellular PR-protein activity was determined in wheat seedlings following foliar application of the purified saponin at 100 µg mL^?1. In vitro peroxidase enzyme activity increased significantly (60 %) in wheat seedlings 48 h after treatment with the purified saponin, demonstrating its role as an elicitor to activate a defence reaction in wheat.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from <Emphasis Type="Italic">Citreicella</Emphasis> sp. SE45

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS–polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including l-alanine amide and l-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K _m and V _max values for d- and l-ACL were 5.3 and 2.17 mM, and 769 and 558 μmol/min.mg protein, respectively. Moreover, the turn over number (K _cat) and catalytic efficiency (K _cat/K _m ) of purified ACL racemase from Citreicella sp. SE45 using l-ACL as a substrate were 465 S^?1 and 214 S^?1mM^?1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Rational design and analysis of an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain for high-efficiency tryptophan production

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG ^fbr ). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.     

Co-expression of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase and catalase in <Emphasis Type="Italic">Escherichia coli</Emphasis> to produce α-ketoglutaric acid by whole-cell biocatalyst

Objectives

To improve the production of α-ketoglutaric acid (α-KG) from l-glutamate by whole-cell biocatalysis.

Results

A novel and highly active l-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg^?1 at pH 6.0, 35 ^oC. The apparent K_m and V_max values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg^?1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H₂O₂, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l^?1 with a conversion rate from l-glutamate of 98.5% after 12 h.

Conclusions

An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.

     

Increased production of plumbagin in <Emphasis Type="Italic">Plumbago indica</Emphasis> root cultures by biotic and abiotic elicitors

Objectives

To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.

Results

Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l^?1 and 10 mM l-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l^?1). Treatments with 100 µM AgNO₃ significantly increased intracellular plumbagin production by up to 7.6 mg g^?1 DW that was 4-fold more than the untreated root cultures (1.9 mg g^?1 DW). In contrast, treatments with 150 mg chitosan l^?1, 5 mM l-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g^?1 DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g^?1 DW, respectively.

Conclusions

Chitosan (150 mg l^?1), l-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.

     

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.

     

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.     

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.