Engineering of human tryptophan hydroxylase 2 for efficient synthesis of 5-hydroxytryptophan.

L-Tryptophan hydroxylation catalyzed by tryptophan hydroxylase (TPH) presents a promising method for synthesizing 5-hydroxytryptophan (5-HTP), yet the limited activity of wild-type human TPH2 restricts its application. A high-activity mutant, MT10 (H318E/H323E), was developed through semi-rational active site saturation testing (CAST) of wild-type TPH2, exhibiting a 2.85-fold increase in kcat/Km over the wild type, thus enhancing catalytic efficiency. Two biotransformation systems were developed, including an in vitro one-pot system and a Whole-Cell Catalysis System (WCCS). In the WCCS, MT10 achieved a conversion rate of only 31.5 % within 32 h. In the one-pot reaction, MT10 converted 50 mM L-tryptophan to 44.5 mM 5-HTP within 8 h, achieving an 89 % conversion rate, outperforming the M1 (NΔ143/CΔ26) variant. Molecular dynamics simulations indicated enhanced interactions of MT10 with the substrate, suggesting improved binding affinity and system stability. This study offers an effective approach for the efficient production of 5-HTP.

L-lysine production by systems metabolic engineering of an NADPH auto-regulated Corynebacterium glutamicum.

Here, the systems metabolic engineering of L-lysine-overproducing Corynebacterium glutamicum is described to create a highly efficient microorganism producer. The key chromosomal mutations associated with L-lysine synthesis were identified based on whole-genome sequencing. The carbon flux was subsequently redirected into the L-lysine synthesis pathway and increased the availability of energy and product transport systems required for L-lysine synthesis. In addition, a promoter library sensitive to intracellular L-lysine concentration was constructed and applied to regulate the NADPH pool dynamically. In the fed-batch fermentation experiment, the L-lysine titer of the final engineered strain was 223.4 ± 6.5 g/L. This study is the first to improve L-lysine production by enhancing ATP supply and NADPH self-regulation to improve the intracellular environment.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

Transcriptomics reveals the effect of ammonia nitrogen concentration on Pseudomonas stutzeri F2 assimilation and the analysis of amtB function.

The biological treatment of wastewater with high concentrations of ammonia nitrogen has become a hot research issue, but there are limited reports on the mechanism of ammonia nitrogen utilization by microorganisms. In this paper, a transcriptomic approach was used to investigate the differences in gene expression at 500.0 mg/L (Amo 500) and 100.0 mg/L (Amo 100) ammonium concentrations to reveal the mechanism of ammonia nitrogen removal from water by Pseudomonas stutzeri F2. The transcriptome data showed 1015 (459 up-regulated and 556 down-regulated) differentially expressed genes with functional gene annotation related to nitrogen source metabolism, glycolysis, tricarboxylic acid cycle, extracellular polysaccharide synthesis, energy conversion and transmembrane transport, revealing the metabolic process of ammonium nitrogen conversion to biological nitrogen in P. stutzeri F2 through assimilation. To verify the effect of ammonium transporter protein (AmtB) of cell membrane on assimilation, a P. stutzeri F2-ΔamtB mutant strain was obtained by constructing a knockout plasmid (pK18mobsacB-ΔamtB), and it was found that the growth characteristics and ammonium removal rate of the mutant strain were significantly reduced at high ammonium concentration. The carbon source components and dissolved oxygen conditions were optimized after analyzing the transcriptome data, and the ammonium removal rate was increased from 41.23% to 94.92% with 500.0 mg/L ammonium concentration. The study of P. stutzeri F2 transcript level reveals the mechanism of ammonia nitrogen influence on microbial assimilation process and improvement strategy, which provides a new strategy for the treatment of ammonia nitrogen wastewater.

Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin.

Phospholipase D (PLD) is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification. However, the efficient heterologous expression of PLD is limited by its cell toxicity. In this study, a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL. A secretory expression system containing the signal peptide SPEstA and the dual-promoter PHpaII-SrfA was established, and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation, 191.30-fold higher than that of the control. Under optimum reaction conditions, a 61.61% conversion ratio and 21.07 g/L of phosphatidylserine production were achieved. Finally, the synthesis system of PL derivates was established, which could efficiently synthesis novel PL derivates. The results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application, and laid the foundation for the biosynthesis of phosphatidylserine and other PL derivates. As far as we know, this work reports the highest level of extracellular PLD expression to date and the enzymatic production of several PL derivates for the first time.

An enzymatic colorimetric whole-cell biosensor for high-throughput identification of lysine overproducers.

L-lysine is a crucial nutrient for both humans and animals, and its main commercial use is as a supplement in animal feed to promote chicken and other animal growth. Fluorescence biosensors based on the transcriptional regulator have been developed for high-throughput screening of L-lysine producers. However, due to its inability to specifically detect lysine, this fluorescent biosensor cannot be employed to screen high-yielding strains. Here, we present a novel technique for observing L-lysine concentrations within individual Corynebacterium glutamicum cells. The transcriptional regulator LysG and its binding site, as well as the phytoene desaturase that catalyzes the synthesis of the red pigment, make up the functional core of the biosensor. The lysine-sensitive mutant LysG(E123Y, E125A), which improved the sensitivity of biosensors, was generated by site-directed saturation mutagenesis. In addition, we increased the lysine-induced chromogenic biosensor response to 320 mM by optimizing the L-lysine export mechanism and the pathway for the synthesis of lycopene precursors. The direct identification of producers with elevated L-lysine accumulation is thus made straightforward by colorimetric screening. Lys-8, a lysine producer with a maximum lysine titer of 316.2 mM, was sorted out based on the biosensor. The enzymatic colorimetric biosensor constructed here is a simple tool with great potential for the development of high-level lysine-producing C. glutamicum.

Pathway engineering facilitates efficient protein expression in Pichia pastoris.

Pichia pastoris has been recognized as an important platform for the production of various heterologous proteins in recent years. The strong promoter AOX1, induced by methanol, with the help of the α-pre-pro signal sequence, can lead to a high expression level of extracellular protein. However, this combination was not always efficient, as protein secretion in P. pastoris involves numerous procedures mediated by several cellular proteins, including folding assisted by endoplasmic reticulum (ER) molecular chaperones, degradation through ubiquitination, and an efficient vesicular transport system. Efficient protein expression requires the cooperation of various intracellular pathways. This article summarizes the process of protein secretion, modification, and transportation in P. pastoris. In addition, the roles played by the key proteins in these processes and the corresponding co-expression effects are also listed. It is expected to lay the foundation for the industrial protein production of P. pastoris. KEY POINTS: • Mechanisms of chaperones in protein folding and their co-expression effects are summarized. • Protein glycosylation modifications are comprehensively reviewed. • Current dilemmas in the overall protein secretion pathway of Pichia pastoris and corresponding solutions are demonstrated.

Industrial production of L-lysine in Corynebacterium glutamicum: Progress and prospects.

L-lysine is one of the amino acids necessary for humans and animals and widely used in food processing, pharmaceutical preparations and feed additives. In recent years, rational design based on systems metabolic engineering and conventional optimization of fermentation parameters have contributed to the high production of L-lysine. As the demand for L-lysine in the world market is increasing year by year, intensive research has been devoted to efficient productivity and economic production costs. This review briefly explains the biosynthesis and regulation mechanism of L-lysine in Corynebacterium glutamicum, and then outlines the construction, scale-up culture, and product separation and purification strategies of L-lysine high-producing strains. In addition, emerging strategies for the breeding and fermentation of C. glutamicum for the production of L-lysine have been emphatically elucidated. In short, the commercialization of L-lysine production requires chassis strains with excellent production performance, efficient fermentation process, and the development of sustainable purification technologies.

Enhancement of l-Pipecolic Acid Production by Dynamic Control of Substrates and Multiple Copies of the pipA Gene in the Escherichia coli Genome.

l-Pipecolic acid is an important rigid cyclic nonprotein amino acid, which is obtained through the conversion of l-lysine catalyzed by l-lysine cyclodeaminase (LCD). To directly produce l-pipecolic acid from glucose by microbial fermentation, in this study, a recombinant Escherichia coli strain with high efficiency of l-pipecolic acid production was constructed. This study involves the dynamic regulation of the substrate concentration and the expression level of the l-lysine cyclodeaminase-coding gene pipA. In terms of substrate concentration, we adopted the l-lysine riboswitch to dynamically regulate the expression of lysP and lysO genes. As a result, the l-pipecolic acid yield was increased about 1.8-fold as compared with the control. In addition, we used chemically inducible chromosomal evolution (CIChE) to realize the presence of multiple copies of the pipA gene on the genome. The resultant E. coli strain XQ-11-4 produced 61 ± 3.4 g/L l-pipecolic acid with a productivity of 1.02 ± 0.06 g/(L·h) and a glucose conversion efficiency (α) of 29.6% in fermentation. This is the first report that discovered multiple copies of pipA gene expression on the genome that improves the efficiency of l-pipecolic acid production in an l-lysine high-producing strain, and these results give us new insight for constructing the other valuable biochemicals derived from l-lysine.

Enhancing ß-alanine production from glucose in genetically modified Corynebacterium glutamicum by metabolic pathway engineering.

To directly produce ß-alanine from glucose by microbial fermentation, a recombinant Corynebacterium glutamicum strain with high efficiency of ß-alanine production was constructed in this study. To do this, the biosynthetic pathway of ß-alanine in an L-lysine-producing strain XQ-5 was modified by enhancing carbon flux in biosynthetic pathway and limiting carbon flux in competitive pathway. This study showed that replacement of L-aspartate kinase (AK) with wild-type AK and disruption of lactate dehydrogenase and alanine/valine aminotransferases increase ß-alanine production because of decreasing the by-products accumulation. Moreover, L-aspartate-α-decarboxylase (ADC) from Bacillus subtilis was designed as the best enzyme for increasing ß-alanine production, and its variant (BsADCE56S/I88M) showed the highest activity for catalyzing L-aspartate to generate ß-alanine. To further increase ß-alanine production, expression level of BsADCE56S/I88M was controlled by optimizing promoter and RBS, indicating that Pgro plus ThirRBS is the best combination for BsADCE56S/I88M expression and ß-alanine production. The resultant strain XQ-5.5 produced 30.7 ± 2.3 g/L of ß-alanine with a low accumulation of lactate (from 5.2 ± 0.14 to 0.2 ± 0.09 g/L) and L-alanine (from 7.6 ± 0.22 to 3.8 ± 0. 32 g/L) in shake-flask fermentation and produced 56.5 ± 3.2 g/L of ß-alanine with a productivity of 0.79 g/(L·h) and the glucose conversion efficiency (α) of 39.5% in feed-batch fermentation. This is the first report of genetically modifying the biosynthetic pathway of ß-alanine that improves the efficiency of ß-alanine production in an L-lysine-producing strain, and these results give us a new insight for constructing the other valuable biochemical. KEY POINTS: • Optimization and overexpression of the key enzyme BsADC increased the accumulation of ß-alanine. • The AK was replaced with wild-type AK to increase the conversion of aspartic acid to ß-alanine. • A 56.5-g/L ß-alanine production in fed-batch fermentation was achieved.

Reconstruction of the Diaminopimelic Acid Pathway to Promote L-lysine Production in Corynebacterium glutamicum.

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

L-valine production in Corynebacterium glutamicum based on systematic metabolic engineering: progress and prospects.

L-valine is an essential branched-chain amino acid that cannot be synthesized by the human body and has a wide range of applications in food, medicine and feed. Market demand has stimulated people's interest in the industrial production of L-valine. At present, the mutagenized or engineered Corynebacterium glutamicum is an effective microbial cell factory for producing L-valine. Because the biosynthetic pathway and metabolic network of L-valine are intricate and strictly regulated by a variety of key enzymes and genes, highly targeted metabolic engineering can no longer meet the demand for efficient biosynthesis of L-valine. In recent years, the development of omics technology has promoted the upgrading of traditional metabolic engineering to systematic metabolic engineering. This whole-cell-scale transformation strategy has become a productive method for developing L-valine producing strains. This review provides an overview of the biosynthesis and regulation mechanism of L-valine, and summarizes the current metabolic engineering techniques and strategies for constructing L-valine high-producing strains. Finally, the opinion of constructing a cell factory for efficiently biosynthesizing L-valine was proposed.

Improvement of NADPH-dependent P450-mediated biotransformation of 7α,15α-diOH-DHEA from DHEA by a dual cosubstrate-coupled system.

Hydroxylation of DHEA to 7α,15α-diOH-DHEA was catalyzed by NADPH-dependent cytochrome P450 monooxygenase from Colletotrichum lini. By adding coenzyme precursor nicotinic acid, the NADPH/NADP ratio was significantly increased, and the 7α,15α-diOH-DHEA molar conversion was enhanced from 37.37% to 50.85%. To improve the availability of intracellular NADPH, a dual cosubstrate-coupled system consisting of nicotinic acid and glucose was investigated in C. lini. Using 20mM nicotinic acid and 15g/L glucose as cosubstrate for NADPH regeneration, the 7α,15α-diOH-DHEA molar conversion was dramatically increased by 74.58%. The conversion course was simultaneously shortened by 30h. Moreover, a fed-batch transformation model was established to diminish DHEA toxicity to C. lini and further increase DHEA concentration. The maximum concentration of DHEA was elevated to 15g/L using a three-batch transformation in a coenzyme regeneration system, and 7α,15α-diOH-DHEA production of 11.21g/L could be achieved after 60h of biotransformation. These results demonstrated that this strategy was promising for steroids hydroxylation.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Haloarchaeon Halogranum amylolyticum.

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1 mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.

Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment.

Steroid medication is used extensively in clinical applications and comprises a large and vital part of the pharmaceutical industry. However, the difficulty of separating 4-androstene-3,17-dione (AD) from 1,4-androstadiene-3,17-dione (ADD) restricts the application of the microbial transformation of phytosterols in the industry. A novel atmospheric and room temperature plasma (ARTP) treatment, which employs helium as the working gas, was used to generate Mycobacterium neoaurum mutants producing large amounts of AD. After treatment of cultures with ARTP, four mutants were selected using a novel screening method with a color assay. Among the mutants, M. neoaurum ZADF-4 was considered the best candidate for industrial application. When the fermentation medium contained 15 g/L phytosterols and was cultivated on a rotary shaker at 160 r/min at 30 °C for 7 d, (6.28±0.11) g/L of AD and (0.82±0.05) g/L of ADD were produced by the ZADF-4 mutant, compared with (4.83±0.13) g/L of AD and (2.34±0.06) g/L of ADD by the original strain, M. neoaurum ZAD. Compared with ZAD, the molar yield of AD increased from 48.3% to 60.3% in the ZADF-4 mutant. This result indicates that ZADF-4 may have potential for industrial production of AD.

Enhancement of steroid hydroxylation yield from dehydroepiandrosterone by cyclodextrin complexation technique.

The cyclodextrins (CDs) complexation technique was performed for the enhancement of hydroxylation yield from dehydroepiandrosterone (DHEA) by Colletotrichum lini ST-1. Using DHEA/methyl-ß-cyclodextrin (M-ß-CD) or DHEA/hydroxypropyl-ß-cyclodextrin (HP-ß-CD) inclusion complexes as substrate (10g/L), the hydroxylation yields were increased by 14.98% and 20.54% respectively, and the biotransformation course was shortened by 12h. X-ray diffractometry, differential scanning calorimetry, and phase solubility analyses showed an inclusion complex was formed between CDs and DHEA at a molar ratio of 1:1, which remarkably increased the solubility of DHEA, and then improved substrate biotransformation efficiency and hydroxylation yield. Meanwhile, results of thermodynamic parameters (ΔG, ΔH, ΔS and Ks) analysis revealed the complexation process was spontaneous and DHEA/CDs inclusion complex was stable. Scanning electron microscopy and transmission electron microscopy showed that the enhancement of DHEA hydroxylation yield also depended on the improvement of cell permeability through interaction between cytomembrane and CDs. These results suggested that the CDs complexation technique was a promising method to enhance steroids hydroxylation yield by increasing steroids solubility and decreasing membrane resistance of substrate and product during biotransformation process.

Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed.

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high µ for cell growth and high q(p) for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l(-1) in 48 h with the yield of 0.051 g g(-1) by applying this strategy, which were 20.5 and 21.4% over the best results controlled by constant agitation speeds.

Fermentation of biodiesel-derived glycerol by Bacillus amyloliquefaciens: effects of co-substrates on 2,3-butanediol production.

Cultivation in glycerol instead of sugars inhibits 2,3-butanediol (2,3-BD) production by Bacillus amyloliquefaciens. In this study, we report that B. amyloliquefaciens readily produces 2,3-BD from biodiesel-derived glycerol in the presence of beet molasses as a co-substrate. Unexpectedly, the molasses stimulated 2,3-BD production and simultaneously reduced the duration of fermentation. Productivity of 2,3-BD was enhanced at the start of fermentation, and yields increased under continuous molasses supply. Subsequently, 2,3-BD production in molasses-supplemented fed-batch culture was observed. Prior to inoculation of fed-batch fermentation culture, 15 g/l of molasses was added to the bioreactor. After 6 h of incubation, the bioreactor was fed with a solution containing 80 % glycerol and 15 % molasses. The 2,3-BD concentration, yield, and productivity significantly improved, reaching 83.3 g/l, 0.42 g/g, and 0.87 g/l·h, respectively. To our knowledge, these results are the highest report for 2,3-BD fermentation from biodiesel-derived glycerol.

[Flow-injection chemiluminescence determination of procaterol hydrochloride].

A new flow injection chemiluminescences method for the determination of procaterol hydrochloride has been developed. It is based on the chemiluminescence reaction of procaterol hydrochloride with Ce (IV)-rhodamine B in sulphuric-chlorhydric acid medium and the experimental fact of the enhancement of chemiluminescence by surfactant CTMAB. The concentration of the analyte shows a good linear relationship with the produced luminescence intensity in the range of 2.0 x 10(-8) g x mL(-1) to 1.0 x 10(-6) g x mL(-1). The detection limit of the proposed method is 6 x 10(-9) g x mL(-1) and the RSD is 1.6% (n = 11) at 2 x 10(-7) g x mL(-1). This result is satisfactory compared with the method mentioned in the pharmacopoeia of People's Republic of China.

[Cloning of the gene encoding a key enzyme involved in production of glycerol in Candida glycerinogenes].

Candida glycerinogenes WL2002-5, an excellent glycerol producer, has been used for industrial scale fermentation of glycerol by an aerobic process. However, our knowledge about glycerol biosynthesis at the molecular level and genetic background of this yeast species lags far behind those of model yeasts such as Saccharomyces cerevisiae et al. In this report, inverse primers, in conjunction with degenerated primers, were used to amplify the NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) encoding gene from C. glycerinogenes. The completed nucleotide sequence of the coding, as well as flanking genomic regions was determined (GenBank accession No. EU186536). DNA sequence analysis revealed the present of the open reading frame (ORF) of 1,167 bp, encoding a polypeptide with 388-amino-acid with a molecular mass of 42,695 Da. The CgGPD did not exhibit significant sequence similarity with others described in other eukaryotic systems by comparative analysis. However, it consisted of two typical functional domains which belong to almost all eukaryotic GPDs: a co-enzyme binding domain in the N-terminal, and a catalytic domain. Moreover, some relevant features involved in initiation, regulation and stress response element of gene transcription were observed in the nucleotide sequence of the 5'-non-coding regions. Heterologous expression of CgGPD gene in S. cerevisiae improved its glycerol production significantly. In conclusion, the functional CgGPD has been cloned and identified successfully from C. glycerinogenes genome.