Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

     

Nutritional requirements for the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137

The amino acid and humic acid requirements of Saccharothrix algeriensis NRRL B-24137 for growth and production of the dithiolopyrrolone antibiotics were studied in a semi-synthetic medium (SSM). Nature and concentration of amino acids and humic acid strongly influenced the growth and dithiolopyrrolone specific production.The highest value of thiolutin (acetyl-pyrrothine) specific production was obtained in the presence of 1 g/l humic acid (336 mg/g DCW), and in the presence of 5 mM l-cystine (309 mg/g DCW) as compared to 19 mg/g DCW obtained with the control. Furthermore, thiolutin production was increased about six-fold, four-fold and three-fold in the presence of l-proline, l-glutamic acid and dl-histidine, respectively. In contrast, the production of thiolutin was reduced by addition of other amino acids such as l-glutamine, dl-ethionine, l-methionine and l-arginine. The highest value of isobutyryl-pyrrothine production was obtained in the presence of 2,6-diaminopimelic acid and l-lysine (7.8 and 1.0 mg/g DCW, respectively). However, the highest value of butanoyl-pyrrothine production was obtained in the presence of humic acid (6.6 mg/g DCW), followed by l-cysteine and l-proline (3.6 and 3.2 mg/g DCW, respectively). In addition, the maximum specific production of senecioyl-pyrrothine (29 mg/g DCW) and tigloyl-pyrrothine (21 mg/g DCW) was obtained in the presence of humic acid. We found that, except for isobutyryl-pyrrothine, production of all dithiolopyrrolones was favoured by addition of l-proline. The maximum specific production was obtained with l-proline at concentrations of 2.50 mM for thiolutin (133 mg/g DCW), 1.25 mM for senecioyl-pyrrothine, tigloyl-pyrrothine and butanoyl-pyrrothine production (29, 23 and 3.9 mg/g DCW, respectively). Production of all dithiolopyrrolones strongly decreased as the l-methionine or dl-ethionine concentration was increased in the culture medium.     

Impact of methyl jasmonate on squalene biosynthesis in microalga Schizochytrium mangrovei

Squalene is an effective antioxidant and a potential chemopreventive agent. In this work, the effect of methyl jasmonate (MJA) on squalene biosynthesis in microalga Schizochytrium mangrovei was investigated. The maximum squalene content (1.17 ± 0.06 mg/g cell dry weight, DW) reached during the next 3 h after MJA treatment (0.1 mM) at 48 h of cultivation, which was 60% higher than that of control. The activity of squalene synthase (SS) increased 2-fold over control at this point. The maximum cholesterol content of 0.45 ± 0.03 mg/g DW was reached at hour 51 when MJA concentration was 0.4 mM, whereas the squalene content was lower at this point. The observations suggested that the increased squalene content was resulted from an increased activity of SS. MJA could be used to regulate the key enzymes in squalene biosynthetic pathway for the increased production of this compound in thraustochytrids. This research also provided novel information on the stimulation effect of methyl jasmonate on the biosynthesis of essential intermediate involved in the primary metabolism in microorganism.     

Biosynthesis of plant-derived ginsenoside Rh2 in yeast via repurposing a key promiscuous microbial enzyme

Ginsenoside Rh2 is a potential anticancer drug isolated from medicinal plant ginseng. Fermentative production of ginsenoside Rh2 in yeast has recently been investigated as an alternative strategy compared to extraction from plants. However, the titer was quite low due to low catalytic capability of the key ginseng glycosyltransferase in microorganisms. Herein, we have demonstrated high-level production of ginsenoside Rh2 in Saccharomyces cerevisiae via repurposing an inherently promiscuous glycosyltransferase, UGT51. The semi-rationally designed UGT51 presented an ~1800-fold enhanced catalytic efficiency (k_cat/K_m) for converting protopanaxadiol to ginsenoside Rh2 in vitro. Introducing the mutant glycosyltransferase gene into yeast increased Rh2 production from 0.0032 to 0.39 mg/g dry cell weight (DCW). Further metabolic engineering, including preventing Rh2 degradation and increasing UDP-glucose precursor supply, increased Rh2 production to 2.90 mg/g DCW, which was more than 900-fold higher than the starting strain. Finally, fed-batch fermentation in a 5-L bioreactor led to production of ~300 mg/L Rh2, which was the highest titer reported.     

Effect of oxygen supply on biomass and helvolic acid production in submerged fermentation of Cordyceps taii

The entomogenous fungus Cordyceps taii, a traditional Chinese medicinal mushroom, exhibits potent important pharmacological effects and it has great potential for health foods and medicine. In this work, the effects of oxygen supply on production of biomass and bioactive helvolic acid were studied in shake-flask fermentation of C. taii mycelia. The value of initial volumetric oxygen transfer coefficient (K_La) within 10.1–33.8 h⁻¹ affected the cell growth, helvolic acid production and expression levels of biosynthetic genes. The highest cell concentration of 17.2 g/L was obtained at 14.3 h⁻¹ of initial K_La. The highest helvolic acid production was 9.6 mg/L at 10.1 h⁻¹ of initial K_La. The expression levels of three genes encoding hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and squalene synthase were down-regulated on day 2 and day 8 but up-regulated on day 14 at an initial K_La value of 10.1 h⁻¹ vs. 33.8 h⁻¹, which well corresponded to the helvolic acid biosynthesis in those conditions. The information obtained would be helpful for improving the biomass and helvolic acid production in large-scale fermentation of C. taii.     

Improvement of acetoin reductase activity enhances bacitracin production by Bacillus licheniformis

Bacitracin fermentation by Bacillus licheniformis in this work showed three characteristics: (1) the extracellular propionate, butyrate, acetoin and 2,3-butanediol accumulates under conditions of low dissolved oxygen (zero after 4 h cultivation), reaching a total content of approximately 11.1 g/L; (2) cell growth occurs quickly subsequent to cell autolysis and the second growth; and (3) there is a low content of 2,3-butanediol, a reduced product of acetoin catalyzed by acetoin reductase, in the culture process. In this study, addition of MnCl₂ (0.3 mg/L) to the production medium increased the acetoin reductase activity, redirected the NADH oxidation partly from the propionate- and butyrate-production pathways to the 2,3-butanediol synthesis pathway, reduced the intracellular NADH/NAD⁺ ratio, and facilitated cell growth, ultimately achieving a 11.6% increase in bacitracin production (1076 U/mL) versus the control. The results provide useful information regarding large-scale bacitracin production by B. licheniformis.     

Changes of ginsenosides in Korean red ginseng (Panax ginseng) fermented by Lactobacillus plantarum M1

To obtain microorganisms for the microbial conversion of ginsenosides in red ginseng powder (RGP), Lactobacillus species (M1–M4 and P1–P4) were isolated from commercial ginseng products. Strain M1 was determined to be L. plantarum by 16S rRNA sequencing. Red ginseng powder (RGP) fermented by L. plantarum M1 had a high total content of ginsenosides (142.4 mg/g) as compared to the control (121.8 mg/g). In particular, the ginsenoside metabolites Rg3, Rg5, Rk1, Compound K (CK), Rh1, and Rg2 showed a high level in the fermented RGP (65.5 mg/g) compared to the control (32.7 mg/g). During fermentation for 7 days, total sugar content decreased from 8.55 mg/g to 4 mg/g, uronic acid content reached its maximum (53.43 μg/g) at 3 days, and total ginsenoside content increased to 176.8 mg/g at 4 days. In addition, ginsenoside metabolites increased from 38.0 mg/g to 83.4 mg/g at 4 days of fermentation. Using everted instestinal sacs of rats, the fermented red ginseng showed a high transport level (10.3 mg of polyphenols/g sac) compared to non-fermented red ginseng (6.67 mg of polyphenols/g sac) after 1 h. These results confirm that fermentation with L. plantarum M1 is very useful for preparing minor ginsenoside metabolites while being safe for foods.     

Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production

Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45 mg/L to 71 mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71 mg/L to 140 mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140 mg/L to 330 mg/L). Through fed-batch fermentation using resting cells, over 1.1 g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date.     

Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium), are thought to be representative plant of Panax species, have important commercial value and are used in worldwide. Panax species produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Dammarane-type ginsenosides dominate over oleanane-type not only in amount but also in structural varieties. Researches shows that the saponins content in American ginseng is higher than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed that protopanaxadiol derived from dammarenediol-II, is a key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American ginseng ginsenoside biosynthesis. Here, we first cloned and identified a P450 gene named PqD12H encoding enzymes catalyzed dammarenediol-II to protopanaxadiol by RT-PCR using degenerate primers designed based on sequence homology. In vitro, the ectopic expression of PqD12H in recombinant WAT21 yeast resulted in protopanaxadiol production after dammarenediol-II was added to the culture medium. In vivo, we established both PgCYP716A47 and PqD12H RNAi transgenic. The RT-PCR and HPLC analysis of the final products of protopanaxadiol and protopanaxatriol showed a result that declined level of protopanaxadiol-type and protopanaxatriol-type ginsenosides. It suggested that the P450 synthase content or expression in American ginseng exceed than in Asian ginseng. The result elucidated the evolution relationship of P450s and the reason of different saponins content among Panax species.     

Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137

The influence of organic acids on growth and dithiolopyrrolone antibiotic production by Saccharothrix algeriensis NRRL B-24137 was studied. The production of dithiolopyrrolones depends upon the nature and concentration of the organic acids in the culture medium. Study of the nature of organic acids showed that the most effective organic acids for thiolutin specific production were maleic, 4-hydroxybenzoic, benzentetracarboxylic, pantothenic, pivalic and pyruvic acids (which yielded almost five-fold over the starting medium) and pimelic acid (more than three-fold). 4-Bromobenzoic acid showed the best production of senecioyl-pyrrothine (59 mg g⁻¹ DCW). Tiglic acid showed the best production of tigloyl-pyrrothine (22 mg g⁻¹ DCW). The highest yield of isobutyryl-pyrrothine (7.6 mg g⁻¹ DCW) was observed in the presence of crotonic acid. Sorbic acid yielded the best production of butanoyl-pyrrothine (26 mg g⁻¹ DCW). Methacrylic, butyric, pyruvic and 4-bromobenzoic acids also exhibited the best production of butanoyl-pyrrothine (27–11-fold).Study of organic acid concentration showed that among the selected organic acids, pimelic acid yielded the highest specific production of thiolutin (91 mg g⁻¹ DCW) at 7.5 mM; and senecioyl-pyrrothine (11 mg g⁻¹ DCW), tigloyl-pyrrothine (9 mg g⁻¹ DCW) and butanoyl-pyrrothine (3.5 mg g⁻¹ DCW) at 5 mM. Pyruvic acid at 1.25 mM enhanced the production of senecioyl-pyrrothine (4.3 mg g⁻¹ DCW). The maximum production of tigloyl-pyrrothine (18.6 mg g⁻¹ DCW) was observed in the presence of tiglic acid at 2.5 mM. Maximum production of isobutyryl-pyrrothine was observed in the presence of 7.5 mM tiglic acid. In addition, methacrylic acid (at 5 mM) and butyric acid (at 2.5 mM) enhanced the production of butanoyl-pyrrothine (26 and 20 times, respectively).The above results can be employed in the optimisation of the culture medium for the production of dithiolopyrrolone in higher quantities.     

Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing

2,3-Butanediol is a promising valuable chemical that can be used in various areas as a liquid fuel and a platform chemical. Here, 2,3-butanediol production in Saccharomyces cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered adh1Δadh2Δadh3Δadh4Δadh5Δgpd1Δgpd2Δ strain overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), S. cerevisiae 2,3-butanediol dehydrogenase (Bdh1), and L. lactis NoxE from a single multigene-expression vector produced 72.9 g/L 2,3-butanediol with the highest yield (0.41 g/g glucose) and productivity (1.43 g/(L·h)) ever reported in S. cerevisiae.     

The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7 g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639 g/(l h).     

Improved 2,3-butanediol production from corncob acid hydrolysate by fed-batch fermentation using Klebsiella oxytoca

Corncob acid hydrolysate, detoxed by sequently boiling, overliming and activated charcoal adsorption, was used for 2,3-butanediol production by Klebsiella oxytoca ACCC 10370. The effects of acetate in hydrolysate and pH on 2,3-butanediol production were investigated. It was found that acetic acid in hydrolysate inhibited the growth of K. oxytoca while benefited the 2,3-butanediol yield. With the increase in acetic acid concentration in medium from 0 to 4 g/l, the lag phase was prolonged and the specific growth rate decreased. The acetic acid inhibition on cell growth can be alleviated by adjusting pH to 6.3 prior to fermentation and a substrate fed-batch strategy with a low initial acetic acid concentration. Under the optimum condition, a maximal 2,3-butanediol concentration of 35.7 g/l was obtained after 60 h of fed-batch fermentation, giving a yield of 0.5 g/g reducing sugar and a productivity of 0.59 g/h l.     

Metabolic engineering of Bacillus sp. for diacetyl production

Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe³⁺ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.     

Stereoselective biocatalytic hydride transfer to substituted acetophenones by the yeast Metschnikowia koreensis

Freely suspended and variously entrapped viable cells of the yeast Metschnikowia koreensis were examined for the asymmetric reduction of prochiral acetophenone. A ketone substrate at 25 mM can be converted (92%) to the corresponding alcohol within 3 h using freely suspended cells [46 mg/mL dry cell weight (DCW)] at pH 9 (Tris buffer, 50 mM), 25 °C, in an agitated reactor (200 rpm). The reaction displayed an excellent stereoselectivity of >99%. Supplementation of the reaction mixture with glucose (20 g/L) greatly enhanced the rate of the bioreduction reaction likely because of improved cofactor recycling in the cells. The cells could successfully reduce various acetophenones substituted with electron withdrawing groups on the phenyl ring, particularly at the para-position compared to ortho- or meta-substituted acetophenones. The ketone reductase of M. koreensis showed Prelog-selectivity as the reaction exclusively yielded (S)-alcohols. The thermostability and the substrate tolerance of the yeast were improved by immobilization in calcium alginate beads. Immobilization reduced the effectiveness factor only slightly.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.