The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.     

Biphenyl‐Type Neolignan Derivatives from the Twigs of Magnolia denudata and Their Anti‐Inflammatory Activity

Two new biphenyl‐type neolignan derivatives, 2‐[2‐(hydroxymethyl)‐1‐benzofuran‐5‐yl]‐4‐(prop‐2‐en‐1‐yl)phenol ( 1 ) and 2′‐ethoxy‐5,5′‐di(prop‐2‐en‐1‐yl)biphenyl‐2‐ol ( 2 ), were isolated from the twigs of Magnolia denudata, together with six known compounds ( 3 – 8 ). The structures of 1 and 2 were determined through extensive 1D‐ and 2D‐NMR and mass‐spectrometric analyses. Magnolol ( 6 ) and honokiol ( 7 ) exhibited potent inhibition (IC₅₀ values=4.4±0.2 and 0.71±0.13 μg/ml, resp.) of O$\rm{{_{2}^{{^\cdot} -}}}$ generation by human nutrophils in response to N‐formyl‐L ‐methionyl‐L ‐leucyl‐L ‐phenylalanine/cytochalasin B (fMLP/CB). In addition, 2‐[2‐(hydroxymethyl)‐1‐benzofuran‐5‐yl]‐4‐(prop‐2‐en‐1‐yl)phenol ( 1 ), 2′‐ethoxy‐5,5′‐di(prop‐2‐en‐1‐yl)biphenyl‐2‐ol ( 2 ), magnolol ( 6 ), and vanillic acid ( 8 ) inhibited fMLP/CB‐induced elastase release with IC₅₀ values=6.4±1.5, 2.4±0.4, 1.5±0.2, and 4.8±0.5 μg/ml, respectively.     

A predictive high‐throughput scale‐down model of monoclonal antibody production in CHO cells

Multi‐factorial experimentation is essential in understanding the link between mammalian cell culture conditions and the glycoprotein product of any biomanufacturing process. This understanding is increasingly demanded as bioprocess development is influenced by the Quality by Design paradigm. We have developed a system that allows hundreds of micro‐bioreactors to be run in parallel under controlled conditions, enabling factorial experiments of much larger scope than is possible with traditional systems. A high‐throughput analytics workflow was also developed using commercially available instruments to obtain product quality information for each cell culture condition. The micro‐bioreactor system was tested by executing a factorial experiment varying four process parameters: pH, dissolved oxygen, feed supplement rate, and reduced glutathione level. A total of 180 micro‐bioreactors were run for 2 weeks during this DOE experiment to assess this scaled down micro‐bioreactor system as a high‐throughput tool for process development. Online measurements of pH, dissolved oxygen, and optical density were complemented by offline measurements of glucose, viability, titer, and product quality. Model accuracy was assessed by regressing the micro‐bioreactor results with those obtained in conventional 3 L bioreactors. Excellent agreement was observed between the micro‐bioreactor and the bench‐top bioreactor. The micro‐bioreactor results were further analyzed to link parameter manipulations to process outcomes via leverage plots, and to examine the interactions between process parameters. The results show that feed supplement rate has a significant effect (P < 0.05) on all performance metrics with higher feed rates resulting in greater cell mass and product titer. Culture pH impacted terminal integrated viable cell concentration, titer and intact immunoglobulin G titer, with better results obtained at the lower pH set point. The results demonstrate that a micro‐scale system can be an excellent model of larger scale systems, while providing data sets broader and deeper than are available by traditional methods. Biotechnol. Bioeng. 2009; 104: 1107–1120. © 2009 Wiley Periodicals, Inc.     

In sito galanthamine extraction during the cultivation of Leucojum aestivum L. shoot culture in two‐phase bubble column cultivation system

Two‐phase bioreactor cultivation system was developed and applied for in sito recovery of extracellular galanthamine during the cultivation of Leucojum aestivum L. shoot culture in a modified column bioreactor system. The inclusion of an external circulation column with adsorbent resin Amberlite XAD‐4 as a second phase, on the 21st day of the beginning of cultivation resulted in 1.25 folds increase in biomass accumulation and maximal amounts of accumulated galanthamine of 6 mg/L (3.1 mg/L intracellular and 2.9 mg/L extracellular). It was demonstrated that the inclusion of a second phase at the cultivation of the L. aestivum shoot culture in a bubble column bioreactor with internal sections redirected the alkaloid metabolism to galanthamine synthesis and inhibits the synthesis of hemanthamine and lycorine type alkaloids. Our research demonstrated that the application of the two‐phase cultivation systems could be an important tool to increase the yields of valuable secondary metabolites in plant tissue culture‐based bioprocess.     

High Density Cultivation of Panax notoginseng Cell Cultures with Methyl Jasmonate Elicitation in a Centrifugal Impeller Bioreactor

True ginseng roots contain “active compounds” called ginsenosides. The enhanced production of useful bioactive ginsenosides by high‐density cell cultures of Panax notoginseng in a self‐developed centrifugal impeller bioreactor (CIB) was achieved by adding methyl jasmonic acid (MJA) during cultivation. The production of the major, individual ginsenosides Rg₁, Re and Rb₁ was significantly enhanced in both 3‐L and 30‐L CIBs. The production titer of Rg₁, Re and Rb₁ ginsenosides in the 30‐L CIB was improved from 42 ± 8, 42 ± 9 and 41 ± 6 mg/L without MJA elicitation, to 104 ± 6, 71 ± 5 and 95 ± 6 mg/L with MJA elicitation, respectively. The ratio of Rb/Rg was slightly improved by MJA treatment in a 3‐L CIB but no apparent difference was observed in a 30‐L CIB. This work is useful for the understanding of the effects of large‐scale production on the individual ginseng saponins produced by plant cell cultures     

Generation of high rapamycin producing strain via rational metabolic pathway‐based mutagenesis and further titer improvement with fed‐batch bioprocess optimization

Rapamycin is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications. Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have rationally designed approaches to generate a rapamycin high producer strain of S. hygroscopicus HD‐04‐S. These have included alleviation of glucose repression, improved tolerance towards lysine and shikimic acid, and auxotrophy of tryptophan and phenylalanine through the application of stepwise UV mutagenesis. The resultant strain produced rapamycin at 450 mg/L in the shake flask scale. These fermentations were further scaled up in 120 and 20,000 L fermentors, respectively, at the pilot plant. Selected fermentation factors including agitation speed, pH, and on‐line supplementation were systematically evaluated. A fed‐batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. The final titer of rapamycin was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor. This work highlights a high rapamycin producing strain derived by mutagenesis and subsequent screening, fermentation optimization of which has now made it feasible to produce rapamycin on an industrial scale by fermentation. The strategies developed here should also be applicable to titer improvement of other important microbial natural products on an industrial scale. Biotechnol. Bioeng. 2010;107: 506–515. © 2010 Wiley Periodicals, Inc.     

Production and scale‐up of a monoclonal antibody against 17‐hydroxyprogesterone

The hybridoma 192 was used to produce a monoclonal antibody (MAb) against 17‐hydroxyprogesterone (17‐OHP), for possible use in screening for congenital adrenal hyperplasia (CAH). The factors influencing the MAb production were screened and optimized in a 2 L stirred bioreactor. The production was then scaled up to a 20 L bioreactor. All of the screened factors (aeration rate, stirring speed, dissolved oxygen concentration, pH, and temperature) were found to significantly affect production. Optimization using the response surface methodology identified the following optimal production conditions: 36.8°C, pH 7.4, stirring speed of 100 rpm, 30% dissolved oxygen concentration, and an aeration rate of 0.09 vvm. Under these conditions, the maximum viable cell density achieved was 1.34 ± 0.21 × 10⁶ cells mL^?1 and the specific growth rate was 0.036 ± 0.004 h^?1. The maximum MAb titer was 11.94 ± 4.81 μg mL^?1 with an average specific MAb production rate of 0.273 ± 0.135 pg cell^?1 h^?1. A constant impeller tip speed criterion was used for the scale‐up. The specific growth rate (0.040 h^?1) and the maximum viable cell density (1.89 × 10⁶ cells mL^?1) at the larger scale were better than the values achieved at the small scale, but the MAb titer in the 20 L bioreactor was 18% lower than in the smaller bioreactor. A change in the culture environment from the static conditions of a T‐flask to the stirred bioreactor culture did not affect the specificity of the MAb toward its antigen (17‐OHP) and did not compromise the structural integrity of the MAb. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.     

Production of recombinant HIV‐1 nef protein using different expression host systems: A techno‐economical comparison

Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno‐economically using HIV‐1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52–83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Evaluation of piggyBac‐mediated CHO pools to enable material generation to support GLP toxicology studies

Generating purified protein for GLP toxicology studies (GLP‐Tox) represents an important and often rate limiting step in the biopharmaceutical drug development process. Toxicity testing requires large amounts of therapeutic protein (>100 g), typically produced in a single 500–2,500 L bioreactor, using the final CHO clonally derived cell line (CDCL). One approach currently used to save time is to manufacture GLP‐Tox material using pools of high‐producing CHO CDCLs instead of waiting for the final CDCL. Recently, we reported CHO pools producing mAb titers >7 g/L using piggyBac‐mediated gene integration (PB CHO pools). In this study, we wanted to leverage high titer PB CHO pools to produce GLP‐Tox material. A detailed product quality attribute (PQA) assessment was conducted comparing PB CHO pools to pooled Top4 CDCLs. Four mAbs were evaluated. First, we found that PB CHO pools expressed all four mAbs at high titers (2.8–4.4 g/L in shake flasks). Second, all four PB CHO pools were aged to 55 generations (Gen). All four PB CHO Pools were found to be suitable over 55 Gen. Finally, we performed bioreactor scale‐up. PB CHO pool titers (3.7–4.8 g/L) were similar or higher than the pooled Top 4 CDCLs in 5 L bioreactors (2.4–4.1 g/L). The PQAs of protein derived from PB CHO pools were very similar to pooled Top 4 CHO CDCLs according to multiple orthogonal techniques including peptide mapping analysis. Taken together, these results demonstrate the technical feasibility of using PB CHO pools to manufacture protein for GLP‐Tox. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1436–1448, 2017     

Optimization of naringenin and p‐coumaric acid hydroxylation using the native E. coli hydroxylase complex,HpaBC

Flavonoids are a growing class of bioactive natural products with distinct and interesting bioactivity both in vitro and in vivo. The extraction of flavonoids from plant sources is limited by their low natural abundance and commonly results in a mixture of products that are difficult to separate. However, due to recent advances, the microbial production of plant natural products has developed as a promising alternative for flavonoid production. Through optimization of media, induction temperature, induction point, and substrate delay time, we demonstrate the highest conversion of naringenin to eriodictyol (62.7 ± 2.7 mg/L) to date, using the native E. coli hydroxylase complex, HpaBC. We also show the first evidence of in vivo HpaBC activity towards the monohydroxylated flavan‐3‐ol afzelechin with catechin product titers of 34.7 ± 1.5 mg/L. This work confirms the wide applicability of HpaBC towards realizing efficient de novo production of various orthohydroxylated flavonoids and flavonoid derived products in E. coli. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:21–25, 2016     

High‐throughput mAb expression and purification platform based on transient CHO

A high‐cell‐density transient transfection system was recently developed in our laboratory based on a CHO‐GS‐KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7‐day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24‐deep‐well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high‐throughput transfection process to a semiautomated protein A purification process. Using a Biomek FX^p liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer‐exchange steps. We optimized both the batch‐binding and the batch elution steps. The length of the batch‐binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 ± 0.05 mg (average concentration of 0.65 ± 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early‐stage discovery effort, at an average yield of 0.79 ± 0.31 mg (average concentration of 1.59 ± 0.63 mg/mL). The combination of parallel high‐yielding transient transfection and semiautomated high‐throughput protein A purification represents a valuable mAb drug discovery tool. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:239–247, 2015     

Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides

Fatty alcohols (FOHs) are important feedstocks in the chemical industry to produce detergents, cosmetics, and lubricants. Microbial production of FOHs has become an attractive alternative to production in plants and animals due to growing energy demands and environmental concerns. However, inhibition of cell growth caused by intracellular FOH accumulation is one major issue that limits FOH titers in microbial hosts. In addition, identification of FOH-specific exporters remains a challenge and previous studies towards this end are limited. To alleviate the toxicity issue, we exploited nonionic surfactants to promote the export of FOHs in Rhodosporidium toruloides, an oleaginous yeast that is considered an attractive next-generation host for the production of fatty acid-derived chemicals. Our results showed FOH export efficiency was dramatically improved and the growth inhibition was alleviated in the presence of small amounts of tergitol and other surfactants. As a result, FOH titers increase by 4.3-fold at bench scale to 352.6 mg/L. With further process optimization in a 2-L bioreactor, the titer was further increased to 1.6 g/L. The method we show here can potentially be applied to other microbial hosts and may facilitate the commercialization of microbial FOH production.     

Stirred tank bioreactor culture combined with serum‐/xenogeneic‐free culture medium enables an efficient expansion of umbilical cord‐derived mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell‐based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non‐invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)‐free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin‐based Cultispher^®S microcarriers and xeno‐free culture medium for the expansion of umbilical cord matrix (UCM)‐derived MSC. This system enabled the production of 2.4 (±1.1) x10⁵ cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)‐fold increase in cell number. The established protocol was then implemented in a stirred‐tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier‐based stirred culture system, using xeno‐free culture medium that suits the intrinsic features of UCM‐derived MSC represents an important step towards a GMP compliant large‐scale production platform for these promising cell therapy candidates.     

An Integrated System for the Bioremediation of Wastewater Containing Xenobiotics and Toxcic Metals

An integrated system for the biotreatment of acidic wastewaters containing both toxic metals and organics is presented. It consists of two bioprocess stages (i) an anaerobic, SRB stage (containing alkaline‐tolerant s ulfate‐ r educing b acteria) that at pH 8 (chosen to acclimatize the bacteria in the biomedium) produces high concentrations of total sulfide ions (more than 400 mg/L) which are added to the wastewater to precipitate the heavy metals out at pH 2 as metal sulfides, and (ii) an aerobic, acidophilic stage containing heterotrophic bacteria (WJB3) that degrade organic xenobiotics. The anaerobic system was comprised of a 4‐L fluidized bed bioreactor with immobilized SRB, a mixing tank, and a precipitation tank. The effluent from the bioreactor with a high concentration of sulfide ions was fed into a mixing tank where model wastewaters containing toxic metals and phenol at pH 2 were also fed at increasing loading rates until free metal ions could be detected in the precipitation tank outlet. Then the effluent from the precipitation tank outlet was fed into a 2.5‐L aerobic bioreactor in which phenol was degraded. In this research, 100 % removal efficiencies were obtained with wastewaters containing more than 400 mg/L metal ions and 900 mg/L phenol at a 6‐h HRT of the mixing tank.     

Economic analysis of pilot‐scale production of B‐phycoerythrin

β‐Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B‐phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two‐phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two‐phase systems in a scaled‐up process. This study analyzed the production of B‐Phycoerythrin using real data obtained during the scale‐up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two‐phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10‐year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B‐phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472–1479, 2016     

Bioprocess development for endospore production by Bacillus coagulans using an optimized chemically defined medium

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%–90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.     

Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5-methyltetrahydrofolate

5-Methyltetrahydrofolate (5-MTHF) is the major form of folate in human plasma and is the only folate form that can penetrate the blood–brain barrier. It has been widely used for the prevention and treatment of various diseases. It is mainly produced by chemical synthesis. However, the low production rate cannot meet the increasing demand. In addition, chemical synthesis is potentially detrimental to the environment. Despite various microorganisms synthetizing 5-MTHF, an efficient 5-MTHF bioproduction approach is lacking because of the tight regulation of the 5-MTHF pathway and limited metabolic flux toward the folic acid pathway. In this study, the 5-MTHF synthetic pathway in Bacillus subtilis was systematically engineered to realize 5-MTHF accumulation and further improve 5-MTHF production. Specifically, the 5-MTHF synthesis pathway with dihydrofolate (DHF) as the precursor was strengthened to shift the metabolic flux to 5-MTHF biosynthesis by replacing the native yitJ gene with Escherichia coli metF, knockout of purU, and overexpressing dfrA. The intracellular level of 5-MTHF increased 26.4-fold, reaching 271.64 µg/L. Next, the 5-MTHF precursor supply pathway was strengthened by co-overexpression of folC, pabB, folE, and yciA. This resulted in a 93.2-fold improvement of the 5-MTHF titer, which reached 960.27 µg/L. Finally, the clustered regularly interspaced short palindromic repeats interference system was used to identify key genes in the competitive and catabolic pathways for repression to further shift the metabolic flux toward 5-MTHF biosynthesis. The repression of genes thyA (existing in the purine metabolic pathway), pheA (existing in the competitive metabolic pathway), trpE (existing in the competitive metabolic pathway), and panB (existing in the pantoate synthesis pathway) significantly increased the titer of 5-MTHF. By repressing the pheA gene, the 5-MTHF titer reached 1.58 mg/L, which was 153.8-fold that of the wild-type strain of B. subtilis 168. Through medium optimization, the 5-MTHF titer reached 1.78 mg/L, which was currently the highest titer of 5-MTHF in B. subtilis. Apart from the highest titer of 5-MTHF, the highest titer of total folates including 5-MTHF, 5-FTHF, folic acid, and THF could reach 3.31 mg/L, which was 8.5-fold that in B. subtilis. To the best of our knowledge, the 5-MTHF and total folate titers reported here are the highest using a Generally regarded as safe (GRAS) bacterium as the production host. Overall, this study provides a good starting point for further metabolic engineering to achieve efficient biosynthesis of 5-MTHF by GRAS bacteria.     

Identification of progesterone and 5α‐dihydroprogesterone (5α‐DHP) in zebras by two‐dimensional high‐performance liquid chromatography and their dependence on the state of reproduction

Because of its low levels in late pregnancy, the relationship of progesterone to pregnancy maintenance in Equidae is not obvious. This study investigated the levels of progesterone (4‐pregnane‐3,20‐dione; P4) and 5α‐dihydroprogesterone (5α‐DHP) during pregnancy in zebras in relation to reproductive state. Blood samples from female zebras (Equus burchelli, E. zebra hartmannae, E. grevyi) were taken at Dvur Kralove Zoo. Progesterone and 5α‐DHP were separated by high‐performance liquid chromatography techniques and detected by cross‐reacting antibodies. Identification of progestins was achieved by comparing the identity of peaks of the samples with a standard. In E. z. hartmannae progesterone, values reached 50 ng/mL at the beginning of pregnancy and dropped to levels below 1 ng/mL during the second half of pregnancy. In contrast, 5α‐DHP increased up to 123 and 183 ng/mL during late pregnancy in E. z. hartmannae and E. burchelli, respectively. In E. grevyi, 5α‐DHP levels of 368 ng/mL were obtained during pregnancy, whereas progesterone values were similar in pregnant and non‐pregnant individuals. These marked differences in the course of progesterone and 5α‐DHP levels point to the importance of 5α‐DHP for pregnancy maintenance in zebras. Zoo Biol 18:325–333, 1999. © 1999 Wiley‐Liss, Inc.