Fed-batch Fermentation Conditions Research Articles

High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation

BackgroundMethane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products.ResultsIn this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high–cell–density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane.ConclusionsThis study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

Optimization of the fermentative production of L-methionine by engineered Escherichia coli

As the only essential amino acid containing elemental sulphur, L-methionine has important physiological and biochemical functions in living organisms. However, the fermentative production of L-methionine has not met the requirements of industrial production because of its low production level. In this paper, the fermentation process of an efficient L-methionine producing strain E. coli W3110ΔIJAHFEBC trc-fliY trc-malY/PAM glyA-22 metF constructed previously was systematically optimized. Based on the optimal initial glucose concentration, the effects of different fed-batch fermentation processes, including DO-Stat, pH-Stat, controlling residual sugar control at different level and feeding glucose with constant rate, on L-methionine fermentation were studied. It was found that the control of glucose concentration greatly affected the fermentation process. Subsequently, an optimal fed-batch fermentation process was developed, where the L-methionine titer was increased to 31.71 g/L, the highest yield reported to date, while the fermentation time was shortened to 68 h. Meanwhile, a fermentation kinetics model under the optimal fed-batch fermentation conditions was established, which fitted well with the biosynthesis process of L-methionine. This study may facilitate further development of the fermentative production of L-methionine.

Metabolic engineering of Escherichia coli for high-level production of the biodegradable polyester monomer 2-pyrone-4,6-dicarboxylic acid

2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable pseudo-aromatic dicarboxylic acid, is a promising building block compound for manufacturing biodegradable polyesters. This study aimed to construct high-performance cell factories enabling the efficient production of PDC from glucose. Firstly, the effective enzymes of the PDC biosynthetic pathway were overexpressed on the chromosome of the 3-dehydroshikimate overproducing strain. Consequently, the one-step biosynthesis of PDC from glucose was achieved. Further, the PDC production was enhanced by multi-copy integration of the key gene PsligC encoding 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and co-expression of Vitreoscilla hemoglobin. Subsequently, the PDC production was substantially improved by redistributing the metabolic flux for cell growth and PDC biosynthesis based on dynamically downregulating the expression of pyruvate kinase. The resultant strain PDC50 produced 129.37 g/L PDC from glucose within 78 h under fed-batch fermentation conditions, with a yield of 0.528 mol/mol and an average productivity of 1.65 g/L/h. The findings of this study lay the foundation for the potential industrial production of PDC.

Selection and improvement of Saccharomyces cerevisiae by direct and mass mating for integrated first and second generation (1G + 2G) ethanol production

The integrated 1G + 2G ethanol production process seems to be a feasible strategy considering the Brazilian scenario, in which sugarcane molasses and bagasse are available substrates in biorefineries. However, such process imposes harsh conditions to yeasts, which must be able to simultaneously deal with several stresses from molasses and hydrolysate. In this work, improved hybrid strains of Saccharomyces cerevisiae with higher resistance for an integrated 1G + 2G ethanol production were obtained by hybridization technique. Initially, 663 wild S. cerevisiae strains were screened in microculture growth assays. Seven strains were pre-selected out of 40 based on their stress multi-tolerance traits. Subsequently, these pre-selected strains underwent evaluation in cell-recycling fed-batch fermentation conditions utilizing a medium containing molasses-bagasse hydrolysate. The two most successful strains from this evaluation were subjected to sporulation, yielding haploid cells. These haploid cells were then subjected to crossbreeding, employing direct and mass mating techniques. From the initial pool of 7 pre-selected strains, 3 hybrid strains were selected due to their enhanced resistance under the demanding conditions of cell recycling fed-batch fermentation, particularly when utilizing a higher osmotic molasses-hydrolysate medium. These hybrid strains consistently exhibited high cell viability rates and effectively accumulated elevated levels of trehalose and glycogen over the course of five fermentation cycles. Remarkably, they maintained ethanol productivity at levels comparable to their respective parental strains. The genotyping molecular identification attested the difference between the hybrids and the parental strains. The 3 hybrids selected seem to present valuable backgrounds for metabolic engineering for lignocellulosic-derived xylose fermentation.

Metabolic engineering of Halomonas bluephagenesis for high-level mevalonate production from glucose and acetate mixture

Mevalonate (MVA) plays a crucial role as a building block for the biosynthesis of isoprenoids. In this study, we engineered Halomonas bluephagenesis to efficiently produce MVA. Firstly, by screening MVA synthetases from eight different species, the two efficient candidate modules, specifically NADPH-dependent mvaESEfa from Enterococcus faecalis and NADH-dependent mvaESLca from Lactobacillus casei, were integrated into the chromosome, leading to the construction of the H. bluephagenesis MVA11. Through the synergetic utilization of glucose and acetate as mixed carbon sources, MVA11 produced 11.2 g/L MVA with a yield of 0.45 g/g (glucose + acetic acid) in the shake flask. Subsequently, 10 beneficial genes out of 50 targets that could promote MVA production were identified using CRISPR interference. The simultaneous repression of rpoN (encoding RNA polymerase sigma-54 factor) and IldD (encoding L-lactate dehydrogenase) increased MVA titer (13.3 g/L) by 19.23% and yield (0.53 g/g (glucose + acetic acid)) by 17.78%, respectively. Furthermore, introducing the non-oxidative glycolysis (NOG) pathway into MVA11 enhanced MVA yield by 12.20%. Ultimately, by combining these strategies, the resultant H. bluephagenesis MVA13/pli-63 produced 13.9 g/L MVA in the shake flask, and the yield increased to 0.56 g/g (glucose + acetic acid), which was the highest reported so far. Under open fed-batch fermentation conditions, H. bluephagenesis MVA13/pli-63 produced 121 g/L of MVA with a yield of 0.42 g/g (glucose + acetic acid), representing the highest reported titer and yield in the bioreactor to date. This study demonstrates that H. bluephagenesis is one of the most favorable chassis for MVA production.

Improved Glutamic Acid Production Capacity of Corynebacterium glutamicum by the ARTP Mutagenesis Method

Glutamic acid is an important amino acid that is used widely in the fields of food, medicine, and agriculture. One of the methods of glutamic acid production is direct microbial fermentation, so the genetic stability and glutamic-acid-producing capacity of the producing strain are the keys to improving glutamic acid concentration. Experiments were carried out using Corynebacterium glutamicum GL−6 as the parental strain, with two iterations of mutagenesis by atmospheric and room temperature plasma (ARTP) and screening with agar plates tolerant to high sugar and malonic acid, and the best strains with stable phenotypes were verified by fermentation in 20 L tanks. The results show that the optimal mutagenesis time of ARTP was 140 s, with lethality and positive mutation rates of 93.0% and 15.6%, respectively. The concentrations of the high-sugar and malonic acid agar plates were 240 g/L and 35 g/L, respectively. A mutant strain, P−45, with improved glutamic acid production capacity and genetic stability, was obtained through two rounds of iterative mutagenesis screening. The concentration of this strain in the Erlenmeyer flasks was 17.7 g/L, which was 18.8% higher than that of the parental strain, GL−6, and could be inherited stably for 10 generations. In the glutamic acid synthesis pathway, the upregulation of the gene encoding citrate synthase (cs), gene encoding isocitrate dehydrogenase (icdh), and gene encoding glutamate dehydrogenase (gdh), and the downregulation of the gene encoding oxoglutarate dehydrogenase complex (odhc) increased the carbon flows of the TCA cycle and its branch metabolic flow to glutamic acid synthesis. P−45 showed a glutamic acid concentration of 147.0 g/L under fed-batch fermentation conditions in 20 L tanks, which was 81.5% higher than the starting strain, GL−6. This study provides a new technical solution for improving microbial metabolites and genetic stability.

Improving solubility and copy number of taxadiene synthase to enhance the titer of taxadiene in Yarrowia lipolytica

Taxadiene is an important precursor for the biosynthesis of highly effective anticancer drug paclitaxel, but its microbial biosynthesis yield is very low. In this study, we employed Yarrowia lipolytica as a microbial host to produce taxadiene. First, a “push–pull” strategy was adopted to increase taxadiene production by 234%. Then taxadiene synthase was fused with five solubilizing tags respectively, leading a maximum increase of 62.3% in taxadiene production when fused with SUMO. Subsequently, a multi-copy iterative integration method was used to further increase taxadiene titer, achieving the maximum titer of 23.7 mg/L in shake flask culture after three rounds of integration. Finally, the taxadiene titer was increased to 101.4 mg/L by optimization of the fed-batch fermentation conditions. This is the first report of taxadiene biosynthesis accomplished in Y. lipolytica, serving as a good example for the sustainable production of taxadiene and other terpenoids in this oleaginous yeast.

Elimination of enzymes catalysis compartmentalization enhancing taxadiene production in Saccharomyces cerevisiae.

Taxadiene is an important precursor in taxol biosynthesis pathway, but its biosynthesis in eukaryotic cell factories is limited, which seriously hinders the biosynthesis of taxol. In this study, it is found that there was the catalysis compartmentalization between two key exogenous enzymes of geranylgeranyl pyrophosphate synthase and taxadiene synthase (TS) for taxadiene synthesis progress, due to their different subcellular localization. Firstly, the enzyme-catalysis compartmentalization was overcome by means of the intracellular relocation strategies of taxadiene synthase, including N-terminal truncation of taxadiene synthase and enzyme fusion of GGPPS-TS. With the help of two strategies for enzyme relocation, the taxadiene yield was increased by 21% and 54% respectively, among them the GGPPS-TS fusion enzyme is more effective. Further, the expression of GGPPS-TS fusion enzyme was improved via the multi-copy plasmid, resulting that the taxadiene titer was increased by 38% to 21.8mg/L at shake-flask level. Finally, the maximum taxadiene titer of 184.2mg/L was achieved by optimization of the fed-batch fermentation conditions in 3L bioreactor, which is the highest reported titer of taxadiene biosynthesis accomplished in eukaryotic microbes. This study provides a successful example for improving biosynthesis of complex natural products by solving the critical problem of multistep enzymes catalysis compartmentalization.

Metabolic Engineering of De Novo Pathway for the Production of 2'-Fucosyllactose in Escherichia coli.

2'-Fucosyllactose (2'-FL), one of the most abundant oligosaccharides in human milk, has gained increased attention owing to its nutraceutical and pharmaceutical potential. However, limited availability and high-cost of preparation have limited its widespread application and in-depth investigation of its potential functions. Here, a modular pathway engineering was implemented to construct an Escherichia coli strain to improve the biosynthesis titer of 2'-FL. Before overexpression of manB, manC, gmd, wcaG, and heterologous expression of futC, genes wcaJ and lacZ encoding UDP-glucose lipid carrier transferase and β-galactosidase, respectively, were inactivated from E. coli BL21 (DE3) with the CRISPR-Cas9 system, which inhibited the production of 2'-FL. The results showed that final shake flask culture yielded a 3.8-fold increase in 2'-FL (0.98g/L) from the engineered strain ELC07. Fed-batch fermentation conditions were optimized in a 3-L bioreactor. The highest titer of 2'-FL (18.22g/L) was obtained, corresponding to a yield of 0.25g/g glycerol and a substrate conversion of 0.88g/g lactose.

Enhanced β-mannanase production by Bacillus licheniformis by optimizing carbon source and feeding regimes

Bacillus licheniformis HDYM-04 was isolated in flax retting water and showed β-mannanase activity. Carbon sources for β-mannanase production, as well as the fermentation conditions and feeding strategy, were optimized in shake flasks. When glucose or konjac powder was used as the carbon source, the β-mannanase activity was 288.13 ± 21.59 U/mL and 696.35 ± 23.47 U/mL at 24 h, respectively, which was approximately 4.4- to 10.68-fold higher than the values obtained with wheat powder. When 0.5% (w/v) glucose and 1% (w/v) konjac powder were added together, maximum enzyme activities of 789.07 ± 25.82 U/mL were obtained, an increase of 13.35% compared to the unoptimized cultures with only 1% (w/v) konjac powder. The enzyme activity decreased in the presence of 1% (w/v) konjac powder, but the highest enzyme activity was 1,533.26 ± 33.74 U/mL, a 1.2-fold increase compared with that in nonoptimized cultures; when 0.5% (w/v) glucose was used, the highest enzyme activity was 966.53 ± 27.84 U/mL, an increase in β-mannanase activity of 38.79% compared with control cultures. In this study, by optimizing fed-batch fermentation conditions, the yield of β-mannanase produced by HDYM-04 was increased, laying the foundation for the industrial application and further research of B. licheniformis HDYM-04.

Life cycle assessment of bioethanol production from Pennisetum sp. using fed‐batch simultaneous saccharification and cofermentation at high solid loadings

Considerable progress has been achieved for the production of bioethanol from lignocellulosic biomass. However, increasing the substrate concentration has been shown to decrease ethanol productivity. Therefore, Saccharomyces cerevisiae (S. cerevisiae) and Pichia sp. were used for ethanol production from glucose and xylose sugars in the present investigation. Furthermore, coculture fermentations were conducted for 13 g/L of sugar (10 g of glucose and 3 g/L of xylose). It was further used for ethanol production from ultrasonication-assisted NaOH (UA-NaOH) pretreated and enzymatically saccharified biomass in batch and fed-batch fermentation conditions. Furthermore, fed-batch fermentation was used for separate hydrolysis and cofermentation (SHCF) and simultaneous saccharification and cofermentation (SSCF) in shake flask conditions. The highest ethanol production of 12.2 and 7.9 g/L was observed for fed-batch SSCF deenanath grass (DG) and hybrid Napier grass (HNG) (Palkonal MBW as the enzyme) biomass (80 g/L), respectively, in shake flask conditions. However, increasing the biomass concentration to 270 g/L produced an ethanol concentration of 77.6 and 51.3 g/L for DG and HNG, respectively, in fed-batch SSCF conditions in a bioreactor. Furthermore, nuclear magnetic resonance studies of the residual biomass of DG revealed lower carbohydrate content, demonstrating the efficiency of the fermentation strategy. Life cycle analysis of DG and HNG revealed DG had a higher yield as well as lower environmental impact than HNG. Thus, the fed-batch SSCF process can be considered a promising technique for biorefinery-based bioethanol production from Pennisetum sp. in the future. Novelty Statement For making lignocellulosic fuel ethanol economically competitive as compared to fossil fuels, it is important to achieve higher titers of ethanol from increased substrate concentration. But an increase in the substrate concentration decreases the ethanol productivity. In this study, the fed-batch simultaneous saccharification and fermentation produced higher titers of about 77.6 g/L ethanol even at higher substrate loadings. Besides, The LCA results demonstrated lower environmental toxicity, indicating the efficiency of the pretreatment and simultaneous saccharification and cofermentation process.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70gl-1 in 500ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5gl-1 , which is 5·6 times higher than before (15·6gl-1 ). O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

Strengthening the TCA cycle to alleviate metabolic stress due to blocking by-products synthesis pathway in Klebsiella pneumoniae.

1,3-Propanediol (1,3-PDO) is an important synthetic monomer for the production of polytrimethylene terephthalate (PTT). Here, we engineered Klebsiella pneumoniae by a multi-strategy to improve 1,3-PDO production and reduce by-products synthesis. First, the 2,3-butanediol (2,3-BDO) synthesis pathway was blocked by deleting the budB gene, resulting in a 74% decrease of 2,3-BDO titer. The synthesis of lactate was decreased by 79% via deleting the ldhA gene, leading to a 10% increase of 1,3-PDO titer. Further, reducing ethanol synthesis by deleting the aldA gene led to a 64% decrease of ethanol titer, and the 1,3-PDO titer and yield on glycerol increased by 12 and 10%, respectively. Strengthening the TCA cycle by overexpressing the mdh gene improved 1,3-PDO synthesis effectively. Under 5-L fed-batch fermentation conditions, compared to wild type strain, the production of 2,3-BDO, lactate and ethanol in the mutant strain decreased by 73, 65 and 50%, respectively. Finally, the production of 1,3-PDO was 73.5g/L with a molar yield of 0.67mol/mol glycerol, improved 16% and 20%, respectively. This work provides a combined strategy for improving 1,3-PDO production by strengthening the TCA cycle to relieve metabolic stress by deleting genes of by-products synthesis, which was also beneficial for the extraction and separation of downstream products.

Efficient Biosynthesis of R-(-)-Linalool through Adjusting the Expression Strategy and Increasing GPP Supply in Escherichia coli.

R-(-)-linalool is widely used in pharmaceutical, agrochemical, and fragrance industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method is urgently needed. To induce the R-(-)-linalool biosynthesis pathway in Escherichia coli, we expressed several heterologous (3R)-linalool synthases (LISs) and then chose a suitable LIS from Streptomyces clavuligerus (bLIS) for further study. The bLIS expression was markedly elevated by using optimized ribosomal binding sites and protein fusion tags. To increase the geranyl diphosphate content, we tested various alterations in prenyltransferases and their mutants. The final strain accumulated 100.1 and 1027.3 mg L-1 R-(-)-linalool under shake flask and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date. This work could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.

High-level soluble expression of human Cu,Zn superoxide dismutase with high activity in Escherichia coli.

As the most important member of antioxidant defense system, human Cu,Zn superoxide dismutase (hCu,Zn-SOD) protects cells against the free radicals produced by aerobic metabolism. hCu,Zn-SOD has been widely used in food, cosmetic and medicine industry due to its health benefits and therapeutic potentials. However, a more extensive application of hCu,Zn-SOD is limited by the challenge of expensive and low production of high-activity hCu,Zn-SOD in large scale. In this study, the codon-optimized hCu,Zn-SOD gene was synthesized, cloned into pET-28a( +) and transformed into Escherichia coli BL21(DE3). After induction with IPTG or lactose, hCu,Zn-SOD was highly expressed as soluble form in LB medium with 800μM Cu2+ and 20μM Zn2+ at 25°C. The recombinant hCu,Zn-SOD was efficiently purified by nickel affinity chromatography. Through optimization of fed-batch fermentation conditions, 342mgpurified hCu,Zn-SOD was obtained from 1 L cultures fermented in a 3-L bioreactor. Furthermore, the recombinant hCu,Zn-SOD retained the enzymatic specific activity of 46,541 U/mg. This study has openedupaneffective avenue for industrial production of hCu,Zn-SOD through microbial fermentation in the future.

Microbial Synthesis of Human-Hormone Melatonin at Gram Scales.

Melatonin is a commercially attractive tryptophan-derived hormone. Here we describe a bioprocess for the production of melatonin using Escherichia coli to high titers. The first engineered strain produced 0.13 g/L of melatonin from tryptophan under fed-batch fermentation conditions. A 4-fold improvement on melatonin titer was further achieved by (1) protein engineering of rate-limiting tryptophan hydroxylase to improve 5-hydroxytryptophan biosynthesis and (2) chromosomal integration of aromatic-amino-acid decarboxylase to limit byproduct formation and to minimize gene toxicity to the host cell. Fermentation optimization improved melatonin titer by an additional 2-fold. Deletion of yddG, a tryptophan exporter, exhibited an additive beneficial effect. The final engineered strain produced ∼2.0 g/L of melatonin with tryptophan supplemented externally and ∼1.0 g/L with glucose as the sole carbon source for tryptophan supply. This study lays the foundation for further developing a commercial melatonin-producing E.coli strain.

Improved 2,3-butanediol yield and productivity from lignocellulose biomass hydrolysate in metabolically engineered Enterobacter aerogenes

We previously engineered Enterobacter aerogenesfor glucose and xylose co-utilization and 2,3-butanediol production. Here, strain EMY-22 was further engineered to improve the 2,3-butanediol titer, productivity, and yield by reducing the production of byproducts. To reduce succinate production, the budABC operon and galP gene were overexpressed, which increased 2,3-butanediol production. For further reduction of succinate and 2-ketogluconate production, maeA was selected and overexpressed in EMY-22. The optimally engineered strain produced 2,3-butanediol for a longer time and showed reduced byproduct formation from sugarcane bagasse hydrolysate under flask cultivation conditions. The engineered strain displayed 66.6, 13.4, and 16.8% improvements in titer, yield, productivity of 2,3-butanediol, respectively, compared to its parental strain under fed-batch fermentation conditions. The data demonstrate that the metabolic engineering to reduce byproduct formation is a promising strategy to improve 2,3-butanediol production from lignocellulosic biomass.

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli.

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L-1 ·h-1 , respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.

Modulating heterologous pathways and optimizing fermentation conditions for biosynthesis of kaempferol and astragalin from naringenin in Escherichia coli.

Kaempferol and astragalin are used as standards to assess the quality of Ginkgo biloba extract and Radix astragali, respectively, and possess numerous biological properties. In this study, we constructed a recombinant strain with a highly efficient biosynthetic pathway of kaempferol by screening key enzyme genes, designing a synthetic fusion enzyme and increasing the gene copy number. By optimizing conversion and fed-batch fermentation conditions, maximal kaempferol production reached 1184.2 ± 16.5mg/L, which represents the highest yield of kaempferol from naringenin reported to date. Based on this result, glycosyltransferase (AtUGT78D2) and an efficient UDP-glucose synthesis pathway were introduced into the recombinant strain to produce astragalin, resulting in maximal astragalin production at 1738.5 ± 24.8mg/L without kaempferol accumulation. The efficient synthesis pathway described in this study for kaempferol and astragalin biosynthesis can be widely used for flavonoid biosynthesis in Escherichia coli.

Presence of protein production enhancers results in significantly higher methanol-induced protein production in Pichia pastoris

BackgroundThe yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Here expression vectors are reported that address the different steps of the transcription–translation–secretion pathway of heterologous protein production.ResultsTranscription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements. Multiple copy strains were selected using a zeocin resistance marker in the expression cassette and showed another sixfold higher yield. Modification of the C-terminal amino acid sequence of the secretion signal did not significantly improve the production yield. Similar data were obtained for the production of another protein, recombinant human interleukin 8. Upscaling to fed-batch fermentation conditions resulted in a twofold increase for reference strains, while for strains with enhancing elements a tenfold improvement was observed.ConclusionsPichia pastoris is used for recombinant protein production in industrial fermentations. By addressing the transcription and translation of mRNA coding for recombinant protein, significant yield improvement was obtained. The yield improvement obtained under microscale conditions was maintained under fed-batch fermentation conditions. These data demonstrate the potential of these expression vectors for large scale application as improved production of proteins has major implications on the economics and sustainability of biocatalyst dependent production processes e.g. for the production of pharmaceuticals and for the bioconversions of complex molecules.