Shake Flask Fermentation Research Articles

Efficient production of ε-poly-L-lysine from cassava bagasse hydrolysate used as carbon source by Streptomyces albulus US3-18.

The production of ε-poly-L-lysine (ε-PL) from cassava bagasse hydrolysate (CBH) by Streptomyces albulus US3-18 was investigated in this study. With 30g/L glucose from CBH, 1.30g/L ε-PL and 10.68g/L biomass were obtained in shake flask fermentation. Interestingly, the two values were increased by 14.0% and 21.5%, respectively, compared to the control (1.14g/L and 8.79g/L). Simultaneously, the activities of four key enzymes of ε-PL synthesis during CBH fermentation were enhanced to varying degrees. In batch fermentation of 5-L bioreactor, 3.39g/L ε-PL and 10.17g/L DCW were harvested with 40g/L glucose from CBH. The combination of fed-batch fermentation with two-stage pH strategy significantly increased ε-PL titer and biomass to 37.41g/L and 41.0g/L, respectively. Moreover, eleven volatile components were detected in CBH by GC-MS, and 6-pentyl-α-pyrone (6PP) was first identified as the most abundant volatile ingredient. The results in CBH fermentation demonstrated that S. albulus US3-18 exhibited high tolerance to these volatile byproducts. Using ICP-MS, the calcium concentration in CBH was determined as 195.0mg/(kghydrolyzate), and cobalt, copper, lead, chromium, mercury and arsenic were not detected. By adding 0.05g/L CaCl2 to M3G medium, ε-PL yield was improved by 28.0%, indicating calcium was one of the factors for the enhanced ε-PL production. The study provides a reference for the efficient production of ε-PL from low-cost agricultural residues.

Efficient production of clerodane and ent-kaurane diterpenes through truncated artificial pathways in Escherichia coli.

The clerodane and ent-kaurane diterpenoids are two typical categories of diterpenoid natural products with complicated polycyclic carbon skeletons and significant pharmacological activities. Despite exciting advances in organic chemistry, access to these skeletons is still highly challenging. Using synthetic biology to engineer microbes provides an innovative alternative to bypass synthetic challenges. In this study, we constructed two truncated artificial pathways to efficiently produce terpentetriene and ent-kaurene, two representative clerodane and ent-kaurane diterpenes, in Escherichia coli. Both pathways depend on the exogenous addition of isoprenoid alcohol to reinforce the supply of IPP and DMAPP via two sequential phosphorylation reactions. Optimization of these constructs provided terpentetriene and ent-kaurene titers of 66 ± 4 mg/L and 113 ± 7 mg/L, respectively, in shake-flask fermentation. The truncated pathways to overproduce clerodane and ent-kaurane skeletons outlined here may provide an attractive route to prepare other privileged diterpene scaffolds.

Adaptive Evolution-Assisted Riboflavin Production of Ashbya gossypii from Cane Molasses

In this study, adaptive evolution was used to improve the adaptation of Ashbya gossypii ATCC 10895 to the substrate (cane molasses) to ensure enhanced substrate utilization and increased riboflavin yield. During the adaptation process, antioxidant activity and riboflavin production ability were evaluated using shake flask fermentation to evaluate the strain response to environmental conditions. After the seventh day of fermentation, results showed that compared with the yield of the parent strain, that of A. gossypii A-24 increased by 97.5%, and the dry cell weight increased by 125%. The A-24 strain was used to further study the cofermentation process of cane molasses and glucose under different ratios. When the ratio of glucose to cane molasses was 1 : 4, the yield of riboflavin was 978 ± 5.37 mg/L, which was 3.7 times that of cane molasses monofermentation.

Development and application of a CRISPR-dCpf1 assisted multiplex gene regulation system in Bacillus amyloliquefaciens LB1ba02

Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which makes its CRISPR tool development lagging far behind other Bacillus species. Here, we adapted a nuclease-deficient mutant dCpf1 (D917A) of Cpf1 and developed a CRISPR/dCpf1 assisted multiplex gene regulation system for the first time in B. amyloliquefaciens LB1ba02. A 73.9-fold inhibition efficiency and an optimal 1.8-fold activation effect at the − 327 bp site upstream of the TSS were observed in this system. In addition, this system achieved the simultaneous activation of the expression of three genes (secE, secDF, and prsA) by designing a crRNA array. On this basis, we constructed a crRNA activation library for the proteins involved in the Sec pathway, and screened 7 proteins that could promote the secretion of extracellular proteins. Among them, the most significant effect was observed when the expression of molecular motor transporter SecA was activated. Not only that, we constructed crRNA arrays to activate the expression of two or three proteins in combination. The results showed that the secretion efficiency of fluorescent protein GFP was further increased and an optimal 9.8-fold effect was observed when SecA and CsaA were simultaneously activated in shake flask fermentation. Therefore, the CRISPR/dCpf1-ω transcription regulation system can be applied well in a restriction-modification system strain and this system provides another CRISPR-based regulation tool for researchers who are committed to the development of genetic engineering and metabolic circuits in B. amyloliquefaciens.

Transcriptome Analysis Reveals Potential Mechanisms of L-Serine Production by Escherichia coli Fermentation in Different Carbon-Nitrogen Ratio Medium.

L-serine is an industrially valuable amino acid that is widely used in the food, cosmetics and pharmaceutical industries. In this study, transcriptome sequencing technology was applied to analyze the changes in gene expression levels during the synthesis of L-serine in Escherichia coli fermentation. The optimal carbon–nitrogen ratio for L-serine synthesis in E. coli was determined by setting five carbon–nitrogen ratios for shake flask fermentation. Transcriptome sequencing was performed on E. coli fermented in five carbon–nitrogen ratio medium in which a total of 791 differentially expressed genes (DEGs) were identified in the CZ4_vs_CZ1 group, including 212 upregulated genes and 579 downregulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these DEGs showed that the effect of an altered carbon–nitrogen ratio on the fermentability of E. coli was mainly focused on metabolic pathways such as GABAergic synapse and the two-component system (TCS) in which the genes playing key roles were mainly gadB, gadA, glsA, glnA, narH and narJ. In summary, these potential key metabolic pathways and key genes were proposed to provide valuable information for improving glucose conversion during E. coli fermentation.

Evaluation of probiotic Lactobacillus plantarum CS fermented culture and its crude enzymes as chicken growth enhancers

Studies have shown that supplementation of important enzymes in poultry feed can improve the growth of birds. However, there were little or no available information on the utilization of such enzymes or the probiotic fermented culture in local feed production in Nigeria. Therefore, the aim of the study was focused on the evaluation of probiotic Lactobacillus plantarum fermented culture and its crude enzymes as chicken growth enhancers. The organism was first screened for amylase and protease producing potential using starch agar and skim milk agar respectively. Thereafter, confirmation of the produced amylase and protease enzymes via shake-flask fermentation at 30oC for 3 days was by 3,5-dinitro salicylic acid and Folin ciocalteu reagents respectively. The produced fermented culture and its recovered crude enzymes (2 %v/v) were respectively supplemented in the locally produced chicken feed for 10weeks. The chicken fed without supplement served as control and all their growths were determined every week. The screening results revealed that L. plantarum CS produced more amylase(29 mm) than protease(19 mm) with a total enzyme yield of 22.50 U/ml and 20.92 U/ml respectively, The chicken fed with supplements containing either the enzymes or the fermented culture grew faster and significantly higher (5 kg /10 wk) than those without the supplement (3.0 kg/10 wk)at p>0.05. The growth enhanced by the fermented culture ((5 kg/10 wk) was more than that stimulated by either the amylase (3.52 kg/10 wk) or the protease (3.86 kg/10 wk), although not significant. Supplementation with protease enzyme resulted in significant robust and faster growth (3.57 kg/ 7 wk) than that with amylase enzyme (3.37 kg/7 wk). These results indicated the great potentials of both L. plantarum fermented culture and its crude enzymes especially protease as chicken growth stimulants.

Expression of a SARS-CoV-2 neutralizing nanobody in Trichoderma reesei

Nanobodies derived from camelid single-chain antibodies have the advantages of being small, simple, highly soluble and stable. Nanobodies can be administered by inhalation and therefore is potentially valuable for the prevention and control of respiratory viruses. Trichoderma reesei is a food-grade protein expression host with a cellulase production capacity of up to 80 g/L, which can be employed for low-cost production of therapeutic proteins. In this study, a codon-optimized SARS-CoV-2 neutralizing nanobody Nb20 was expressed in T. reesei under a strong constitutive promoter Pcdna1. Nb20 protein was fused downstream of the N-terminal fragment of cellobiohydrolase Ⅰ, and the fusion protein can be intracellularly cleaved by the KEX2 protease to release Nb20. In a shake-flask fermentation using glucose medium, 47.4 mg/L Nb20 was detected in the culture after 48 h of cultivation. The expressed Nb20 showed the ability to interact with the receptor-binding domain of SARS-CoV-2 spike protein, suggesting that it can be used for the neutralization of SARS-CoV-2. The results indicate that T. reesei has the potential for recombinant production of nanobodies.

Production of protease with Bacillus megaterium DSM32: Partial characterisation of the enzyme and modelling of the production

Abstract Proteases hold an important position in today's world commercial enzyme market. Among various microbial producer genera, Bacillus is leading the commercial protease production. However, industry is still actively looking for new microbial protease producers with distinctive properties. Therefore, this study was undertaken for the evaluation of protease production by Bacillus megaterium DSM 32 strain in terms of its protease productivity, calculation of various production kinetics, partial characterisation of the enzyme, and modelling the protease production process. As results, the highest protease activity, specific cellular protease production rate, and protease productivity were calculated as 255.42 U mL−1, 36.2514 U g−1, and 16.1313 U mL−1 h−1, respectively, in shake flask fermentations. Partial characterisation studies showed that the enzyme has 45 °C and pH 8 as optimum working conditions, and its activity increased by 24% with the addition of 5 mM Mn+2 to the reaction medium. Additionally, the enzyme showed high stability and kept almost full activity in a cell-free medium for 20 days at 4 °C. Furthermore, modified Gompertz model provided the best fit in describing protease production with the lowest error and high fit values.

Global Metabolic Rewiring of Yeast Enables Overproduction of Sesquiterpene (+)-Valencene.

(+)-Valencene is a bioactive sesquiterpene that can be used for flavoring and fragrances, and microbial production provides an alternative sustainable access. However, the complexity of cellular metabolism makes it challenging for its high-level production. Here, we report the global rewiring cellular metabolism for de novo production of (+)-valencene in yeast Saccharomyces cerevisiae by engineering central metabolism, mevalonate pathway, and sesquiterpenoid synthase. In particular, we show that metabolic transformation can help accelerate the strain construction process and multiple copy expression of sesquiterpenoid synthase is essential for boosting the metabolic flux for product synthesis with enhanced supply of precursors. The engineered strain produced 1.2 g/L (+)-valencene under fed-batch fermentation in shake flasks, which was increased by 549-fold and demonstrated great potential of the yeast cell factory for (+)-valencene production.

Meroterpenoids Possibly Produced by a Bacterial Endosymbiont of the Tropical Basidiomycete Echinochaete brachypora.

A mycelial culture of the African basidiomycete Echinochaete cf. brachypora was studied for biologically active secondary metabolites, and four compounds were isolated from its crude extract derived from shake flask fermentations, using preparative high-performance liquid chromatography (HPLC). The pure metabolites were identified using extensive nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). Aside from the new metabolites 1-methoxyneomarinone (1) and (E)-3-methyl-5-(-12,13,14-trimethylcyclohex-10-en-6-yl)pent-2-enoic acid (4), the known metabolites neomarinone (2) and fumaquinone (4) were obtained. Such compounds had previously only been reported from Actinobacteria but were never isolated from the cultures of a fungus. This observation prompted us to evaluate whether the above metabolites may actually have been produced by an endosymbiontic bacterium that is associated with the basidiomycete. We have indeed been able to characterize bacterial 16S rDNA in the fungal mycelia, and the production of the metabolites stopped when the fungus was sub-cultured on a medium containing antibacterial antibiotics. Therefore, we have found strong evidence that compounds 1–4 are not of fungal origin. However, the endofungal bacterium was shown to belong to the genus Ralstonia, which has never been reported to produce similar metabolites to 1–4. Moreover, we failed to obtain the bacterial strain in pure culture to provide final proof for its identity. In any case, the current report is the first to document that polyporoid Basidiomycota are associated with endosymbionts and constitutes the first report on secondary metabolites from the genus Echinochaete.

Dynamic Regulation of Transporter Expression to Increase L-Threonine Production Using L-Threonine Biosensors

The cytotoxicity of overexpressed transporters limits their application in biochemical production. To overcome this problem, we developed a feedback circuit for L-threonine production that uses a biosensor to regulate transporter expression. First, we used IPTG-induced rhtA regulation, L-threonine exporter, to simulate dynamic regulation for improving L-threonine production, and the results show that it had significant advantages compared with the constitutive overexpression of rhtA. To further construct a feedback circuit for rhtA auto-regulation, three L-threonine sensing promoters, PcysJ, PcysD, and PcysJH, were characterized with gradually decreasing strength. The dynamic expression of rhtA with a threonine-activated promoter considerably increased L-threonine production (21.19 g/L) beyond that attainable by the constitutive expression of rhtA (8.55 g/L). Finally, the autoregulation method was used in regulating rhtB and rhtC to improve L-threonine production and achieve a high titer of 26.78 g/L (a 161.01% increase), a yield of 0.627 g/g glucose, and a productivity of 0.743 g/L/h in shake-flask fermentation. This study analyzed in detail the influence of dynamic regulation and the constitutive expression of transporters on L-threonine production. For the first time, we confirmed that dynamically regulating transporter levels can efficiently promote L-threonine production by using the end-product biosensor.

Metabolic engineering of the malonyl-CoA pathway to efficiently produce malonate in Saccharomyces cerevisiae

Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation.

Identification of the sesquiterpene synthase AcTPS1 and high production of (–)-germacrene D in metabolically engineered Saccharomyces cerevisiae

BackgroundThe sesquiterpene germacrene D is a highly promising product due to its wide variety of insecticidal activities and ability to serve as a precursor for many other sesquiterpenes. Biosynthesis of high value compounds through genome mining for synthases and metabolic engineering of microbial factories, especially Saccharomyces cerevisiae, has been proven to be an effective strategy. However, there have been no studies on the de novo synthesis of germacrene D from carbon sources in microbes. Hence, the construction of the S. cerevisiae cell factory to achieve high production of germacrene D is highly desirable.ResultsWe identified five putative sesquiterpene synthases (AcTPS1 to AcTPS5) from Acremonium chrysogenum and the major product of AcTPS1 characterized by in vivo, in vitro reaction and NMR detection was revealed to be (–)-germacrene D. After systematically comparing twenty-one germacrene D synthases, AcTPS1 was found to generate the highest amount of (–)-germacrene D and was integrated into the terpene precursor-enhancing yeast strain, achieving 376.2 mg/L of (–)-germacrene D. Iterative engineering was performed to improve the production of (–)-germacrene D, including increasing the copy numbers of AcTPS1, tHMG1 and ERG20, and downregulating or knocking out other inhibitory factors (such as erg9, rox1, dpp1). Finally, the optimal strain LSc81 achieved 1.94 g/L (–)-germacrene D in shake-flask fermentation and 7.9 g/L (–)-germacrene D in a 5-L bioreactor, which is the highest reported (–)-germacrene D titer achieved to date.ConclusionWe successfully achieved high production of (–)-germacrene D in S. cerevisiae through terpene synthase mining and metabolic engineering, providing an impressive example of microbial overproduction of high-value compounds.

The Expression Modulation of the Key Enzyme Acc for Highly Efficient 3-Hydroxypropionic Acid Production.

3-Hydroxypropionic acid (3-HP) is a promising high value-added chemical. Acetyl-CoA carboxylase (Acc) is a vital rate-limiting step in 3-HP biosynthesis through the malonyl-CoA pathway. However, Acc toxicity in cells during growth blocks its ability to catalyze acetyl-CoA to malonyl-CoA. The balancing of Acc and malonyl-CoA reductase (MCR) expression is another an unexplored but key process in 3-HP production. To solve these problems, in the present study, we developed a method to mitigate Acc toxicity cell growth through Acc subunits (AccBC and DtsR1) expression adjustment. The results revealed that cell growth and 3-HP production can be accelerated through the adjustment of DtsR1 and AccBC expression. Subsequently, the balancing Acc and MCR expression was also employed for 3-HP production, the engineered strain achieved the highest titer of 6.8 g/L, with a high yield of 0.566 g/g glucose and productivity of 0.13 g/L/h, in shake-flask fermentation through the malonyl-CoA pathway. Likewise, the engineered strain also had the highest productivity (1.03 g/L/h) as well as a high yield (0.246 g/g glucose) and titer (up to 38.13 g/L) in fed-batch fermentation, constituting the most efficient strain for 3-HP production through the malonyl-CoA pathway using a cheap carbon source. This strategy might facilitate the production of other malonyl-CoA-derived chemical compounds in the future.

Enhanced Phenazine-1-Carboxamide Production in Pseudomonas chlororaphis H5△fleQ△relA through Fermentation Optimization

Phenazine-1-carboxamide (PCN) is effective to control many plant pathogens, and improving PCN production would be of great significance in promoting its development as a biopesticide. This study was conducted to improve the PCN production of Pseudomonas chlororaphis H5△fleQ△relA through fermentation optimization in both shake flask and bioreactor. The PCN production of H5△fleQ△relA was improved from 2.75 ± 0.23 g/L to 5.51 ± 0.17 g/L by medium optimization in shake flask using Plackett-Burman design, the path of steepest ascent experiment and central composite design. Then, PCN production reached 8.58 ± 0.25 g/L through optimizing pH in 1 L bioreactor. After pH optimization, the transcriptional levels of ccoO_2 and ccoQ_2 genes related to microbial aerobic respiration were significantly upregulated, and the relative abundance of 3-oxo-C14-HSL was significantly enhanced 15-fold, and these changes were vital for cell activity and metabolites production. Furthermore, the PCN production reached 9.58 ± 0.57 g/L after optimization of the fed-batch fermentation strategy in 1 L bioreactor. Finally, the fermentation scale-up of the optimal medium and optimal feeding strategy were conducted in 30 L bioreactor at the optimal pH, and their PCN production reached 9.17 g/L and 9.62 g/L respectively, which were comparable to that in 1 L bioreactor. In this study, the high PCN production was achieved from the shake-flask fermentation to 30 L bioreactor, and the optimal feeding strategy improved PCN production in bioreactor without increasing total glycerol compared with in shake flask. It provides promising pathways for the optimization of processes for the production of other phenazines.

Exopolysaccharides production from marine Bacillus strains and their antioxidant and bio-flocculant capacities.

Microbial exopolysaccharides (EPS) have gained high scientific concern due to their exceptional physicochemical features and high industrial applicability. Owing to their biotechnological importance, the present study was designed to screen and isolate the EPS-producing Bacillus strains based on their growth potential on specific media and colony morphologies. The bacterial isolates Bacillus subtilis Bs1-01, Bacillus licheniformis Bl1-02, and Bacillus brevis Bb1-04 showed excellent EPS production due to their shortened lag phase and abundant biomass production. Shake-flask fermentation valued the maximum production yield of 50.19 ± 1.14g/L by Bl1-02 after 72h incubation (about 3.40 times higher than that of Bacillus thuringiensis Bt1-05). The basic component analysis revealed the improved amount of total carbohydrate, reducing sugar ends, and protein contents by Bl1-02 strain. Structural characteristics and functional groups of the EPS characterized by Fourier transform infrared spectroscopy demonstrated that all EPS were in close agreement to each other due to the presence of similar chemical bonds and functional groups. EPS from Bl1-02 strain showed stronger and more stable bio-emulsifying and hygroscopicity activities (12.23%). The crude EPS exhibited potent antioxidant properties which were examined against reducing potential (H2O2 scavenging) and total antioxidant tests. Among bio-flocculation activities of EPS at different concentrations, Bs1-01 strain produced EPS at a concentration of 60mg/mL was observed to show the maximum value of 79.20%. In conclusion, the EPS from marine Bacillus strains showed excellent functional properties suggesting potential industrial applications that demand separate investigations.

METABOLIC PATHWAY MODIFICATION FOR PRODUCTION OF XYLITOL FROM GLUCOSE IN ESCHERICHIA COLI

Glucose is a cheap and readily available substrate for production of large-scale chemicals. Synthesis of xylitol, a high demand chemical in global market is currently done by using xylose, which contributes to its high operational cost. Studies on production of xylitol from glucose have explored several approaches, from sequential fermentation to multiple and single gene expression. Xylitol-5-phosphate dehydrogenase (XPDH), is an enzyme that enables conversion of glucose to xylitol in a single step fermentation. This study explores conversion of xylitol from glucose in E. coli by the expression of xpdh from Clostridium difficile with modifications in metabolic pathways to enhance xylitol production. The xpdh gene was carried by pACYC-Duet-1 expression vector and induced by the addition of IPTG. Initial screening of E. coli expressing xpdh (NA116) was done by shake-flask fermentation for 24 hours and its metabolites were analyzed by HPLC. NA116 was able to produce 0.273 g/L xylitol from 4.33 g/L consumed glucose in 24 hours. Further metabolic pathway modification to eliminate competing pathways yielded four mutants, NA207 (∆rpiA), NA208 (∆rpiB), NA209 (∆pgi) and NA211 (∆rpi∆Apgi). Screening of mutants for xylitol production showed that highest xylitol production from glucose was achieved by NA211 with almost double the amount of the original strain, 0.585 g/L. This showed successful xylitol conversion from glucose in a single fermentation in E. coli with improved yield through metabolic pathway modification.

Development of a novel platform for recombinant protein production in Corynebacterium glutamicum on ethanol

Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion, but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield. Here, we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer. First, we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture. The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter. Next, through transcriptional engineering that alters transcription factor binding sites (TFBSs) and the first transcribed sequence, the truncated promoter PA256 with a dramatically higher transcription level was generated. When producing the superfolder green fluorescent protein (sfGFP) under 1% ethanol conditions, PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36. This superior expression mode was further validated using two secreted proteins, camelid antibody fragment (VHH) and endoxylanase (XynA). Furthermore, utilizing CRISPRi technology, ethanol utilization blocking strains were created, and PA256 was shown to be impaired in the phosphotransacetylase (PTA) knockdown strains, indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation. Finally, this platform was applied to produce the “de novo design” protein NEO-2/15, and by introducing the N-propeptide of CspB, NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation. To the best of our knowledge, this is the first report of NEO-2/15 secretory overexpression.

Heterologous expression and physicochemical characteristics identification of Kunitz protease inhibitor in Brassica napus.

A Kunitz protease inhibitor gene (RTI; rti) was cloned from rapeseed and expressed in a Pichia pastoris expression system for the first time. After isolation and purification, the physical and chemical characteristics of the inhibitor were analyzed. The results showed that the induced expression level of the recombinant RTI reached 628mg/L, and the specific activity of the inhibitor reached 69.6TIU/mg protein at the shake flask fermentation level; the recombinant RTI retained more than 70% inhibitory activity between 30 and 90°C and more than 80% inhibitory activity between pH 2.0-11.0. The metal ions Cu2+ and CO2+ and the organic reagents methanol, ethanol, acetone, and chloroform inhibit its activity. The recombinant RTI interacts with trypsin in a noncompetitive manner and has a strong and specific inhibitory effect on trypsin, a typical Kunitz trypsin inhibitor from plants. Combined with its good physical and chemical properties, recombinant RTI has the potential to be developed into an insect resistance protein.

Engineering Saccharomyces cerevisiae for efficient production of glucaric acid

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.