Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl-β- d -1-thiogalactopyranoside concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. Based on a comprehensive data set of about 150 bench-scale bioreactor cultivations in fed-batch mode and choosing the cutinase yield as objective, it was shown that relative secretion performance for bioprocesses remains very similar, irrespective of the applied SP enabling Sec-mediated cutinase secretion. However, to achieve the maximal absolute cutinase yield, careful adjustment of bioprocess conditions was found to be necessary. A model-based, two-step multiple regression approach resembled the collected data in a comprehensive way. The corresponding results suggest that the choice of the heterologous Sec SP and its interaction with the adjusted exponential feeding profile is highly relevant to maximize absolute cutinase yield in this study. For example, the impact of Sec SP is high at low growth rates and low at high growth rates. However, promising Sec SPs could be inferred from less complex batch cultivations. The extensive data were also evaluated in terms of cutinase productivity, highlighting the well-known trade-off between yield and productivity in bioprocess development in detail. Conclusively, only the right combination of target protein, Sec SP, and bioprocess conditions is the key to success.     

FeedER: a feedback-regulated enzyme-based slow-release system for fed-batch cultivation in microtiter plates

With the advent of modern genetic engineering methods, microcultivation systems have become increasingly important tools for accelerated strain phenotyping and bioprocess engineering. While these systems offer sophisticated capabilities to screen batch processes, they lack the ability to realize fed-batch processes, which are used more frequently in industrial bioprocessing. In this study, a novel approach to realize a feedback-regulated enzyme-based slow-release system (FeedER), allowing exponential fed-batch for microscale cultivations, was realized by extending our existing Mini Pilot Plant technology with a customized process control system. By continuously comparing the experimental growth rates with predefined set points, the automated dosage of Amyloglucosidase enzyme for the cleavage of dextrin polymers into d-glucose monomers is triggered. As a prerequisite for stable fed-batch operation, a constant pH is maintained by automated addition of ammonium hydroxide. We show the successful application of FeedER to study fed-batch growth of different industrial model organisms including Corynebacterium glutamicum, Pichia pastoris, and Escherichia coli. Moreover, the comparative analysis of a C. glutamicum GFP producer strain, cultivated under microscale batch and fed-batch conditions, revealed two times higher product yields under slow growing fed-batch operation. In summary, FeedER enables to run 48 parallel fed-batch experiments in an automated and miniaturized manner, and thereby accelerates industrial bioprocess development at the screening stage.

     

Explore or exploit? A model-based screening strategy for PETase secretion by Corynebacterium glutamicum

Extracellular production of target proteins simplifies downstream processing due to obsolete cell disruption. However, optimal combinations of a heterologous protein, suitable signal peptide, and secretion host can currently not be predicted, resulting in large strain libraries that need to be tested. On the experimental side, this challenge can be tackled by miniaturization, parallelization, and automation, which provide high-throughput screening data. These data need to be condensed into a candidate ranking for decision-making to focus bioprocess development on the most promising candidates. We screened for Bacillus subtilis signal peptides mediating Sec secretion of two polyethylene terephthalate degrading enzymes (PETases), leaf-branch compost cutinase (LCC) and polyester hydrolase mutants, by Corynebacterium glutamicum. We developed a fully automated screening process and constructed an accompanying Bayesian statistical modeling framework, which we applied in screenings for highest activity in 4-nitrophenyl palmitate degradation. In contrast to classical evaluation methods, batch effects and biological errors are taken into account and their uncertainty is quantified. Within only two rounds of screening, the most suitable signal peptide was identified for each PETase. Results from LCC secretion in microliter-scale cultivation were shown to be scalable to laboratory-scale bioreactors. This work demonstrates an experiment-modeling loop that can accelerate early-stage screening in a way that experimental capacities are focused to the most promising strain candidates. Combined with high-throughput cloning, this paves the way for using large strain libraries of several hundreds of strains in a Design–Build–Test–Learn approach.     

Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation

Wild-type Corynebacterium glutamicum produced 0.6 g l⁻¹ xylitol from xylose at a productivity of 0.01 g l⁻¹ h⁻¹ under oxygen deprivation. To increase this productivity, the pentose transporter gene (araE) from C. glutamicum ATCC31831 was integrated into the C. glutamicum R chromosome. Consequent disruption of its lactate dehydrogenase gene (ldhA), and expression of single-site mutant xylose reductase from Candida tenuis (CtXR (K274R)) resulted in recombinant C. glutamicum strain CtXR4 that produced 26.5 g l⁻¹ xylitol at 3.1 g l⁻¹ h⁻¹. To eliminate possible formation of toxic intracellular xylitol phosphate, genes encoding xylulokinase (XylB) and phosphoenolpyruvate-dependent fructose phosphotransferase (PTS^fru) were disrupted to yield strain CtXR7. The productivity of strain CtXR7 increased 1.6-fold over that of strain CtXR4. A fed-batch 21-h CtXR7 culture in mineral salts medium under oxygen deprivation yielded 166 g l⁻¹ xylitol at 7.9 g l⁻¹ h⁻¹, representing the highest bacterial xylitol productivity reported to date.     

Metabolic engineering to improve 1,5-diaminopentane production from cellobiose using β-glucosidase-secreting Corynebacterium glutamicum

Microbial production of 1,5-diaminopentane (DAP) from renewable feedstock is a promising and sustainable approach for the production of polyamides. In this study, we constructed a β-glucosidase (BGL)-secreting Corynebacterium glutamicum and successfully used this strain to produce DAP from cellobiose and glucose. First, C. glutamicum was metabolically engineered to produce l -lysine (a direct precursor of DAP), followed by the coexpression of l -lysine decarboxylase and BGL derived from Escherichia coli and Thermobifida fusca YX (Tfu0937), respectively. This new engineered C. glutamicum strain produced 27 g/L of DAP from cellobiose in CGXII minimal medium using fed-batch cultivation. The yield of DAP was 0.43 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose), which is the highest yield reported to date. These results demonstrate the feasibility of DAP production from cellobiose or cellooligosaccharides using an engineered C. glutamicum strain.     

Glucose-controlled -isoleucine fed-batch production with recombinant strains of Corynebacterium glutamicum

-Isoleucine was produced in a fed-batch bioprocess with -leucine auxotrophic Corynebacterium glutamicum strains developed by genetic engineering. An efficient supply with nutrients was achieved by applying closed-loop control of glucose as the main carbon source, with a model-based, parameter-adaptive control strategy. This control strategy is based on an extended, semi-continuous Kalman filter for process identification and a minimum variance controller. The lab scale fed-batch process with C. glutamicum SM1 and C. glutamicum DR17 pECM3::ilvA38 was characterized with respect to biomass, product and by-product accumulation. A differential analysis of growth, specific productivities, and selectivities was performed to characterize the carbon flow over process time. Characterization of -isoleucine transport steps across the cell membrane resulted in a balance of -isoleucine transport over process time. Up to an extracellular -isoleucine concentration of 140 mM the cytosolic -isoleucine, provided by the biosynthesis, was quantitatively excreted into the medium via the export carrier system. Optimized feeding profiles for -leucine and phosphate in correlation with the on-line estimated glucose consumption were achieved up to the pilot scale (300-1 stirred tank reactor). The maximum -isoleucine concentration was 150 mM (21 g l⁻¹) with a space-time yield of 4.3 mmol l⁻¹ h⁻¹. With a 98% closed carbon balance the selectivity for isoleucine was 14%, for biomass 13%, and for CO₂ 68%.     

Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF ^317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF ^317A –bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.     

Engineering <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for the production of pyruvate

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD⁺-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN produced about 190 mM pyruvate with a Y _P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g_(CDW)⁻¹ h⁻¹ (i.e., 0.08 g g_(CDW) ⁻¹ h⁻¹) in the production phase.     

Parallelized disruption of prokaryotic and eukaryotic cells via miniaturized and automated bead mill

The application of integrated microbioreactor systems is rapidly becoming of more interest to accelerate strain characterization and bioprocess development. However, available high‐throughput screening capabilities are often limited to target extracellular compounds only. Consequently, there is a great demand for automated technologies allowing for miniaturized and parallel cell disruption providing access to intracellular measurements. In this study, a fully automated bead mill workflow was developed and validated for four different industrial platform organisms: Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Aspergillus niger. The workflow enables up to 48 parallel cell disruptions in microtiter plates and is applicable at‐line to running lab‐scale cultivations. The resulting cell extracts form the basis for quantitative omics studies where no rapid metabolic quenching is required (e.g., genomics and proteomics).     

Enhanced production of α-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum

The fed-batch culture system was employed to enhance production of α-ketoglutarate (α-KG) by the strainsof Corynebacterium glutamicum, whose genes encoding the key enzymes responsible for the biosynthesis of L-glutamate from α-KG were deleted. In a shake flask fermentation, C. glutamicum JH110 in which the 3 genes, gdh (encoding glutamate dehydrogenase), gltB (encoding glutamate synthase), and aceA (encoding isocitrate lyase) were disrupted showed the highest production of α-KG (12.4 g/L) compared to the strains JH102 (gdh mutant), JH103 (gltB mutant), and JH107 (gdh gltB double mutant). In the fed-batch cultures using a 5 L-jar fermenter, the strain JH107 produced more α-KG (19.5 g/L), but less glutamic acid (23.3 g/L) than those produced by the parent strain HH109, as well as JH102. The production of α-KG was significantly enhanced and the accumulation of glutamicacid was minimized by the ammonium-limited fed-batch cultures employing C. glutamicum JH107. Further improvement of α-KG production by the strain JH107 was achieved through the ammonium-limited fed-batch culture with the feeding of molasses, and the levels of α-KG and glutamic acid produced were 51.1 and 0.01 g/L, respectively.     

Cre/loxP-mediated deletion system for large genome rearrangements in Corynebacterium glutamicum

Genome rearrangement is an increasingly important technique to facilitate the understanding of genome functions. A Cre/loxP-mediated deletion system for large-scale genome rearrangements in Corynebacterium glutamicum was developed. By comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes, distinct 14.5-kb and 56-kb regions not essential for cell survival were identified and targeted for deletion. By homologous recombination, loxP sites were integrated at each end of the target region. Deletions between the two chromosomal loxP sites in the presence of Cre recombinase were highly efficient. Accurate deletion was observed in all 96 Cre-expressing strains tested. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of 11 and 58 predicted ORF(s), respectively, upon the deletion of the14.5-kb and 56-kb regions, the cells still exhibited normal growth under standard laboratory conditions. Based on the precision of its deletion, the Cre/loxP system provides a new, efficient genome rearrangement technique for studying C. glutamicum.     

Extracellular overexpression of recombinant <Emphasis Type="Italic">Thermobifida fusca</Emphasis> cutinase by alpha-hemolysin secretion system in <Emphasis Type="Italic">E. coli</Emphasis> BL21(DE3)

Background  

Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.     

D-lactic acid production by a genetically engineered strain <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Based on its ability to produce lactic acid from glucose in mineral salt medium under anaerobic conditions, genetic modifications on Corynebacterium glutamicum Res 167 were carried out with the aim of producing optical pure D-lactic acid, involving the knockout of L-lactate dehydrogenase gene from C. glutamicum and the heterologous expression of D-lactate dehydrogenase gene from Lactobacillus bulgaricus into C. glutamicum. D-lactic acid production of the genetically engineered strain C. glutamicum Res 167Δldh/ldhA was 17.92 g/l (optical purity higher than 99.9%) after 16 h fermentation, which was 32.25% higher than the lactic acid production of the parental strain.     

Chromogranin B: intra‐ and extra‐cellular mechanisms to regulate catecholamine storage and release,in catecholaminergic cells and organisms

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under‐expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic‐stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over‐expression of CHGB was achieved by transduction of a CHGB‐expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic‐triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic‐stimulated catecholamine release by ~ 72%. One conserved‐region CHGB peptide inhibited nicotinic‐triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi‐directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra‐cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

     

Improved phloroglucinol production by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.     

双功能尿苷酰转移/去除酶GlnD在谷氨酸棒杆菌JNR中的功能

【目的】通过改造谷氨酸棒杆菌JNR中双功能尿苷酰转移/去除酶GlnD,减弱尿苷酰去除酶的活性,增强NH_4~+的转运和利用,提高L-精氨酸的合成。【方法】本文对来源于谷氨酸棒杆菌的突变菌株JNR中的双功能尿苷酰转移/去除酶GlnD进行整合突变,采用同源重组的方法将H_(414)和D_(415)位点突变为两个丙氨酸AA,在此菌株的基础上过量表达PII蛋白GlnK,并对其进行尿苷酰化研究,离子色谱检测摇瓶发酵过程中NH4+的浓度,并对最终的改造菌株进行连续流加发酵分析。【结果】该双功能尿苷酰转移/去除酶在谷氨酸棒杆菌中成功进行整合突变,有效减弱了尿苷酰去除酶的活性;同时过表达PII蛋白GlnK,其酰基化程度明显增强。摇瓶发酵结果表明菌株L4消耗NH_4~+增加,L-精氨酸产量为36.2±1.2 g/L,比对照菌株L3高出22.7%。5-L发酵罐实验结果显示改造菌株L4的L-精氨酸的产量为52.2 g/L,较野生型菌株L0提高了25.3%。【结论】谷氨酸棒杆菌合成L-精氨酸的过程中氮源是必不可少的。减弱GlnD尿苷酰去除酶的活性后,胞内尿苷酰化的GlnK-UMP增加,GlnK-UMP与氮转录调控因子AmtR结合,转运至胞内的NH_4~+浓度提高,促使L-精氨酸产量显著提高。     

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.     

Enhanced production of neoagarobiose from agar with Corynebacterium glutamicum producing exo-type and endo-type β-agarases

Neoagarobiose (NA2) derived from agar marine biomass is a rare reagent that acts as an anti-melanogenesis reagent and moisturizer. Here, for the economical manufacturing of NA2, we developed the co-secretory production system of endo-type β-agarases (DagA) and exo-type β-agarases (EXB3) in Corynebacterium glutamicum. For this purpose, we first developed a secretory system of DagA via Tat pathway. To improve the secretion efficiency, we coexpressed two Tat pathway components (TatA and TatC), and to improve the purity of secreted DagA in the culture supernatant, two endogenous protein genes (Cg2052 and Cg1514) were removed. Using the engineered strain (C. glutamicum SP002), we confirmed that DagA as high as 1.53 g l^-1 was successfully produced in the culture media with high purity (72.7% in the supernatant protein fraction). Next, we constructed the expression system (pHCP-CgR-DagA-EXB3) for the simultaneous secretion of EXB3 via Sec-pathway together with DagA, and it was clearly confirmed that DagA and EXB3 were successfully secreted as high as 54% and 24.5%, respectively. Finally, using culture medium containing DagA and EXB3, we successfully demonstrated the conversion of high-concentration agar (40 g l^-1) into NA2 via a two-stage hydrolysis process.