Application of Corynebacterium glutamicum engineering display system in three generations of biorefinery.

The fermentation production of platform chemicals in biorefineries is a sustainable alternative to the current petroleum refining process. The natural advantages of Corynebacterium glutamicum in carbon metabolism have led to C. glutamicum being used as a microbial cell factory that can use various biomass to produce value-added platform chemicals and polymers. In this review, we discussed the use of C. glutamicum surface display engineering bacteria in the three generations of biorefinery resources, and analyzed the C. glutamicum engineering display system in degradation, transport, and metabolic network reconstruction models. These engineering modifications show that the C. glutamicum engineering display system has great potential to become a cell refining factory based on sustainable biomass, and further optimizes the inherent properties of C. glutamicum as a whole-cell biocatalyst. This review will also provide a reference for the direction of future engineering transformation.

Application of RecET-Cre/loxP system in Corynebacterium glutamicum ATCC14067 for L-leucine production.

OBJECTIVE: To explore the RecET-Cre/loxP system for chromosomal replacement of promoter and its application on enhancement L-leucine production in Corynebacterium glutamicum (C. glutamicum) ATCC14067. RESULTS:  The RecET-Cre/loxP system was used to achieve the chromosomal replacement of promoter in C. glutamicum ATCC14067 to adjust the metabolic flux involving the L-leucine synthetic pathway. First, leuAr_13032 from C. glutamicum ATCC13032 which carried two mutations was overexpressed to release enzyme feedback inhibition. Then, comparing different mutations in ilvBNC gene clusters, the results indicated that ilvBNC_CP was most effective to enhance the metabolic flux of pyruvate towards L-leucine synthesis. The promoters of pck, odx and pyk2 were overexpressed under the strong promoter Peftu or Psod to improve the supply of pyruvate. Besides, the promoter PilvBNC was employed to dynamically control the transcription level of icd due to its attenuation mechanism by responding to the concentration of L-leucine. The final engineered strain produced 14.05 g L-leucine/L in flask cultivation. CONCLUSION:  The RecET-Cre/loxP system is effective for gene manipulation in C. glutamicum ATCC14067. Besides, the results demonstrate the potential of C. glutamicum ATCC14067 for L-leucine production and provide new targets and strategies for strain development.

Multiple cellular responses guarantee yeast survival in presence of the cell membrane/wall interfering agent sodium dodecyl sulfate.

Sodium dodecyl sulfate (SDS), a representative anionic surfactant, is a commonly used reagent in studies of the cell membrane and cell wall. However, the mechanisms through which SDS affects cellular functions have not yet been fully examined. Thus, to gain further insights into the cellular functions and responses to SDS, we tested a haploid library of Saccharomyces cerevisiae single-gene deletion mutants to identify genes required for tolerance to SDS. After two rounds of screening, we found 730 sensitive and 77 resistant mutants. Among the sensitive mutants, mitochondrial gene expression; the mitogen-activated protein kinase signaling pathway; the metabolic pathways involved in glycoprotein, lipid, purine metabolic process, oxidative phosphorylation, cellular amino acid biosynthesis and pentose phosphate pathway were found to be enriched. Additionally, we identified a set of transcription factors related to SDS responses. Among the resistant mutants, disruption of ribosome biogenesis and translation alleviated SDS-induced cytotoxicity. Collectively, our results provided new insights into the mechanisms through which SDS regulates the cell membrane or cell wall.

Construction and screening of a glycosylphosphatidylinositol protein deletion library in Pichia pastoris.

BACKGROUND: Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. In this study, the functions of potential GPI proteins in Pichia pastoris were explored by gene knockout approaches. RESULTS: Through an extensive knockout of GPI proteins in P. pastoris, a single-gene deletion library was constructed for 45 predicted GPI proteins. The knockout of proteins may lead to the activation of a cellular response named the 'compensatory mechanism', which is characterized by changes in the content and relationship between cell wall polysaccharides and surface proteins. Among the 45 deletion strains, five showed obvious methanol tolerance, four owned high content of cell wall polysaccharides, and four had a high surface hydrophobicity. Some advantages of these strains as production hosts were revealed. Furthermore, the deletion strains with high surface hydrophobicity were used as hosts to display Candida antarctica lipase B (CALB). The strain gcw22Δ/CALB-GCW61 showed excellent fermentation characteristics, including a faster growth rate and higher hydrolytic activity. CONCLUSIONS: This GPI deletion library has some potential applications for production strains and offers a valuable resource for studying the precise functions of GPI proteins, especially their putative functions.

Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

Genome-wide screening of Saccharomyces cerevisiae deletion mutants reveals cellular processes required for tolerance to the cell wall antagonist calcofluor white.

We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants to identify nonessential genes associated with increased sensitivity to or resistance against the cell wall antagonist calcofluor white. Through a genome-wide screen, we isolated 537 strains that had an altered growth rate relative to wild type, of which 485 showed increased sensitivity and 52 showed increased resistance to calcofluor white. The MAPK signaling pathway, N-glycan biosynthesis, endocytosis, vacuole acidification, autophagy, and the sulfur relay system were identified as being associated with calcofluor white sensitivity. Resistance genes were mainly involved in chitin metabolism and the RIM101 pathway or encoded several components of the ESCRT complexes or related to cysteine and methionine metabolism and RNA degradation. Further investigation indicated a clear global response network that S. cerevisiae relies on in the presence of the cell wall antagonist calcofluor white, which may help us to understand fungal cell wall remodeling and the mechanisms of toxicity of calcofluor white with respect to eukaryotic cells.

Deletion of the GCW13 gene derepresses Gap1-dependent uptake of amino acids in Pichia pastoris grown on methanol as the sole carbon source.

In Pichia pastoris, most of the Glycosylphosphatidylinositol (GPI)-anchored proteins are of unknown function. Gcw13, one of these GPI-anchored proteins, was found to exert an inhibitory effect on the growth of the histidine auxotrophic P. pastoris strain GS115 on methanol as the sole carbon source. To investigate the biological function of Gcw13, RNA sequencing (RNA-Seq) was performed to compare the difference of gene expression between GS115 and GCW13-deletion strain D13. RNA-Seq analysis showed that, in strain D13, the expression of genes involved in the methanol utilization pathway or peroxisome biogenesis was not changed, and a high proportion of genes involved in the biosynthesis of amino acids were down-regulated, whereas GAP1, which encodes a general amino acid permease, was significantly up-regulated. Besides, the intracellular concentrations of various amino acids were significantly higher in D13 than that in GS115. We also observed that deletion of GCW13 resulted in more Gap1 presented on the cell surface and more active uptake of the toxic proline analogue l-azetidine-2-carboxylate acid (AzC). These results suggest that Gcw13 suppresses the expression of GAP1 and facilitates the endocytosis of Gap1 on methanol, resulting in decreasing Gap1-dependent uptake of amino acids in P. pastoris, which might contribute to the poor growth of GS115 on methanol.

RNA-Seq analysis of global transcriptomic changes suggests a roles for the MAPK pathway and carbon metabolism in cell wall maintenance in a Saccharomyces cerevisiae FKS1 mutant.

FKS1 encodes a ß-1,3-glucan synthase, which is a key player in cell wall assembly in Saccharomyces cerevisiae. Here we analyzed the global transcriptomic changes in the FKS1 mutant to establish a correlation between the changes in the cell wall of the FKS1 mutant and the molecular mechanism of cell wall maintenance. These transcriptomic profiles showed that there are 1151 differentially expressed genes (DEGs) in the FKS1 mutant. Through KEGG pathway analysis of the DEGs, the MAPK pathway and seven pathways involved in carbon metabolism were significantly enriched. We found that the MAPK pathway is activated for FKS1 mutant survival and the synthesis of cell wall components are reinforced in the FKS1 mutant. Our results confirm that the FKS1 mutant has a ß-1,3-glucan defect that affects the cell wall and partly elucidate the molecular mechanism responsible for cell wall synthesis. Our greater understanding of these mechanisms helps to explain how the FKS1 mutant survives, has useful implications for the study of similar pathways in other fungi, and increases the theoretical foundation for the regulation of the cell wall in S. cerevisiae.

Improved production and characterization of Volvariella volvacea Endoglucanase 1 expressed in Pichia pastoris.

Endoglucanase 1 (EG1) isolated from the straw mushroom has great potential in the textile and paper industries. Improving EG1 expression level will add to its value for industrial applications. In this study, we employed two combined strategies to enhance the expression quantity of EG1, which are increase the copy number of EG1 and enhance the folding and secretion efficiency of EG1 in the endoplasmic reticulum by overexpress HAC1. Multiple plasmids, which contains four copies of EG1, were constructed by isocaudamers, resulted a recombinant strain with EG1 activity up to 39.6 U/mL, 262% higher than that measured in the strain containing only a single copy. A significant increase in activity (151%) was found when eight copies of EG1 was introduced into a different host, compared with a host harboring four copies. Further overexpression of the HAC1 transcription factor in the host harboring eight EG1 copies led to activity of 91.9 U/mL, which is 619% higher than that measured in the original strain. Finally, EG1 activity of 650.1 U/mL was achieved in a 3-L scaled-up fed-batch fermenter and the protein yield was 4.05 g/L. The characteristics of recombinant EG1 were also investigated, the optimal values for enzyme activity were 60 °C and pH 5.0, which yielded a catalytic efficiency of 312.9 mL mg-1min-1 using carboxymethyl cellulose(CMC) as the substrate.

Synthetic biology approaches to access renewable carbon source utilization in Corynebacterium glutamicum.

Corynebacterium glutamicum (C. glutamicum), an important industrial workhorse, is capable of efficiently producing a variety of value-added chemicals and fuels beyond amino acids. C. glutamicum has a broad natural substrate spectrum and can simultaneously utilize various carbon sources in blends. The substrate spectrum of C. glutamicum has been further extended by detailed knowledge of carbon core metabolism and well-established genetic tools and engineering strategies. At present, many pathways have been successfully engineered in C. glutamicum for access to alternative renewable sources to produce natural or non-natural products, making C. glutamicum a promising and favorable microbial cell factory. In this review, we mainly focus on synthetic biology and metabolic engineering strategies for developing synthetic strains that grow on renewable sources to produce the target products. At the same time, we also explore the promotion and future challenges of existing synthetic biology platforms for industrial platform microorganism metabolic engineering efforts.

Accurate analysis of fusion expression of Pichia pastoris glycosylphosphatidylinositol-modified cell wall proteins.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. The GPI-modified cell wall proteins GCW21, GCW51, and GCW61 of Pichia pastoris were chosen as anchoring proteins to construct co-expression strains in P. pastoris GS115. The hydrolytic activity and the amount of Candida antarctica lipase B (CALB) displayed on cell surface increased significantly following optimization of the fusion gene dosage and combination of the homogeneous or heterogeneous cell wall proteins. Maximum CALB hydrolytic activity was achieved at 4920 U/g dry cell weight in strain GS115/CALB-GCW (51 + 51 + 61 + 61) after 120 h of methanol induction. Changes in structural morphology and the properties of the cell surfaces caused by co-expression of fusion proteins were observed by transmission electron microscopy (TEM) and on plates containing cell-wall-destabilizing reagent. Our results suggested that both the outer and inner cell layers were significantly altered by overexpression of GPI-modified cell wall proteins. Interestingly, quantitative analysis of the inner layer components showed an increase in ß-1,3-glucan, but no obvious changes in chitin in the strains overexpressing GPI-modified cell wall proteins.

Enzymatic Process Enhances the Flavour Profile and Increases the Proportion of Esters in Citrus Essential Oils.

Citrus essential oils (CEOs) are important flavors in the food and confectionary industries. A lipase process was proposed for enhancing the flavor profiles and increasing the proportions of esters in CEOs. The effects of the enzymatic process were explored by detecting the constituents of the CEOs of American sweet orange oil (ASO) and Brazil mandarin oil (BMO) through GC/MS and sensory evaluation by a trained panel, and positive effects were confirmed by both methods. A further eleven kinds of CEOs were treated via the lipase process and increments of 10 - 1170% were achieved in the proportions of esters, which were mostly ethyl esters. Enhancement in fruity odor, especially the top note, was demonstrated by all CEOs after enzymatic processing. All CEOs were tested for antimicrobial activities, and only ASO displayed fairly ideal antimicrobial activities. Meanwhile, modified ASO showed a certain increase in antimicrobial activities. This methodology might be considered a sustainable route for acquiring 'natural' essential oils with enhanced flavor profiles and simultaneously enhancing the comprehensive utilization of citrus fruits.

Improving the catalytic characteristics of lipase-displaying yeast cells by hydrophobic modification.

Lipase-displaying yeast cells are a promising alternative to the conventional immobilised lipases for organic bioconversions. However, the hydrophilic characteristics of the yeast cell surface may impede efficient immobilisation. Herein, we tested three methods to enhance the hydrophobicity of the surface of Candida antarctica lipase B-displaying Pichia pastoris cells, co-displaying a fungal hydrophobin, coating with ionic liquids, and adding decane as a hydrophobic carbon source during fermentation. Modified cells showed higher surface hydrophobicity and superior esterification of C6-C18 saturated fatty acids in hydrophobic solvents. When used for biodiesel synthesis, modified cells exhibited an improved initial reaction rate and equilibrium fatty acid methyl ester yield. We systematically discuss the influence of cell surface hydrophobicity on the catalytic properties, and the results provide guidance for improving the catalytic efficiency and operational characteristics of lipase-displaying yeast cells for organic bioconversions.

Monomeric Corynebacterium glutamicum N-acetyl glutamate kinase maintains sensitivity to L-arginine but has a lower intrinsic catalytic activity.

N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine, and L-arginine-sensitive NAGK typically has hexameric architecture. Defining the relationship between this architecture and L-arginine inhibition can provide a foundation to identify the key amino acids involved in the allosteric regulation network of L-arginine. In the present study, the key amino acids in the N-terminal helix (N-helix) of Corynebacterium glutamicum (Cg) NAGK required for hexamer formation were determined using structural homology modeling and site-directed mutagenesis. It was also verified that hexameric architecture is required for the positive cooperativity of inhibition by L-arginine and for efficient catalysis, but that it is not the determinant of inhibition by L-arginine. Monomeric mutants retained a similar sensitivity to L-arginine as the hexameric form, indicating that monomers contain an independent, sensitive signal transduction network of L-arginine to mediate allosteric regulation. Mutation studies of CgNAGKs also revealed that amino acid residues 18-23 of the N-helix are required for inhibition by L-arginine, and that E19 may be an essential amino acid influencing the apparent affinity of L-arginine. Collectively, these studies may illuminate the basic mechanism of metabolic homeostasis of C. glutamicum.

Mutational analysis to identify the residues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum.

N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine that is inhibited by its end product L-arginine in Corynebacterium glutamicum (C. glutamicum). In this study, the potential binding sites of arginine and the residues essential for its inhibition were identified by homology modeling, inhibitor docking, and site-directed mutagenesis. The allosteric inhibition of NAGK was successfully alleviated by a mutation, as determined through analysis of mutant enzymes, which were overexpressed in vivo in C. glutamicum ATCC14067. Analysis of the mutant enzymes and docking analysis demonstrated that residue W23 positions an arginine molecule, and the interaction between arginine and residues L282, L283, and T284 may play an important role in the remote inhibitory process. Based on the results of the docking analysis of the effective mutants, we propose a linkage mechanism for the remote allosteric regulation of NAGK activity, in which residue R209 may play an essential role. In this study, the structure of the arginine-binding site of C. glutamicum NAGK (CgNAGK) was successfully predicted and the roles of the relevant residues were identified, providing new insight into the allosteric regulation of CgNAGK activity and a solid platform for the future construction of an optimized L-arginine producing strain.

Quantification analysis of yeast-displayed lipase.

We describe a method for quantification of displayed lipase on yeast cell surface. The strategy uses an organophosphonate ester to irreversibly inhibit the active lipase and release a detectable fluorescent group. The amount of displayed lipase can be represented as "g/g cell" or "molecules/cell". The results obtained correlated well with those obtained by existing methods. Therefore, this method is credible and will provide a powerful tool to promote research of lipase yeast surface display.

Displaying Candida antarctica lipase B on the cell surface of Aspergillus niger as a potential food-grade whole-cell catalyst.

Aspergillus niger is a recognized workhorse used to produce food processing enzymes because of its extraordinarily high protein-producing capacity. We have developed a new cell surface display system de novo in A. niger using expression elements from generally recognized as safe certified microorganisms. Candida antarctica lipase B (CALB), a widely used hydrolase, was fused to an endogenous cell wall mannoprotein, CwpA, and functionally displayed on the cell surface. Localization of CALB was confirmed by enzymatic assay and immunofluorescence analysis using laser scanning confocal microscopy. After induction by maltose for 45 h, the hydrolytic activity and synthesis activity of A. niger mycelium-surface displayed CALB (AN-CALB) reached 400 and 240 U/g dry cell, respectively. AN-CALB was successfully used as a whole-cell catalyst for the enzymatic production of ethyl esters from a series of fatty acids of different chain lengths and ethanol. In a solvent-free system, AN-CALB showed great synthetic activity and afforded high substrate mole conversions, which amounted to 87 % for ethyl hexanoate after 2 h, 89 % for ethyl laurate after 2 h, and 84 % for ethyl stearate after 3 h. These results suggested that CwpA can act as an efficient anchoring motif for displaying enzyme on A. niger, and AN-CALB is a robust, green, and cost-effective alternative food-grade whole-cell catalyst to commercial lipase.

Development of the IMP biosensor for rapid and stable analysis of IMP concentrations in fermentation broth.

IMP (inosinic acid) is a crucial intermediate in the purine metabolic pathway and is continuously synthesized in all cells. Besides its role as a precursor for DNA and RNA, IMP also plays a critical or essential role in cell growth, energy storage, conversion, and metabolism. In our study, we utilized the circularly permuted fluorescent protein (cpFP) and IMP dehydrogenase to screen and develop the IMP biosensor, IMPCP1. By introducing a mutation in the catalytically active site of IMPCP1, from Cys to Ala, we disrupted its ability to catalyze IMP while retaining its capability to bind to IMP without affecting the IMP concentration in the sample. To immobilize IMPCP1, we employed the SpyCatcher/SpyTag system and securely attached it to Magarose-Epoxy, resulting in the development of the IMP rapid test kit, referred to as IMPTK. The biosensor integrated into IMPTK offers enhanced stability, resistance to degradation activity, and specific recognition of IMP. It is also resistant to peroxides and temperature changes. IMPTK serves as a rapid and stable assay for analyzing IMP concentrations in fermentation broth. Within the linear range of IMP concentrations, it can be utilized as a substitute for HPLC. The IMPTK biosensor provides a reliable and efficient alternative for monitoring IMP levels, offering advantages such as speed, stability, and resistance to environmental factors.

Screening for glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant expression on the cell surface.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by ß-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.