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Microbial Cell Factories: Production of Amino Acids Using Microorganisms

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A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: closed (31 October 2022) | Viewed by 32884

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Dr. Hisashi Kawasaki

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Guest Editor

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Interests: microbial cell factory; membrane transport; transporter; channel; electrophysiology

Dr. Yoshihiro Usuda

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Guest Editor

Research and Business Planning Department, Ajinomoto Co., Inc. 15-1, Kyobashi 1, Chuo-ku, Tokyo 104-8315, Japan
Interests: microbial cell factory; metabolic engineering; metabolic modeling; bioinformatics

Special Issue Information

Dear Colleagues,

Amino acid production using microbial cells of Corynebacterium glutamicum, a glutamic acid-producing microorganism, was discovered in 1956. This epoch-making discovery led to the industrial production of monosodium glutamate (MSG) for use as seasoning. Subsequently, the range of target products has expanded, from glutamic acids to other amino acids for animal nutrition, pharmaceutical use, and cosmetics. This technology has contributed to the creation of new fermentation industries via the production of a wide variety of useful products that did not previously exist on the market.

As for microbial producers, some species such as Escherichia coli and C. glutamicum have been developed as microbial cell factories through metabolic engineering. Genome analysis and systems biological approaches have contributed to determination of the mechanism of amino acid overproduction in microbial cell factories. Recently, synthetic biology methods, including experimental robot automation and artificial intelligence-based metabolic pathway design, are improving microbial cell factories and taking them to the next level.

For this Special Issue, you are invited to submit either review articles or original research articles on any aspect of microbial cell factories for producing amino acids, which can include achievements using microbial producers, mechanisms underlying how microbial cell factories overproduce amino acids revealed by recent technology, and the latest technologies for analyzing or designing microbial cell factories.

Dr. Hisashi Kawasaki
Dr. Yoshihiro Usuda
Guest Editors

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25 pages, 3028 KiB

Open AccessArticle

H⁺-Translocating Membrane-Bound Pyrophosphatase from Rhodospirillum rubrum Fuels Escherichia coli Cells via an Alternative Pathway for Energy Generation

by Evgeniya A. Malykh, Liubov I. Golubeva, Ekaterina S. Kovaleva, Mikhail S. Shupletsov, Elena V. Rodina, Sergey V. Mashko and Nataliya V. Stoynova

Microorganisms 2023, 11(2), 294; https://doi.org/10.3390/microorganisms11020294 - 23 Jan 2023

Cited by 3 | Viewed by 2030

Abstract

Inorganic pyrophosphatases (PPases) catalyze an essential reaction, namely, the hydrolysis of PP_i, which is formed in large quantities as a side product of numerous cellular reactions. In the majority of living species, PP_i hydrolysis is carried out by soluble cytoplasmic PPase (S-PPases) with the released energy dissipated in the form of heat. In Rhodospirillum rubrum, part of this energy can be conserved by proton-pumping pyrophosphatase (H⁺-PPase^Rru) in the form of a proton electrochemical gradient for further ATP synthesis. Here, the codon-harmonized gene hppa^Rru encoding H⁺-PPase^Rru was expressed in the Escherichia coli chromosome. We demonstrate, for the first time, that H⁺-PPase^Rru complements the essential native S-PPase in E. coli cells. ¹³C-MFA confirmed that replacing native PPase to H⁺-PPase^Rru leads to the re-distribution of carbon fluxes; a statistically significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes was found compared with wild-type E. coli MG1655. Such a flux re-distribution can indicate the presence of an additional method for energy generation (e.g., ATP), which can be useful for the microbiological production of a number of compounds, the biosynthesis of which requires the consumption of ATP. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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21 pages, 2550 KiB

Open AccessArticle

“Force-From-Lipids” Dependence of the MscCG Mechanosensitive Channel Gating on Anionic Membranes

by Yoshitaka Nakayama, Paul R. Rohde and Boris Martinac

Microorganisms 2023, 11(1), 194; https://doi.org/10.3390/microorganisms11010194 - 12 Jan 2023

Cited by 1 | Viewed by 2765

Abstract

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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16 pages, 1581 KiB

Open AccessArticle

The Production of γ-Aminobutyric Acid from Free and Immobilized Cells of Levilactobacillus brevis Cultivated in Anaerobic and Aerobic Conditions

by Teresa Zotta, Immacolata Faraone, Marilisa Giavalisco, Eugenio Parente, Ludovica Lela, Livia Vanessa Storti and Annamaria Ricciardi

Microorganisms 2022, 10(11), 2184; https://doi.org/10.3390/microorganisms10112184 - 3 Nov 2022

Cited by 4 | Viewed by 1578

Abstract

γ-aminobutyric acid (GABA) has several beneficial effects on human health. GABA may be produced via chemical synthesis or through microbial metabolism, and Levilactobacillus brevis is recognized as a GABA-producing species. In this study, 11 Lvb. brevis strains were screened for GABA production, and the best producers were selected to verify the effect of aerobic (AE) and respiratory (RS) cultivations on growth parameters, biomass, and GABA accumulation. Lvb. brevis LB12 was then used to evaluate the combined effect of the incubation atmosphere (anaerobiosis vs. aerobiosis), cell protection (free vs. immobilized cells), and cell recycling (fresh vs. starved cells) on GABA production. Glutamate (GLU) consumption and GABA accumulation were detected by Thin-layer Chromatography (TLC) and RP-HPLC analyses. The ability to produce GABA was widespread among the strains. AE and RS growth improved biomass production, but oxygen availability impaired GLU to GABA conversion, and the anaerobically growing cells had the highest GABA productivity. Immobilized strains had lower efficiency in both GLU uptake and conversion compared to free cells, probably due to the poor diffusion in alginate beads. The use of resting cells allowed further GABA production without the cultivation step, but cell activity was exhausted after three cycles of reutilization. Lvb. brevis LB12 is an excellent GABA producer, and AE cultivation can be exploited to improve the final cell density; however, the conditions for boosting GLU to GABA conversion and cell regeneration need to be further investigated. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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19 pages, 2555 KiB

Open AccessArticle

Bioprocess Engineering, Transcriptome, and Intermediate Metabolite Analysis of L-Serine High-Yielding Escherichia coli W3110

by Chenyang Wang, Qinyu Li, Peng Zhou, Xiaojia Chen, Jiping Shi and Zhijun Zhao

Microorganisms 2022, 10(10), 1927; https://doi.org/10.3390/microorganisms10101927 - 28 Sep 2022

Cited by 3 | Viewed by 2290

Abstract

L-serine is widely used in the food, cosmetic, and pharmaceutical industries. However, the complicated metabolic network and regulatory mechanism of L-serine production lead to the suboptimal productivity of the direct fermentation of L-serine and limits its large-scale industrial production. In this study, a high-yield L-serine production Escherichia coli strain was constructed by a series of defined genetic modification methodologies. First, L-serine-mediated feedback inhibition was removed and L-serine biosynthetic pathway genes (serA^fr, serC, and serB) associated with phosphoglycerate kinase (pgk) were overexpressed. Second, the L-serine conversion pathway was further examined by introducing a glyA mutation (K229G) and deleting other degrading enzymes based on the deletion of initial sdaA. Finally, the L-serine transport system was rationally engineered to reduce uptake and accelerate L-serine export. The optimally engineered strain produced 35 g/L L-serine with a productivity of 0.98 g/L/h and a yield of 0.42 g/g glucose in a 5-L fermenter, the highest productivity and yield of L-serine from glucose reported to date. Furthermore, transcriptome and intermediate metabolite of the high-yield L-serine production Escherichia coli strain were analyzed. The results demonstrated the regulatory mechanism of L-serine production is delicate, and that combined metabolic and bioprocess engineering strategies for L-serine producing strains can improve the productivity and yield. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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12 pages, 2224 KiB

Open AccessArticle

Identification and Molecular Characterization of the Operon Required for L-Asparagine Utilization in Corynebacterium glutamicum

by Koichi Toyoda, Riki Sugaya, Akihiro Domon, Masako Suda, Kazumi Hiraga and Masayuki Inui

Microorganisms 2022, 10(5), 1002; https://doi.org/10.3390/microorganisms10051002 - 10 May 2022

Cited by 1 | Viewed by 3055

Abstract

Understanding the metabolic pathways of amino acids and their regulation is important for the rational metabolic engineering of amino acid production. The catabolic pathways of L-asparagine and L-aspartate are composed of transporters for amino acid uptake and asparaginase and aspartase, which are involved in the sequential deamination to fumarate. However, knowledge of the catabolic genes for asparagine in bacteria of the Actinobacteria class has been limited. In this study, we identified and characterized the ans operon required for L-Asn catabolism in Corynebacterium glutamicum R. The operon consisted of genes encoding a transcriptional regulator (AnsR), asparaginase (AnsA2), aspartase (AspA2), and permease (AnsP). The enzymes and permease encoded in the operon were shown to be essential for L-Asn utilization, but another asparaginase, AnsA1, and aspartase, AspA1, were not essential. Expression analysis revealed that the operon was induced in response to extracellular L-Asn and was transcribed as a leaderless mRNA. The DNA-binding assay demonstrated that AnsR acted as a transcriptional repressor of the operon by binding to the inverted repeat at its 5′-end region. The AnsR binding was inhibited by L-Asn. This study provides insights into the functions and regulatory mechanisms of similar operon-like clusters in related bacteria. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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15 pages, 1981 KiB

Open AccessArticle

Engineering of Escherichia coli Glyceraldehyde-3-Phosphate Dehydrogenase with Dual NAD⁺/NADP⁺ Cofactor Specificity for Improving Amino Acid Production

by Ekaterina A. Slivinskaya, Natalia S. Plekhanova, Irina B. Altman and Tatiana A. Yampolskaya

Microorganisms 2022, 10(5), 976; https://doi.org/10.3390/microorganisms10050976 - 6 May 2022

Cited by 7 | Viewed by 4610

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the central metabolism of microbial cells. GAPDHs differ in cofactor specificity and use NAD⁺, NADP⁺, or both cofactors, reducing them to NADH and NADPH, respectively. Sufficient NADPH supply is one of the critical factors required for synthesis of the amino acids l-lysine, l-threonine, and l-proline in industrially important Escherichia coli-based producer strains. E. coli cells have NAD⁺-dependent glycolytic GAPDH. One reasonable approach to increase NADPH formation in cells is to change the specificity of the GAPDH from NAD⁺ to NADP⁺. In this study, we modified the cofactor specificity of E. coli GAPDH by amino acid substitutions at positions 34, 188 and 189. Several mutant enzymes with dual NAD⁺/NADP⁺ cofactor specificity were obtained, and their kinetic parameters were determined. Overexpression of the genes encoding the resulting mutant GAPDHs with dual cofactor specificity in cells of l-lysine-, l-threonine-, and l-proline-producing E. coli strains led to a marked increase in the accumulation of the corresponding amino acid in the culture medium. This effect was more pronounced when cultivating on xylose as a carbon source. Other possible applications of the mutant enzymes are discussed. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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22 pages, 2704 KiB

Open AccessArticle

Metabolic Engineering of Corynebacterium glutamicum for Sustainable Production of the Aromatic Dicarboxylic Acid Dipicolinic Acid

by Lynn S. Schwardmann, Aron K. Dransfeld, Thomas Schäffer and Volker F. Wendisch

Microorganisms 2022, 10(4), 730; https://doi.org/10.3390/microorganisms10040730 - 29 Mar 2022

Cited by 10 | Viewed by 4320

Abstract

Dipicolinic acid (DPA) is an aromatic dicarboxylic acid that mediates heat-stability and is easily biodegradable and non-toxic. Currently, the production of DPA is fossil-based, but bioproduction of DPA may help to replace fossil-based plastics as it can be used for the production of polyesters or polyamides. Moreover, it serves as a stabilizer for peroxides or organic materials. The antioxidative, antimicrobial and antifungal effects of DPA make it interesting for pharmaceutical applications. In nature, DPA is essential for sporulation of Bacillus and Clostridium species, and its biosynthesis shares the first three reactions with the L-lysine pathway. Corynebacterium glutamicum is a major host for the fermentative production of amino acids, including the million-ton per year production of L-lysine. This study revealed that DPA reduced the growth rate of C. glutamicum to half-maximal at about 1.6 g·L⁻¹. The first de novo production of DPA by C. glutamicum was established by overexpression of dipicolinate synthase genes from Paenibacillus sonchi genomovar riograndensis SBR5 in a C. glutamicum L-lysine producer strain. Upon systems metabolic engineering, DPA production to 2.5 g·L⁻¹ in shake-flask and 1.5 g·L⁻¹ in fed-batch bioreactor cultivations was shown. Moreover, DPA production from the alternative carbon substrates arabinose, xylose, glycerol, and starch was established. Finally, expression of the codon-harmonized phosphite dehydrogenase gene from P. stutzeri enabled phosphite-dependent non-sterile DPA production. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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21 pages, 10114 KiB

Open AccessArticle

The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production

by Christine Hook, Natalya Eremina, Petr Zaytsev, Daria Varlamova and Nataliya Stoynova

Microorganisms 2022, 10(3), 647; https://doi.org/10.3390/microorganisms10030647 - 17 Mar 2022

Cited by 6 | Viewed by 3228

Abstract

Amino acid transport systems perform important physiological functions; their role should certainly be considered in microbial production of amino acids. Typically, in the context of metabolic engineering, efforts are focused on the search for and application of specific amino acid efflux pumps. However, in addition, importers can also be used to improve the industrial process as a whole. In this study, the protein CycA, which is known for uptake of nonpolar amino acids, was characterized from the viewpoint of regulating its expression and range of substrates. We prepared a cycA-overexpressing strain and found that it exhibited high sensitivity to branched-chain amino acids and their structural analogues, with relatively increased consumption of these amino acids, suggesting that they are imported by CycA. The expression of cycA was found to be dependent on the extracellular concentrations of substrate amino acids. The role of some transcription factors in cycA expression, including of Lrp and Crp, was studied using a reporter gene construct. Evidence for the direct binding of Crp to the cycA regulatory region was obtained using a gel-retardation assay. The enhanced import of named amino acids due to cycA overexpression in the l-isoleucine-producing strain resulted in a significant reduction in the generation of undesirable impurities. This work demonstrates the importance of uptake systems with respect to their application in metabolic engineering. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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14 pages, 3010 KiB

Open AccessArticle

Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline

by Akira Nishimura, Yurie Takasaki, Shota Isogai, Yoichi Toyokawa, Ryoya Tanahashi and Hiroshi Takagi

Microorganisms 2021, 9(9), 1902; https://doi.org/10.3390/microorganisms9091902 - 7 Sep 2021

Cited by 2 | Viewed by 2327

Abstract

Awamori, the traditional distilled alcoholic beverage of Okinawa, Japan, is brewed with the yeast Saccharomyces cerevisiae. During the distillation process after the fermentation, enormous quantities of distillation residues containing yeast cells must be disposed of, and this has recently been recognized as a major problem both environmentally and economically. Proline, a multifunctional amino acid, has the highest water retention capacity among amino acids. Therefore, distillation residues with large amounts of proline could be useful in cosmetics. Here, we isolated a yeast mutant with high levels of intracellular proline and found a missense mutation (Gln79His) on the PRO1 gene encoding the γ-glutamyl kinase Pro1, a limiting enzyme in proline biosynthesis. The amino acid change of Gln79 to His in Pro1 resulted in desensitization to the proline-mediated feedback inhibition of GK activity, leading to the accumulation of proline in cells. Biochemical and in silico analyses showed that the amino acid residue at position 79 is involved in the stabilization of the proline binding pocket in Pro1 via a hydrogen-bonding network, which plays an important role in feedback inhibition. Our current study, therefore, proposed a possible mechanism underlying the feedback inhibition of γ-glutamyl kinase activity. This mechanism can be applied to construct proline-accumulating yeast strains to effectively utilize distillation residues. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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14 pages, 1183 KiB

Open AccessReview

Microbial Production Potential of Pantoea ananatis: From Amino Acids to Secondary Metabolites

by Yoshihiro Usuda, Yousuke Nishio, Gen Nonaka and Yoshihiko Hara

Microorganisms 2022, 10(6), 1133; https://doi.org/10.3390/microorganisms10061133 - 31 May 2022

Cited by 12 | Viewed by 3504

Abstract

Pantoea ananatis, a gram-negative bacterium belonging to the Erwiniaceae family, is a well-known phytopathogen isolated from many ecological niches and plant hosts. However, this bacterium also provides us with various beneficial characteristics, such as the growth promotion of their host plants and increased crop yield. Some isolated non-pathogenic strains are promising for the microbial production of useful substances. P. ananatis AJ13355 was isolated as an acidophilic bacterium and was used as an excellent host to produce L-glutamic acid under acidic conditions. The genome sequence of P. ananatis AJ13355 was determined, and specific genome-engineering technologies were developed. As a result, P. ananatis was successfully used to construct a bacterial strain that produces cysteine, a sulfur-containing amino acid that has been difficult to produce through fermentation because of complex regulation. Furthermore, by heterologous expression including plant-derived genes, construction of a strain that produces isoprenoids such as isoprene and linalool as secondary metabolites was achieved. P. ananatis is shown to be a useful host for the production of secondary metabolites, as well as amino acids, and is expected to be used as a platform for microbial production of bioactive substances, aromatic substances, and other high-value-added substances of plant origin in the future. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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