Recombinant Corynebacterium Glutamicum Research Articles

Mannosidase-inhibiting iminosugar production by recombinant Corynebacterium glutamicum harboring the 1-deoxynojirimycin biosynthetic gene cluster

The iminosugar class of carbohydrate-active enzyme inhibitors has therapeutic applications in metabolic syndrome conditions, viral infections and cancer. Compared to chemical synthesis, microbial iminosugar production has benefits of cost, sustainability and optimization. In this study, the 1-deoxynojirimycin (DNJ) biosynthetic gene cluster from Bacillus velezensis MBLB0692, and its individual genes, were cloned into Corynebacterium glutamicum (Cg). Characterizations of the encoded aminotransferase GabT1, phosphatase Yktc1, and dehydrogenase GutB1, were performed with purified enzymes and whole cell biocatalysts bearing individual and clustered (TYB) genes. GabT1 showed a variable pattern in its half-reaction with a slow turnover. GutB1 was an alkaline dehydrogenase with a broad substrate specificity and no divalent ion dependency while the zinc-dependent phosphatase Yktc1 had substrate specificity that was both pH- and ion-dependent. The CgYktc1 and CgGutB1 whole cells were viable biocatalysts with wider ranges of substrates than their enzyme counterparts. The CgTYB cells produced mannosidase-inhibiting iminosugars corresponding to mannojirimycin dehydrate (162 m/z) and deoxymannojirimycin (164 m/z). Mannosidase inhibitors have been found to be effective in treating orphan diseases, cancer and viral infections, and their biosynthesis by recombinant C. glutamicum can be optimized for industrial production and novel drug development.

Corynebacterium glutamicum cell factory design for the efficient production of cis, cis-muconic acid

Cis, cis-muconic acid (MA) is widely used as a key starting material in the synthesis of diverse polymers. The growing demand in these industries has led to an increased need for MA. Here, we constructed recombinant Corynebacterium glutamicum by systems metabolic engineering, which exhibit high efficiency in the production of MA. Firstly, the three major degradation pathways were disrupted in the MA production process. Subsequently, metabolic optimization strategies were predicted by computational design and the shikimate pathway was reconstructed, significantly enhancing its metabolic flux. Finally, through optimization and integration of key genes involved in MA production, the recombinant strain produced 88.2 g/L of MA with the yield of 0.30 mol/mol glucose in the 5 L bioreactor. This titer represents the highest reported titer achieved using glucose as the carbon source in current studies, and the yield is the highest reported for MA production from glucose in Corynebacterium glutamicum. Furthermore, to enable the utilization of more cost-effective glucose derived from corn straw hydrolysate, we subjected the strain to adaptive laboratory evolution in corn straw hydrolysate. Ultimately, we successfully achieved MA production in a high solid loading of corn straw hydrolysate (with the glucose concentration of 83.56 g/L), resulting in a titer of 19.9 g/L for MA, which is 4.1 times higher than that of the original strain. Additionally, the glucose yield was improved to 0.33 mol/mol. These provide possibilities for a greener and more sustainable production of MA.

Enhancement of polymyxin B1 production by an artificial microbial consortium of Paenibacillus polymyxa and recombinant Corynebacterium glutamicum producing precursor amino acids

Polymyxin B, produced by Paenibacillus polymyxa, is used as the last line of defense clinically. In this study, exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of polymyxin B analogs of P. polymyxa CJX518-AC (PPAC) from 0.15 g/L and 61.8 % to 0.33 g/L and 79.9 %, respectively. The co-culture of strain PPAC and recombinant Corynebacterium glutamicum-leu01, which produces high levels of threonine, leucine, and isoleucine, increased polymyxin B1 production to 0.64 g/L. When strains PPAC and C. glu-leu01 simultaneously inoculated into an optimized medium with 20 g/L peptone, polymyxin B1 production was increased to 0.97 g/L. Furthermore, the polymyxin B1 production in the co-culture of strains PPAC and C. glu-leu01 increased to 2.21 g/L after optimized inoculation ratios and fermentation medium with 60 g/L peptone. This study provides a new strategy to improve polymyxin B1 production.

Identification of α-isopropylmalate synthase mutants capable of overproducing L-leucine in Corynebacterium glutamicum

The enzyme α-isopropylmalate synthase (α-IPMS) catalyzes the first step of L-leucine biosynthesis and is regulated via feedback inhibition by L-leucine. The gene of α-IPMS variant from strain ARTP-L04 was cloned and sequenced. Interestingly, amino acid mutations of Gly92Asp, Ile162Val, Arg494His and Gly526Asp occurred in the α-IPMS variant. The enzyme of α-IPMS variant was characterized, and exhibited higher resistance to feedback inhibition by L-leucine. In the presence of 20 mM L-leucine, the activity of the α-IPMS variant was still retained at over 50%. The α-IPMS variant almost removed feedback inhibition by L-leucine, while there was no significant difference in specific activities of the α-IPMS variant and wild-type α-IPMS. For the α-IPMS variant, the capacity to overproduce L-leucine was evaluated by recombinant Corynebacterium glutamicum strains. Interestingly, expression of the α-IPMS variant could significantly enhance L-leucine production, especially co-expressed with acetohydroxyacid synthase (accumulating 7.79 g/L L-leucine). Collectively, our findings provided valuable insights into further development in genetic engineering for L-leucine production.

Enhanced production of itaconic acid from enzymatic hydrolysate of lignocellulosic biomass by recombinant Corynebacteriumglutamicum

Itaconic acid (IA) is a value-added chemical currently produced by Aspergillus terreus from edible glucose and starch but not from inedible lignocellulosic biomass owing to the high sensitivity to fermentation inhibitors present in the hydrolysate of lignocellulosic biomass. To produce IA from lignocellulosic biomass, a gram-positive bacterium, Corynebacterium glutamicum, with a high tolerance to fermentation inhibitors was metabolically engineered to express a fusion protein composed of cis-aconitate decarboxylase from A.terreus responsible for IA formation from cis-aconitate and a maltose-binding protein (malE) from Escherichia coli. The codon-optimized cadA_malE gene was expressed in C.glutamicum ATCC 13032, and the resulting recombinant strain produced IA from glucose. IA concentration increased 4.7-fold by the deletion of the ldh gene encoding lactate dehydrogenase. With the Δldh strain HKC2029, an 18-fold higher IA production was observed from enzymatic hydrolysate of kraft pulp as a model lignocellulosic biomass than from glucose (6.15 and 0.34 g/L, respectively). The enzymatic hydrolysate of kraft pulp contained various potential fermentation inhibitors involved in furan aldehydes, benzaldehydes, benzoic acids, cinnamic acid derivatives, and aliphatic acid. Whereas cinnamic acid derivatives severely inhibited IA production, furan aldehydes, benzoic acids, and aliphatic acid improved IA production at low concentrations. The present study suggests that lignocellulosic hydrolysate contains various potential fermentation inhibitors; however, some of them can serve as enhancers for microbial fermentation likely due to the changing of redox balance in the cell.

Fine-tuning the cell morphology of Corynebacterium glutamicum via dual-valve regulation for enhanced hyaluronic acid production

Enhanced synthesis of hyaluronic acid (HA) with recombinant Corynebacterium glutamicum as production host is achieved in this work. Hyaluronan synthase (HAS), which is a membrane protein acting as a key enzyme in HA biosynthesis, impacts both HA yield and its molecular weight. Cell morphology, which includes size, shape, and surface area, has a large impact on the expression and activity of HAS. Therefore, deliberate regulation of cell morphology holds the potential to enhance HA production. Here, we constructed three modules, namely the transporter module, the morphology tuning module and the HA synthesis module. The transporter module contains a strong constitutive promoter Ptuf and arabinose transport protein used to control the maximum amount of inducer entering the cell, thus reducing excessive cell deformation. The morphology tuning module contains an arabinose-inducible weak promoter PBAD and a cell-division-relevant gene used to sense intracellular inducer concentrations and achieve different degrees of change in cell size. These two modules work together, described as a dual-valve regulation, to achieve fine-tuning of cell morphology, resulting in a 1.87-fold increase in cell length and a 2.08-fold increase in cell membrane. When combined with the HA synthesis module, the HA titer reached 16.0 g/L, which is 1.6 times the yield reported in the previous morphology-engineered strain. Hence, for the first time, a morphologically engineered strain resulting in both high cell density and HA titer was constructed.

Production of Trehalose from Maltose by Whole Cells of Permeabilized Recombinant Corynebacterium glutamicum

Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a stable and nonreducing disaccharide; can be used as sweetener, stabilizer, and humectant; and has many applications in the food, pharmaceutical, and cosmetic industries. Trehalose production from maltose catalyzed by trehalose synthase (TreS) is simple and economically feasible for industrial-scale application. Reducing the cost and enhancing the efficiency of TreS synthesis and the conversion of maltose to trehalose is critical for trehalose production. In this study, the homologous TreS was constitutively overexpressed in Corynebacterium glutamicum ATCC13032 by removing the repressor gene lacIq fragment in the plasmid, and TreS expression could be exempt from the inducer addition and induction process. For cell permeabilization, Triton X-100 was used as a permeabilization agent, and the treatment time was 3 h. In the conversion system, the permeabilized cells of recombinant C. glutamicum were used as biocatalysts, 300 g/L maltose was used as a substrate, and 173.7 g/L trehalose was produced within 12 h under 30 °C and pH 7.0 conditions. In addition, the whole-cell biocatalysts showed promising reusability. This study provides a safe, convenient, practical, and low-cost pathway for the production of trehalose.

Construction of multi-strain microbial consortia producing amylase, serine and proline for enhanced bioconversion of food waste into lipopeptides

The availability of precursor amino acids affects the production of lipopeptides. Its yield reached to 517.73 mg/L in the co-culture of Bacillus amyloliquefaciens HM618 with recombinant Corynebacterium glutamicum producing high-level serine. In the three-strain artificial consortium (TSAC) containing strains HM618 and engineered C. glutamicum producing serine and proline, the levels of turin A, fengycin, and surfactin reached 294.60, 171.12, and 108.90 mg/L, respectively. The total lipopeptide (574.62 mg/L) in TSAC was 1.97-fold higher than that in the pure culture strain HM618. To efficiently utilize starch in food waste (FW), engineered Pichia pastoris, which produces amylase, was introduced into TSAC to construct a four-strain artificial consortium (FSAC) for the bioconversion of FW into lipopeptides. Under an FW medium, the two-strain co-culture, TSAC, and FSAC had significantly enhanced lipopeptide production and amylase activity. Therefore, a mutualistic symbiosis in the artificial consortia of strains HM618 and engineered strains supplying amino acid precursors is beneficial to improve the lipopeptides co-production.

Improved the lipopeptide production of Bacillus amyloliquefaciens HM618 under co-culture with the recombinant Corynebacterium glutamicum producing high-level proline

The application of antibacterial lipopeptides is limited by high cost and low yield. Herein, the exogenous L-proline significantly improved lipopeptide production by Bacillus amyloliquefaciens HM618. A recombinant Corynebacterium glutamicum producing high levels of proline using genetically modifying proB and putA was used to establish consortium, to improve lipopeptide production of strain HM618. Compared to a pure culture, the levels of iturin A, fengycin, and surfactin in consortium reached 67.75, 39.32, and 37.25 mg L-1, respectively, an increase of 3.19-, 2.05-, and 1.63-fold over that produced by co-cultures of B. amyloliquefaciens and recombinant C. glutamicum with normal medium. Commercial amylase and recombinant Pichia pastoris with a heterologous amylase gene were used to hydrolyze kitchen waste. A three-strain consortium with recombinant P. pastoris and C. glutamicum increased the lipopeptide production of strain HM618 in medium containing KW. This work provides new strategies to improve lipopeptide production by B. amyloliquefaciens.

Enhanced production of \u03b3-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation

BackgroundBio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics.ResultsUnder aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture.ConclusionsOur results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives.Graphical

Enhancing β-alanine production from glucose in genetically modified Corynebacterium glutamicum by metabolic pathway engineering.

To directly produce β-alanine from glucose by microbial fermentation, a recombinant Corynebacterium glutamicum strain with high efficiency of β-alanine production was constructed in this study. To do this, the biosynthetic pathway of β-alanine in an L-lysine-producing strain XQ-5 was modified by enhancing carbon flux in biosynthetic pathway and limiting carbon flux in competitive pathway. This study showed that replacement of L-aspartate kinase (AK) with wild-type AK and disruption of lactate dehydrogenase and alanine/valine aminotransferases increase β-alanine production because of decreasing the by-products accumulation. Moreover, L-aspartate-α-decarboxylase (ADC) from Bacillus subtilis was designed as the best enzyme for increasing β-alanine production, and its variant (BsADCE56S/I88M) showed the highest activity for catalyzing L-aspartate to generate β-alanine. To further increase β-alanine production, expression level of BsADCE56S/I88M was controlled by optimizing promoter and RBS, indicating that Pgro plus ThirRBS is the best combination for BsADCE56S/I88M expression and β-alanine production. The resultant strain XQ-5.5 produced 30.7 ± 2.3g/L of β-alanine with a low accumulation of lactate (from 5.2 ± 0.14 to 0.2 ± 0.09g/L) and L-alanine (from 7.6 ± 0.22 to 3.8 ± 0. 32g/L) in shake-flask fermentation and produced 56.5 ± 3.2g/L of β-alanine with a productivity of 0.79g/(L·h) and the glucose conversion efficiency (α) of 39.5% in feed-batch fermentation. This is the first report of genetically modifying the biosynthetic pathway of β-alanine that improves the efficiency of β-alanine production in an L-lysine-producing strain, and these results give us a new insight for constructing the other valuable biochemical. KEY POINTS: • Optimization and overexpression of the key enzyme BsADC increased the accumulation of β-alanine. • The AK was replaced with wild-type AK to increase the conversion of aspartic acid to β-alanine. • A 56.5-g/L β-alanine production in fed-batch fermentation was achieved.

Production of Cadaverine in Recombinant Corynebacterium glutamicum Overexpressing Lysine Decarboxylase (ldcC) and Response Regulator dr1558.

In this study, the response regulator DR1558 from Deinococcus radiodurans was overexpressed in recombinant Corynebacterium glutamicum with lysine decarboxylase (ldcC). The recombinant C. glutamicum strain overexpressing dr1558 and ldcC produced 5.9g/L of cadaverine by flask cultivation, whereas the control strain overexpressing only ldcC produced 4.5g/L of cadaverine. To investigate the mechanism underlying the effect of DR1558, the expression levels of genes related to central metabolism and lysine-biosynthesis were analyzed by quantitative-real time polymerase chain reaction. The results showed that phosphoenolpyruvate carboxykinase (pck) was downregulated, and pyruvate kinase (pyk) and other lysine biosynthesis genes were upregulated. Furthermore, in fed-batch fermentation, C. glutamicum coexpressing dr1558 produced 25.14g/L of cadaverine, a 1.25-fold increase in concentration relative to the control. These results suggested that the heterologous expression of dr1558 may improve the production of biorefinery products by recombinant C. glutamicum.

Enhanced cadaverine production by recombinant Corynebacterium glutamicum with a heterologous DR1558 regulator at low pH condition

Corynebacterium glutamicum, industrially producing various bio-based organic acids, is often cultivated under abiotic stress conditions, such as low pH, which can reduce both cell growth and the yield of the target compound. Here, a response regulator from Deinococcus radiodurans, DR1558, was introduced into a recombinant C. glutamicum strain expressing lysine decarboxylase (cadA) to enhance cadaverine production at acidic pHs. During batch cultivation under acidic conditions, 6.4 g/L of cadaverine was produced by the recombinant C. glutamicum strain expressing cadA and dr1558; this yield was 1.7-fold higher than that produced by a recombinant C. glutamicum strain expressing only cadA. Transcriptional analysis revealed altered expression levels of stress defense- and cadaverine biosynthesis-related genes in the recombinant C. glutamicum strain expressing dr1558. During fed-batch cultivation, the recombinant C. glutamicum strain expressing cadA and dr1558 showed a 2.4-fold increase in cadaverine production compared to that produced by the recombinant C. glutamicum strain expressing only cadA. The cell growth of C. glutamicum expressing both cadA and dr1558 increased markedly during fed-batch cultivation at acidic pH. These results indicated that the response regulator dr1558 altered the expression of genes involved in metabolic pathways and stress defense mechanisms in C. glutamicum.

Highly efficient biosynthesis of l-ornithine from mannitol by using recombinant Corynebacterium glutamicum

Mannitol is a promising six-carbon sugar alcohol that is widely found in macroalgae. The potential of mannitol as a renewable raw material is of interest due to the advantages of ocean farms. Herein, the biobased production of l-ornithine from mannitol was resoundingly demonstrated for the first time in engineered Corynebacterium glutamicum S9114 through the deletion of the mannitol repressor MtlR. By modulating the expression of mtlD and reinforcing the fructose metabolic pathway, we generated the strain MTL13 that produced 54.56 g/L of l-ornithine with a yield of 0.47 g/g on mannitol. These results illustrate the robust conversion from mannitol to l-ornithine using engineered Corynebacterium glutamicum, providing a reference for the biobased production of additional chemicals from mannitol.

Dynamic Control of 4-Hydroxyisoleucine Biosynthesis by Modified l-Isoleucine Biosensor in Recombinant Corynebacterium glutamicum.

4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene ido in Corynebacterium glutamicum. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O2, makes such de novo biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-PbrnFE was researched. The native PbrnFE promoter of natural Ile biosensor was still weak even under Ile induction. Through tetA dual genetic selection, several modified stronger PbrnFEN promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of ido expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9-74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing ido by the strong PtacM promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different PbrnFEN-controlled odhI decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O2 supply in ST04 by different combinations of PbrnFEN-odhI and PbrnFEN-vgb improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by PbrnFE7. This titer was higher than those of all the static metabolic engineered C. glutamicum strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL de novo biosynthesis in C. glutamicum.

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed “starch-mannose-fermentation” biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.

Optimization of ribosomal binding site sequences for gene expression and 4-hydroxyisoleucine biosynthesis in recombinant corynebacterium glutamicum

4-Hydroxyisoleucine (4-HIL) has potential value for treating diabetes. α-Ketoglutarate (α-KG)-dependent l-isoleucine dioxygenase (IDO) can convert l-isoleucine (Ile) into 4-HIL. In our previous study, 4-HIL was de novo synthesized from glucose by expressing the ido gene in Corynebacterium glutamicum strain SN01, an Ile producer, and neither Ile nor α-KG was added. In this study, ribosomal binding site (RBS) engineering was applied for gene expression and 4-HIL biosynthesis in C. glutamicum. The 18 tested RBS sequences showed greatly differing strengths for expressing ido, and 8.10–104.22 mM 4-HIL was produced. To supply the cosubstrate α-KG at different levels, the odhI gene was then expressed using the RBS sequences of high, medium, and low strength in the above mentioned optimal strain SF01 carrying R8-ido. However, 4-HIL production decreased to varying amounts, and in some strains, the α-KG was redirected into l-glutamate synthesis. Next, the O2 supply was further enhanced in three ido-odhI coexpressing strains by overexpressing the vgb gene, and 4-HIL production changed dramatically. 4-HIL (up to 119.27 ± 5.03 mM) was produced in the best strain, SF08, suggesting that the synchronic supply of cosubstrates α-KG and O2 is critical for the high-yield production of 4-HIL. Finally, the avtA gene and the ldhA-pyk2 cluster were deleted separately in SF08 to reduce pyruvate-derived byproducts, and 4-HIL production increased to 122.16 ± 5.18 and 139.82 ± 1.56 mM, respectively, indicating that both strains were promising candidates for producing 4-HIL. Therefore, fine-tuning ido expression and the cosubstrates supply through RBS engineering is a useful strategy for improving 4-HIL biosynthesis in C. glutamicum.

Scale-down studies for the scale-up of a recombinant Corynebacterium glutamicum fed-batch fermentation: loss of homogeneity leads to lower levels of cadaverine production.

BACKGROUNDThe loss of efficiency and performance of bioprocesses on scale‐up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) that occur at the larger scale in a bench‐scale two‐compartment reactor [plug flow reactor (PFR) + stirred tank reactor (STR)] using the cadaverine‐producing recombinant Corynebacterium glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT. The new scale‐down strategy developed here studied the effect of increasing the magnitude of fermentation gradients by considering not only the average cell residence time in the PFR (), but also the mean frequency at which the bacterial cells entered the PFR () section of the two‐compartment reactor.RESULTSOn implementing this strategy the cadaverine production decreased on average by 26%, 49% and 59% when the was increased from 1 to 2 min and then 5 min respectively compared to the control fermentation. The carbon dioxide productivity was highest (3.1‐fold that of the control) at a of 5 min, but no losses were observed in biomass production. However, the population of viable but non‐culturable cells increased as the magnitude of fermentation gradients was increased. The new scale‐down approach was also shown to have a bigger impact on fermentation performance than the traditional one.CONCLUSIONThis study demonstrated that C. glutamicum DM1945 Δact3 Ptuf‐ldcC_OPT physiological response was a function of the magnitude of fermentation gradients simulated. The adaptations of a bacterial cell within a heterogeneous environment ultimately result in losses in fermentation productivity as observed here. © 2019 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Multi-Step Enzymatic Synthesis of 1,9-Nonanedioic Acid from a Renewable Fatty Acid and Its Application for the Enzymatic Production of Biopolyesters.

1,9-Nonanedioic acid is one of the valuable building blocks for producing polyesters and polyamides. Thereby, whole-cell biosynthesis of 1,9-nonanedioic acid from oleic acid has been investigated. A recombinant Corynebacterium glutamicum, expressing the alcohol/aldehyde dehydrogenases (ChnDE) of Acinetobacter sp. NCIMB 9871, was constructed and used for the production of 1,9-nonanedioic acid from 9-hydroxynonanoic acid, which had been produced from oleic acid. When 9-hydroxynonanoic acid was added to a concentration of 20 mM in the reaction medium, 1,9-nonanedioic acid was produced to 16 mM within 8 h by the recombinant C. glutamicum. The dicarboxylic acid was isolated via crystallization and then used for the production of biopolyester by a lipase. For instance, the polyesterification of 1,9-nonanedioic acid and 1,8-octanediol in diphenyl ether by the immobilized lipase B from Candida antarctica led to formation of the polymer product with the number-average molecular weight (Mn) of approximately 21,000. Thereby, this study will contribute to biological synthesis of long chain dicarboxylic acids and their application for the enzymatic production of long chain biopolyesters.

Fermentative Production of N-Alkylated Glycine Derivatives by Recombinant Corynebacterium glutamicum Using a Mutant of Imine Reductase DpkA From Pseudomonas putida.

Sarcosine, an N-methylated amino acid, shows potential as antipsychotic, and serves as building block for peptide-based drugs, and acts as detergent when acetylated. N-methylated amino acids are mainly produced chemically or by biocatalysis, with either low yields or high costs for co-factor regeneration. Corynebacterium glutamicum, which is used for the industrial production of amino acids for decades, has recently been engineered for production of N-methyl-L-alanine and sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Here, mutation of an amino acyl residue in the substrate binding site of DpkA (DpkAF117L) led to an increased specific activity for reductive alkylamination of glyoxylate using monomethylamine and monoethylamine as substrates. Introduction of DpkAF117L into the production strain accelerated the production of sarcosine and a volumetric productivity of 0.16 g L−1 h−1 could be attained. Using monoethylamine as substrate, we demonstrated N-ethylglycine production with a volumetric productivity of 0.11 g L−1 h−1, which to the best of our knowledge is the first report of its fermentative production. Subsequently, the feasibility of using rice straw hydrolysate as alternative carbon source was tested and production of N-ethylglycine to a titer of 1.6 g L−1 after 60 h of fed-batch bioreactor cultivation could be attained.