As the only essential amino acid containing elemental sulphur, L-methionine has important physiological and biochemical functions in living organisms. However, the fermentative production of L-methionine has not met the requirements of industrial production because of its low production level. In this paper, the fermentation process of an efficient L-methionine producing strain E. coli W3110ΔIJAHFEBC trc-fliY trc-malY/PAM glyA-22 metF constructed previously was systematically optimized. Based on the optimal initial glucose concentration, the effects of different fed-batch fermentation processes, including DO-Stat, pH-Stat, controlling residual sugar control at different level and feeding glucose with constant rate, on L-methionine fermentation were studied. It was found that the control of glucose concentration greatly affected the fermentation process. Subsequently, an optimal fed-batch fermentation process was developed, where the L-methionine titer was increased to 31.71 g/L, the highest yield reported to date, while the fermentation time was shortened to 68 h. Meanwhile, a fermentation kinetics model under the optimal fed-batch fermentation conditions was established, which fitted well with the biosynthesis process of L-methionine. This study may facilitate further development of the fermentative production of L-methionine.

L-tryptophan is an essential amino acid that is widely used in food, medicine and feed sectors. L-tryptophan can be produced through fermentation, and the main producing strains are engineered Escherichia coli and Corynebacterium glutamicum, which are constructed by rational design methods based on metabolic engineering and synthetic biology. However, due to the long metabolic pathway, complex and unclear regulatory mechanism for L-tryptophan production in microbial cells, the production efficiency and robustness of L-tryptophan producing strains are still low. In this connection, irrational design methods such as laboratory adaptive evolution, are often applied to improve the performance of L-tryptophan producing strains. This review summarizes the recent progress on biosynthesis metabolism of L-tryptophan and its regulation, the construction and optimization of L-tryptophan producing strains, and fermentative production of L-tryptophan, and prospects future development perspective. This review may facilitate research and development for fermentative production of L-tryptophan.

Curcumin is a multitargeting nutraceutical with numerous health benefits, however, its efficacy is limited due to poor aqueous solubility and reduced bioavailability. While nano-formulation has emerged as an alternative to encounter such issues, it often involves use of toxic solvents. Microbial synthesis may be an innovative solution to address this lacuna. Present study, for the first time, reports exploitation of Aureobasidium pullulans RBF4A3 for production of nano-curcumin. For this purpose, Aureobasidium pullulans RBF4A3 was inoculated in YPD media along with curcumin (0.1 mg/mL) and incubated for 24 h, 48 h, and 72 h. Subsequently, residual sugar, biomass, EPS concentration, curcumin concentration, and curcumin nanoparticle size were measured. As a result, nano-curcumin with an average particle size of 31.63 nm and enhanced aqueous solubility was obtained after 72 h. Further, investigations suggested that pullulan, a reducing polysaccharide, played a significant role in curcumin nano-formulation. Pullulan-mediated nano-curcumin formulation, with an average particle size of 24 nm was achieved with conversion rate of around 59.19 %, suggesting improved aqueous solubility. Additionally, the anti-oxidant assay of the resulting nano-curcumin was around 53.7 % per μg. Moreover, kinetics and thermodynamic studies of pullulan-based nano-curcumin revealed that it followed first-order kinetics and was favored by elevated temperature for efficient bio-conversion. Also, various physico-chemical investigations like FT-IR, NMR, and XRD reveal that pullulan backbone remains intact while forming curcumin nanoparticle. This study may open up new avenues for synthesizing nano-polyphenols through a completely green and solvent free process with plausible diverse applications.

In the fermentative production of compounds by using microorganisms, control of the transporter activity responsible for substrate uptake and product efflux, in addition to intracellular metabolic modification, is important from a productivity perspective. However, there has been little progress in analyses of the functions of microbial membrane transporters, and because of the difficulty in finding transporters that transport target compounds, only a few transporters have been put to practical use. Here, we constructed a Corynebacterium glutamicum-derived transporter expression library (CgTP-Express library) with the fusion partner gene mstX and used a peptide-feeding method with the dipeptide L-Ala-L-Ala to search for alanine exporters in the library. Among 39 genes in the library, five candidate alanine exporters (NCgl2533, NCgl2683, NCgl0986, NCgl0453, and NCgl0929) were found; expression of NCgl2533 increased the alanine concentration in cell culture. The CgTP-Express library was thus effective for finding a new transporter candidate.

Indole is produced in nature by diverse organisms and exhibits a characteristic odor described as animal, fecal, and floral. In addition, it contributes to the flavor in foods, and it is applied in the fragrance and flavor industry. In nature, indole is synthesized either from tryptophan by bacterial tryptophanases (TNAs) or from indole-3-glycerol phosphate (IGP) by plant indole-3-glycerol phosphate lyases (IGLs). While it is widely accepted that the tryptophan synthase α-subunit (TSA) has intrinsically low IGL activity in the absence of the tryptophan synthase β-subunit, in this study, we show that Corynebacterium glutamicum TSA functions as a bona fide IGL and can support fermentative indole production in strains providing IGP. By bioprospecting additional bacterial TSAs and plant IGLs that function as bona fide IGLs were identified. Capturing indole in an overlay enabled indole production to titers of about 0.7 g L-1 in fermentations using C. glutamicum strains expressing either the endogenous TSA gene or the IGL gene from wheat.

The potential for the use of rice bran, an agricultural waste, as a substrate in the manufacture of gellan gum was examined. Using a standard strain of Sphingomonas paucimobilis (ATCC 31461) and rice bran substrate, gellan gum was produced under optimized conditions. The optimal yield of gellan gum using rice bran substrate was found to be 11.96 g L-1 with 5% glucose, 10% inoculum, and a mixing speed of 300 rpm. Native gum was found to have a consistency index of 2.00 Pa.sn. The viscosity of the gum was found to be extremely stable when exposed to thermal stress. Concerning the rheological characteristics, the Herschel-Bulkley model offered a more realistic representation of the flow characteristics of gum solutions. The synthesized gums were mostly composed of glucose, rhamnose, and glucuronic acid. The acetic acid content of gellan gums was 2.95%, while the molecular weight was 2.88 × 105 Da. Characterization of native gellan gums by UV-Vis spectroscopy, SEM, TEM and FTIR spectroscopy is also presented.

N-alkylated amino acids are intermediates of natural biological pathways and can be found incorporated in peptides or have physiological roles in their free form. The N-ethylated amino acid l-theanine shows taste-enhancing properties and health benefits. It naturally occurs in green tea as major free amino acid. Isolation of l-theanine from Camilla sinensis shows low efficiency, and chemical synthesis results in a racemic mixture. Therefore, biochemical approaches for the production of l-theanine gain increasing interest. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of l-theanine from monoethylamine and carbon sources glucose, glycerol, or xylose using heterologous enzymes from Methylorubrum extorquens for l-theanine production and heterologous enzymes from Caulobacter crescentus for growth with xylose. l-Theanine (15.4 mM) accumulated in shake flasks with minimal medium containing monoethylamine and glucose, 15.2 mM with glycerol and 7 mM with xylose. Fed-batch bioreactor cultures yielded l-theanine titers of 10 g L-1 with glucose plus xylose, 17.2 g L-1 with glycerol, 4 g L-1 with xylose, and 21 g L-1 with xylose plus glycerol, respectively. To the best of our knowledge, this is the first l-theanine process using P. putida and the first compatible with the use of various alternative carbon sources.

L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of γ-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type Escherichia coli for one-step fermentative production of theanine from sugars and ethylamine. First, the synthetic pathway of theanine was conducted by heterologous introduction of a novel GMAS from Paracoccus aminovorans. A xylose-induced T7 RNA polymerase-P T7 promoter system was used to enhance and control gmas gene expression. Next, the precursor glutamate pool was increased by overexpression of native citrate synthase and introduction of glutamate dehydrogenase from Corynebacterium glutamicum. Then, in order to push more carbon flux towards theanine synthesis, the tricarboxylic acid cycle was interrupted and pyruvate carboxylase from C. glutamicum was introduced as a bypath supplying oxaloacetate from pyruvate. Finally, an energy-conserving phosphoenolpyruvate carboxykinase from Mannheimia succiniciproducens was introduced to increase ATP yield for theanine synthesis. After optimizing the addition time and concentration of ethylamine hydrochloride in the fed-batch fermentation, the recombinant strain TH11 produced 70.6 ​g/L theanine in a 5-L bioreactor with a yield and productivity of 0.42 ​g/g glucose and 2.72 ​g/L/h, respectively. To our knowledge, this is the first report regarding the pathway engineering of E. coli for fermentative production of theanine. The high production capacity of recombinant strain, combined with the easy processes, will hold attractive industrial application potential for the future.

Fermentative production of L-cysteine has been established using Escherichia coli. In that procedure, thiosulfate is a beneficial sulfur source, whereas repressing sulfate utilization. We first found that thiosulfate decreased transcript levels of genes related to sulfur assimilation, particularly whose expression is controlled by the transcription factor CysB. Therefore, a novel approach, i.e. increment of expression of genes involved in sulfur-assimilation, was attempted for further improvement of L-cysteine overproduction. Disruption of the rppH gene significantly augmented transcript levels of the cysD, cysJ, cysM and yeeE genes (≥1.5-times) in medium containing sulfate as a sole sulfur source, probably because the rppH gene encodes mRNA pyrophosphohydrolase that triggers degradation of certain mRNAs. In addition, the ΔrppH strain appeared to preferentially uptake thiosulfate rather than sulfate, though thiosulfate dramatically reduced expression of the known sulfate/thiosulfate transporter complexes in both ΔrppH and wild-type cells. We also found that both YeeE and YeeD are required for the strain without the transporters to grow in the presence of thiosulfate as a sole sulfur source. Therefore, yeeE and yeeD are assigned as genes responsible for thiosulfate uptake (tsuA and tsuB, respectively). In final, we applied the ΔrppH strain to the fermentative production of L-cysteine. Disruption of the rppH gene enhanced L-cysteine biosynthesis, as a result, a strain producing approximately twice as much L-cysteine as the control strain was obtained.

Here, proteins involved in sulfur-containing amino acid uptake in Escherichia coli strains were investigated with the aim of applying the findings in fermentative amino acid production. A search of genes in an l-methionine auxotrophic strain library suggested YecSC as the putative transporter of l-cystathionine. l-Methionine production increased by 15% after amplification of yecSC in producer strains. A candidate protein responsible for l-cysteine uptake was also found by experimentation with multicopy suppressor E. coli strains that recovered from growth defects caused by l-cysteine auxotrophy. Based on the results of an uptake assay, growth using l-cysteine as a sole sulfur source, and sensitivity to l-cysteine toxicity, we proposed that YeaN is an l-cysteine transporter. l-Cysteine production increased by 50% as a result of disrupting yeaN in producer strain. The study of amino acid transporters is valuable to industrialized amino acid production and also sheds light on the role of these transporters in sulfur assimilation.