Accumulation Of 5-aminolevulinic Acid By Corynebaererium Glutamicum Using Heme Biosynthesis Pathway

5-Aminolevulinic acid (ALA), a nonprotein amino acid, is a significant intermediate involved in the tetrapyrrole biosynthesis of compounds, such as porphyrin, heme, chlorophyll, and vitamin B12, which extensively exists in bacteria, algae, plants, and animals. ALA has recently drawn increasing attention as a photodynamic chemical, which has been widely applied in medical and agricultural fields. ALA is applied for photodynamic therapy (PDT) in gastroenterology, urology, and dermatology and is used in tumor-localizing and photodynamic therapy for various cancers. Moreover, the application of low concentrations of ALA for agricultural purposes increases the tolerance of plants to low temperatures and high salt concentrations and is a biodegradable herbicide and insecticide.ALA is mainly synthesized through chemical methods. However, the chemical synthesis of ALA is complicated, difficult and generates relatively low yields. The concentration of researchers is on using microbial cell factories to synthesize ALA, because such systems are environmentally safe, economical, and sustainable. This will be the future development trend. For example, microbes such as Rhodobacter sphaeroides and Escherichia coli were engineered to produce ALA through metabolic engineering and genetic engineering, and most of these studies focused on the condensation reaction of succinyl-CoA and glycine that is catalyzed by ALA synthase (C4 pathway). However, the precursor of hippuric acid and succinic acid are synthesized through chemical methods, which results in the high production cost of biotransformation of ALA. In addition, the cell growth is inhibited when the concentration of glycine is higher than 1.7 g/L, and the study of ALA biotransformation is complicated. In these studies of C4 pathway, the expensive LB medium is applied, which is the bottleneck of the industrialized ALA biotransformation. Currently, the C5 biosynthetic pathway was engineered mE. coli to achieve yields of 4.13 g/L using batch fermentation. The direct synthesis of ALA from cheap glucose by the C5 pathway is an important advantage over the C4 pathway. However, E. coli is one typical strain but not one safe strain. At the same time, the precursor of C5 pathway is glutamate, and the safe strain Corynebacterium glutamicum could largely secrete glutamate. Thus, it not only reduces the production cast, but also simplifies the fermentation.C. glutamicum is used for large-scale industrial production of the flavor enhancer L-glutamate and several other amino acids. Recent studies demonstrate the potential of C. glutamicum to produce a variety of other commercially interesting compounds such as organic acids, diamines, and bio fuels. Because of its importance to industrial biotechnology, C. glutamicum serves as a prominent model organism for studying prokaryotic metabolism and its regulation as well as providing a subject for applying the tools and concepts of synthetic biology. Because L-glutamate is a precursor of ALA, and glutamate-producing C. glutamicum is generally regarded as safe, we reasoned that it might serve as an ideal host for the production of ALA.The iron-containing tetrapyrrole heme plays an essential role as a cofactor for various enzymes, in particular, for those involved in the electron transport chain. Although the synthesis of heme is required to capture energy through respiration, an excess of this critical cofactor is toxic to bacteria. The mechanism of the biosynthesis of heme from its first precursor, ALA, is highly conserved among organisms. However, the biosynthesis of ALA is regulated at different levels depending on species and may be subject to feedback inhibition by heme. The key genes gltX, hemA, and hemL, which participate in the synthesis of ALA from glutamate, were identified in the genome of C. glutamicum using in silico techniques, including sequence alignments and the identification of domains shared with those of their functionally verified counterparts. However, the sequences of these C. glutamicum genes are only 31.53%,25.32%, and 47.05% identical to those of GluRS, HemA, and HemL, respectively, of E. coli, and the functions of the former are unknown.In our study, the key genes gltX, hemA, and hemL, which participate in the synthesis of ALA from glutamate, were identified in the genome of C. glutamicum using in silico techniques, and then were further demonstrated by heterologous complementation and overexpression experiments. These results suggest that these putative gltX, hemA, and hemL genes encode glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase, respectively. Overexpression of gltX and hemL did not improve the accumulation of ALA in C. glutamicum. However, overexpression of hemA produced increased levels of ALA, indicating that the conversion of glutamyl-tRNA to glutamate-1-semialdehyde catalyzed by HemA is likely a rate-limiting step. Coexpression of hemA and hemL in native host led to the accumulation of ALA, suggesting the potential of C. glutamicum to produce ALA for research and commercial purposes. To improve ALA production, we constructed recombinant C. glutamicum strains expressing hemA and hemL derived from different organisms. These data reveal that the activity of the Salmonella arizona mutant hemAM was active in C. glutamicum and that its coexpression with hemL from E. coli greatly increased ALA production. The low yield of acetate and lactate will benefit ALA production by C. glutamicum. To optimize culture conditions to increase ALA synthesis, we compared the gene expression levels by transcriptome analysis. Transcriptome data indicated that the dissolved oxygen level and Fe2+ concentration had major effects on ALA synthesis. To address this question, we determined Fe2+ concentrations and DO levels and found that ALA production increased in proportion to the decrease in the concentration of Fe2+, and decreased DO levels were associated with increased ALA production. In addition, the downstream pathway of heme biosynthesis was firstly inhibited using small molecules such as levulinic acid and other inhibitors or introducing genetic modifications such as the addition of the degradation tag. These results suggest that the inhibition of downstream metabolic pathway of ALA affected cell metabolism and that this strategy increased ALA production. Small-scale flask cultures of engineered C. glutamicum produced 1.79 g/Lof ALA.Overexpression of hemA and hemL, which encode glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase, respectively, in C. glutamicum produces ALA, although whether ALA accumulation causes unintended effects on the host is unknown. It is proposed that the lower rates of growth and glucose consumption of SEAL may be explained by the presence of inhibitory concentrations of porphyrins and heme. Here we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL. Metabolic pathway such as glyco lysis was inhibited, but the tricarboxylic acid cycle, the pentose phosphate pathway, and respiratory metabolism were stimulated. It was proposed that reduced glycolysis redirected metabolic flux from glyco lysis to the NADPH-generating oxidative component of the PPP. The increased generation of NADPH was probably provided for the ALA synthesis which needs NADPH as the cofactor. The improved respiration rate likely increased the flux of the TCA cycle, which decreased overflow metabolism, including decreased production of acetate. The enhanced respiration rate and the TCA cycle glucose-carbon flux likely increases energy production mediated by the electron transport chain. Moreover, the transcriptional levels of certain genes involved in heme biosynthesis were up-regulated, and the data implicate the two-component system HrrSA in the regulation of heme synthesis. These results demonstrate that excess heme can be regulated by the HrrSA system, indicating indirectly that the accumulation of heme influenced cellular metabolism. In addition, the regulation of the downstream heme biosynthesis is indicated to be the important key to prevent the accumulation of intermediates to produce ALA based on the published papers and the analysis of transcriptional data. It is concluded that the overexpression of hemA and hemL by C. glutamicum produced a profound influence on these primary metabolic pathways that interacted and contributed complementary functions. This study not only aids our understanding of how the gene expression underlying metabolic pathways is regulated with the expression of hemA and hemL, but also provides an excellent reference for the design and further improvement of ALA production.Since the two-component system is involved in the regulation of heme synthesis, we studied the two-component system of C. glutamicum. The iron-containing tetrapyrrole heme is a co factor of the protein components of the electron transport chain that drives aerobic and anaerobic respiration. Certain central metabolic pathways and enzymes require heme for activity, although excess heme is toxic because of its reactive nature. In C. glutamicum, the two-component system HrrSA and ChrSA play a central role in the control of heme homeostasis to overcome heme toxicity. When heme is existent, HrrSA promotes heme degradation and the synthesis of the heme-containing cytochrome bcl-aa3 supercomplex, and decreases heme synthesis, while ChrSA activates the divergently located operon hrtBA which encodes a putative heme exporter. In order to reduce the level of cellular heme, the genes hmuO and hrtBA were over-expressed. When the inhibitor was added, the ALA accumulation was up to about 1000 mg/L with higher growth and glucose consumption.In summary, we firstly identified several key genes gltX, hemA, and hemL involved in heme biosynthesis of C. glutamicum and demonstrated their function in complementation and overexpression experiments. Recombinant C. glutamicum strains that produce ALA were constructed by expressing hemA and hemL from different organisms. Using a strategy that included transcriptome analysis, addition of ALAD inhibitors or genomic modifications, we engineered C. glutamicum strain to produce 1.79 g/Lof ALA. Further, we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL. The ALA accumulation was also increased with higher growth and glucose consumption by the two-component system of C. glutamicum.