| L-tryptophan is an essential aromatic amino acid for humans and animals which can be used as food additive, infusion liquids, pellagra treatment, sleep induction and nutritional therapy. It can synthesize serotonin, nicotinic acid, pigment, alkaloid, coenzyme, indoleacetic acid and so on. Nowadays, the worldwide demand for L-tryptophan is above ten thousand tons and is increased by almost10%every year. In the medical and pharmacological field, L-tryptophan is widely used in amino acid injection, essential amino acid Pharmaceuticals and hydrolyzed protein additives. The conversion product of L-tryptophan, serotonin, could improve sleep quality, and can be used for antihypertensive, antidepressant and treatment of pellagra. As a food additive, L-tryptophan can enhance the utilization efficiency of the vegetable protein. It can balance the amino acids in the feed and promote the growth of livestock by adding L-tryptophan in the feed.In the past, L-tryptophan was mainly produced by the chemical synthesis and protein hydrolysis, but these methods were limited for the sources of material, the long and complex process, which were gradually eliminated. With the advantage of low-cost and environmental friendly, microbial L-tryptophan production method has been widely used. The microbial production method comprises microbial transformation, enzymatic methods and direct fermentation.For microbial transformation method, sugars are used as carbon source, and precursors of L-tryptophan, such as anthranilate and indole are simultaneously added into the medium. However, the conversion rate will decreased when the concentration of precursors in the conversion solution are too high. In addition, it is not conducive to lower the cost due to the utilization of expensive precursors. In the method of enzymatic synthesis, L-tryptophan is produced using L-tryptophan biosynthetic enzymes of microorganism. It can take advantage of the chemical synthesis precursors as the raw materials, and has the advantages of high product concentration, high purity, less by-product, and easily operation, and therefore it is a low-cost L-tryptophan industrialized production method. However, this method requires high activity of enzymes and high concentration of the substrates to promote the reaction, as a result, the reaction balance is not easy to manipulate. Direct fermentation method uses excellent L-tryptophan-producing strains such as Corynebacterium glutamicum and Escherichia coli to produce L-tryptophan with cheap raw material such as glucose as carbon source. This method has been studied for a long time, but it had not been reached the titer of industrial requirement until now. High production of L-tryptophan is limited by the long biosynthetic pathway from glucose to L-tryptophan, the relatively low metabolic flow in normal circumstances, the requirements of a variety of precursors and the relatively complex regulation mechanism in L-tryptophan biosynthetic pathway.In recent years, with the development of metabolic engineering, transcription genomics and synthetic biology, through the analysis of metabolic pathways, overexpression of an endogenous gene or the introduction of exogenous genes, and elimination of competition branch, several engineered strains for industrial production of the particular target compound have been constructed. Metabolic engineering has been widely used in microbial synthesis of amino acids, organic acids, terpenoids, polyhydroxyalkanoate and biofuels. E. coli, because of its easy cultivation, clear genetic background and simple genetic manipulation, has been widely used in large-scale synthesis of valuable compounds.According to the limiting factors in L-tryptophan synthesis pathway, to generate an E. coli that overproduces and excretes L-tryptophan, the following manipulation was done:First, trpR gene, which encodes a tryptophan transcriptional repressor, was knocked out to eliminate transcription regulation of the genes in L-tryptophan pathway. Second, trpE and aroG, encoding component I of anthranilate synthase and DAHP synthase, respectively, were overexpressed after site-directed mutations to remove the feedback inhibited. Third, tktA gene, encoding a transketolase in pentose phosphate pathway. Otherwise, we knocked out ptsG, which encodes the11BC component of glucose-specific phosphoenolpyruvate: carbohydrate phosphotransferase (PTS) system, to provide more PEP. Finally, we knocked out the gene tnaA, which encodes a tryptophanase that catalyzes the reaction of L-tryptophan back into indole. The resulting L-tryptophan-synthetic strain GPT1001was able to produce1.3g l-1L-tryptophan in batch cultivation and was therefore used as base strain for further experiment.The expression of tryptophan biosynthesis operon was negatively regulated by the attenuator. However, simply removal of the attenuator probably cannot reach a sufficient expression of the tryptophan operon genes. Therefore it is essential to improve the expression of genes in tryptophan operon at the same time of inactivating the attenuator. Therefore we constructed recombinant E. coli GPT1002by inactivating the tryptophan attenuator and replacing the original trppromoter of tryptophan operon with a novel promoter cluster consisted of five core-tac-promoters aligned in tandem (5CPtacs promoter cluster) in one step. Strain GPT1002exhibited1.67-9.29times higher transcription of tryptophan operon genes than the control GPT1001. In addition, this strain accumulated1.70g l-1L-tryptophan after36h batch cultivation. Bioreactor fermentation experiments showed that GPT1002could produce10.15g l-1L-tryptophan.In E. coli, there are three tryptophan permeases, AroP, TnaB, and Mtr. To study the function of individual permease, we knocked out these three genes aroP, tnaB, and mtr separately in L-tryptophan producing strain GPT101. Then three mutants were subjected to L-tryptophan uptake assay, and it showed knocking out of tnaB decreased the L-tryptophan utilization from0.058g l-1h-1to0.054l-1h-1per OD600. And then the L-tryptophan permease mutants were compared with respect to their growth under L-tryptophan accumulating conditions. The result is consistent with the L-tryptophan uptake assay, and proved that tnaB is the main transporter responsible for the L-tryptophan uptake in E. coli.To further understand the tryptophan permease function, we constructed the double mutants of L-tryptophan permeases. Cultivation of these mutants showed that they all grew poorly comparing to the control. GPT1014, which possesses aroP and tnaB double inactivation, showed highest L-tryptophan production of2.44g l-1,19.02%higher than tnaB single mutant GPT1012, and32.6%higher than the control. And then, we constructed the triple mutant of L-tryptophan permeases GPT1017. In spite of containing mtr, GPT1017exhibited restored cell growth compare to the double mutant of tryptophan permeases. The L-tryptophan production of GPT1017was2.79g l-1in batch cultivation,51.6%higher than the control strain GPT1002. In fermentor, the maximum L-tryptophan production of strain GPT1017reached16.3g l-1at66h. Finally, we performed RT-PCR analysis of three key genes, citrate synthase, glucose-6-phosphate dehydrogenase, and glucosephosphate isomerase in E. coli, respectively. These genes showed decreased transcription in all mutants, especially in GPT1015and GPT1016, in which they were down-regulated to0.01-0.08fold of the level in GPT1002. However, the transcription of gltA, zwf and pgi were restored in tryptophan permeases deficient mutant GPT1017.Polyhydroxybutyrate (PHB), the best known polyhydroxyalkanoates (PHA) has been believed to change intracellular metabolic flow and oxidation/reduction state, as well as enhance stress resistance of the host. In this study, a PHB biosynthesis pathway, which contains phaCAB operon genes from Ralstonia eutropha, was introduced into an L-tryptophan producing Escherichia coli strain GPT1002. The expression of the PHB biosynthesis genes resulted in PHB accumulation inside the cells and improved the L-tryptophan production. RT-PCR analysis showed that the transcription of tryptophan operon genes in GPT2000increased by1.9-4.3times compared with the control. Xylose was then added into the medium as co-substrate to enhance the precursor supply for PHB biosynthesis. The PHB accumulation in this strain reached17.25%(w/w), the highest polymer accumulation among all tested strains. For L-tryptophan production, the mixture of16g l-1glucose and4g1-1xylose was the best. Under this condition, the highest L-tryptophan production,2.24±0.41g l-1was obtained. In addition, the secretion of acetate in the medium was also increased with the increased xylose proportion in the medium. Moreover, we obtained14.4g l-1L-tryptophan production and9.7%PHB (w/w) accumulation in GPT2000 via fed-batch cultivation.Finally, a method of integrating random-copy genes into E. coli genome was carried out. By utilizing the condition-replicon oriR6Ky, and the kanamycin resistance gene and the GFP as a reporter gene, integration plasmid pTKG was constructed. After FLP/FRT recombination, it showed that1-12copies of the kan-trc-gfp gene were successfully integrated into the genome. Considering the recovery incubation time may affect the integration copy number, we examined the impact of different recovery incubation time on random integration copy numbers. It showed1h is the best incubation time, and increasing or decreasing the time would reduce the maximum integration copy number. By integration random copies of aroK gene encoding shimikate kinase in the genome of the L-tryptophan-producing strain GPT102T and performed batch fermentation, we found that three copies of aroK in the genome result in the highest production of L-tryptophan per unit of cell dry weight (CDW).In this study, we firstly construct a base L-tryptophan production strains GPT1001. By use of one-step of tryptophan attenuator inactivation and promoter swapping to generate E. coli GPT1002, the L-tryptophan production was improved further. And then, the knocking out analysis of L-tryptophan permease on the production of L-tryptophan was carried out carefully. In addition, an L-tryptophan and PHB co-producing strain was constructed for the first time. The PHB accumulation was verified to improve L-tryptophan production and tryptophan transcription level. Finaly, we designed a novel method of random copies of gene integration into genome which could be widely used in metabolic engineering. |
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