Overexpression Of Rate-limiting Enzymes Research Articles

Flux redistribution of central carbon metabolism for efficient production of l-tryptophan in Escherichia coli.

Microbial production of l-tryptophan (l-trp) has received considerable attention because of its diverse applications in food additives and pharmaceuticals. Overexpression of rate-limiting enzymes and blockage of competing pathways can effectively promote microbial production of l-trp. However, the biosynthetic process remains suboptimal due to imbalanced flux distribution between central carbon and tryptophan metabolism, presenting a major challenge to further improvement of l-trp yield. In this study, we redistributed central carbon metabolism to improve phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) pools in an l-trp producing strain of Escherichia coli for efficient l-trp synthesis. To do this, a phosphoketolase from Bifidobacterium adolescentis was introduced to strengthen E4P formation, and the l-trp titer and yield increased to 10.8 g/L and 0.148 g/g glucose, respectively. Next, the phosphotransferase system was substituted with PEP-independent glucose transport, meditated by a glucose facilitator from Zymomonas mobilis and native glucokinase. This modification improved l-trp yield to 0.164 g/g glucose, concomitant with 58% and 40% decreases of acetate and lactate accumulation, respectively. Then, to channel more central carbon flux to the tryptophan biosynthetic pathway, several metabolic engineering strategies were applied to rewire the PEP-pyruvate-oxaloacetate node. Finally, the constructed strain SX11 produced 41.7 g/L l-trp with an overall yield of 0.227 g/g glucose after 40 h fed-batch fermentation in 5-L bioreactor. This is the highest overall yield of l-trp ever reported from a rationally engineered strain. Our results suggest the flux redistribution of central carbon metabolism to maintain sufficient supply of PEP and E4P is a promising strategy for efficient l-trp biosynthesis, and this strategy would likely also increase the production of other aromatic amino acids and derivatives.

Metabolic engineering of Pseudomonas putida KT2440 for high-yield production of protocatechuic acid

Protocatechuic acid (PCA) has been widely utilized in conventional pharmaceutical, cosmetic and functional food industries. Currently, chemical synthesis and solvent extraction are the main methods for commercial production, indicating several disadvantages. In this study, we developed a method for the biosynthesis of PCA in Pseudomonas putida KT2440 in high yield. First, we developed constitutive promoters with different expression intensities for fine-tuned gene expression. Second, we improved the biosynthesis of “natural” PCA in P. putida KT2440 via multilevel metabolic engineering strategies: overexpression of rate-limiting enzymes, removal of negative regulators, attenuation of pathway competition, and enhancement of precursor supply. Finally, by further bioprocess engineering efforts, the best-producing strain reached a titer of 12.5 g/L PCA from glucose at 72 h in a shake flask and 21.7 g/L in fed-batch fermentation without antibiotic pressure. This was the highest PCA titer from glucose using metabolically engineered microbial cell factories reported to date.

Improving squalene production by enhancing the NADPH/NADP+ ratio, modifying the isoprenoid-feeding module and blocking the menaquinone pathway in Escherichia coli

BackgroundSqualene is currently used widely in the food, cosmetics, and medicine industries. It could also replace petroleum as a raw material for fuels. Microbial fermentation processes for squalene production have been emerging over recent years. In this study, to study the squalene-producing potential of Escherichia coli (E. coli), we employed several increasing strategies for systematic metabolic engineering. These include the expression of human truncated squalene synthase, the overexpression of rate-limiting enzymes in isoprenoid pathway, the modification of isoprenoid-feeding module and the blocking of menaquinone pathway.ResultsHerein, human truncated squalene synthase was engineered in Escherichia coli to create a squalene-producing bacterial strain. To increase squalene yield, we employed several metabolic engineering strategies. A fivefold squalene titer increase was achieved by expressing rate-limiting enzymes (IDI, DXS, and FPS) involved in the isoprenoid pathway. Pyridine nucleotide transhydrogenase (UdhA) was then expressed to improve the cellular NADPH/NADP+ ratio, resulting in a 59% increase in squalene titer. The Embden–Meyerhof pathway (EMP) was replaced with the Entner–Doudoroff pathway (EDP) and pentose phosphate pathway (PPP) to feed the isoprenoid pathway, along with the overexpression of zwf and pgl genes which encode rate-limiting enzymes in the EDP and PPP, leading to a 104% squalene content increase. Based on the blocking of menaquinone pathway, a further 17.7% increase in squalene content was achieved. Squalene content reached a final 28.5 mg/g DCW and 52.1 mg/L.ConclusionsThis study provided novel strategies for improving squalene yield and demonstrated the potential of producing squalene by E. coli.

Metabolic engineering for the production of l-phenylalanine in Escherichia coli.

As one of the three proteinogenic aromatic amino acids, l-phenylalanine is widely applied in the food, chemical and pharmaceutical industries, especially in production of the low-calorie sweetener aspartame. Microbial production of l-phenylalanine has become attractive as it possesses the advantages of environmental friendliness, low cost, and feedstock renewability. With the progress of metabolic engineering, systems biology and synthetic biology, production of l-phenylalanine from glucose in Escherichia coli with relatively high titer has been achieved by improving the intracellular levels of precursors, alleviating transcriptional repression and feedback inhibition of key enzymes, increasing the export of l-phenylalanine, engineering of global regulators, and overexpression of rate-limiting enzymes. In this review, successful metabolic engineering strategies for increasing l-phenylalanine accumulation from glucose in E. coli are described. In addition, perspectives for further improvement of production of l-phenylalanine are discussed.

Metabolic engineering for improving L-tryptophan production in Escherichia coli.

L-Tryptophan is an important aromatic amino acid that is used widely in the food, chemical, and pharmaceutical industries. Compared with the traditional synthetic methods, production of L-tryptophan by microbes is environmentally friendly and has low production costs, and feed stocks are renewable. With the development of metabolic engineering, highly efficient production of L-tryptophan in Escherichia coli has been achieved by eliminating negative regulation factors, improving the intracellular level of precursors, engineering of transport systems and overexpression of rate-limiting enzymes. However, challenges remain for L-tryptophan biosynthesis to be cost-competitive. In this review, successful and applicable strategies derived from metabolic engineering for increasing L-tryptophan accumulation in E. coli are summarized. In addition, perspectives for further efficient production of L-tryptophan are discussed.

Enhanced butyric acid production in Clostridium tyrobutyricum by overexpression of rate-limiting enzymes in the Embden-Meyerhof-Parnas pathway

•Clostridium tyrobutyricum is an excellent microorganism for bio-based butyric acid production. However, the main obstacles for its industrialization are low butyric acid concentration and productivity. This study indicated that overexpression of rate-limiting enzymes in the Embden-Meyerhof-Parnas pathway, including 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA), in C. tyrobutyricum ATCC 25755 could enhance its intracellular NADH and ATP levels and resistance to butyric acid and glucose. The results of fed-batch fermentation by ATCC 25755/pfkA + pykA showed that the butyrate concentration and productivity reached 48.2 g/L and 0.50 g/L h, which increased 38.1% and 38.9% as compared with the wild-type, respectively. The increased butyric acid tolerance and production could be due to the higher intracellular NADH and ATP levels by overexpression of pfkA and pykA in C. tyrobutyricum ATCC 25755. Furthermore, ATCC 25755/pfkA+pykA exhibited higher glucose tolerance. Consequently, the productivity of butyrate was further increased by 64.0% in batch fermentation using high concentration of glucose (120 g/L).

Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.