Pengfei Gu

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose.

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA(M) from S. arizona on ALA production (2052mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.

One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli.

BackgroundL-tryptophan is an aromatic amino acid widely used in the food, chemical and pharmaceutical industries. In Escherichia coli, L-tryptophan is synthesized from phosphoenolpyruvate and erythrose 4-phosphate by enzymes in the shikimate pathway and L-tryptophan branch pathway, while L-serine and phosphoribosylpyrophosphate are also involved in L-tryptophan synthesis. In order to construct a microbial strain for efficient L-tryptophan production from glucose, we developed a one step tryptophan attenuator inactivation and promoter swapping strategy for metabolic flux optimization after a base strain was obtained by overexpressing the tktA, mutated trpE and aroG genes and inactivating a series of competitive steps.ResultsThe engineered E. coli GPT1002 with tryptophan attenuator inactivation and tryptophan operon promoter substitution exhibited 1.67 ~ 9.29 times higher transcription of tryptophan operon genes than the control GPT1001. In addition, this strain accumulated 1.70 g l-1 L-tryptophan after 36 h batch cultivation in 300-mL shake flask. Bioreactor fermentation experiments showed that GPT1002 could produce 10.15 g l-1 L-tryptophan in 48 h.ConclusionsThe one step inactivating and promoter swapping is an efficient method for metabolic engineering. This method can also be applied in other bacteria.

Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli

N-acetylneuraminic acid (NeuAc) has recently drawn much attention owing to its wide applications in many aspects. Besides extraction from natural materials, production of NeuAc was recently focused on enzymatic synthesis and whole-cell biocatalysis. In this study, we designed an artificial NeuAc biosynthetic pathway through intermediate N-acetylglucosamine 6-phosphate in Escherichia coli. In this pathway, N-acetylglucosamine 2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) were heterologously introduced into E. coli from Synechocystis sp. PCC6803 and Saccharomyces cerevisiae EBY100, respectively. By derepressing the feedback inhibition of glucosamine-6-phosphate synthase, increasing the accumulation of N-acetylglucosamine and pyruvate, and blocking the catabolism of NeuAc, we were able to produce 1.62 g l⁻¹ NeuAc in recombinant E. coli directly from glucose. The NeuAc yield reached 7.85g l⁻¹ in fed-batch fermentation. This process offered an efficient fermentative method to produce NeuAc in microorganisms using glucose as carbon source and can be optimized for further improvement.

A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies.

Direct optimization of the metabolic pathways on the chromosome requires tools that can fine tune the overexpression of a desired gene or optimize the combination of multiple genes. Although plasmid-dependent overexpression has been used for this task, fundamental issues concerning its genetic stability and operational repeatability have not been addressed. Here, we describe a rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies (CIGMC), which uses the flippase from the yeast 2-μm plasmid. Using green fluorescence protein as a model, we verified that the fluorescent intensity was in accordance with the integration copy number of the target gene. When a narrow-host-range replicon, R6K, was used in the integrative plasmid, the maximum integrated copy number of Escherichia coli reached 15. Applying the CIGMC method to optimize the overexpression of single or multiple genes in amino acid biosynthesis, we successfully improved the product yield and stability of the production. As a flexible strategy, CIGMC can be used in various microorganisms other than E. coli.

A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome

Homologous recombination-mediated genome engineering has been broadly applied in prokaryotes with high efficiency and accuracy. However, this method is limited in realizing larger-scale genome editing with numerous genes or large DNA fragments because of the relatively complicated procedure for DNA editing template construction. Here, we describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial gene (s) in a homologous recombination-independent manner and without the use of selective marker. Our study show that CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step that does not require homologous DNA template. It is thus a novel and powerful tool for bacterial genomes reducing and possesses the potential for accelerating the genome evolution.

Tunable switch mediated shikimate biosynthesis in an engineered non-auxotrophic Escherichia coli

Shikimate is a key intermediate in the synthesis of neuraminidase inhibitors. Compared with traditional methods, microbial production of shikimate has the advantages of environmental friendliness, low cost, feed stock renewability, and product selectivity and diversity. Despite these advantages, shikimate kinase I and II respectively encoded by aroK and aroL are inactivated in most shikimate microbial producers, thus requiring the addition of aromatic compounds during the fermentation process. To overcome this problem, we constructed a non-auxotrophic, shikimate-synthesising strain of Escherichia coli. By inactivation of repressor proteins, blocking of competitive pathways and overexpression of key enzymes, we increased the shikimate production of wild-type E. coli BW25113 to 1.73 g/L. We then designed a tunable switch that can conditionally decrease gene expression and substituted it for the original aroK promoters. Expression of aroK in the resulting P-9 strain was maintained at a high level during the growth phase and then reduced at a suitable time by addition of an optimal concentration of inducer. In 5-L fed-batch fermentation, strain P-9 produced 13.15 g/L shikimate without the addition of any aromatic compounds. The tunable switch developed in this study is an efficient tool for regulating indispensable genes involved in critical metabolic pathways.

Novel technologies provide more engineering strategies for amino acid-producing microorganisms.

Traditionally, amino acid-producing strains were obtained by random mutagenesis and subsequent selection. With the development of genetic and metabolic engineering techniques, various microorganisms with high amino acid production yields are now constructed by rational design of targeted biosynthetic pathways. Recently, novel technologies derived from systems and synthetic biology have emerged and open a new promising avenue towards the engineering of amino acid production microorganisms. In this review, these approaches, including rational engineering of rate-limiting enzymes, real-time sensing of end-products, pathway optimization on the chromosome, transcription factor-mediated strain improvement, and metabolic modeling and flux analysis, were summarized with regard to their application in microbial amino acid production.