Efficient production of an antitumor precursor actinocin and other medicinal molecules from kynurenine pathway in Escherichia coli.

Kynurenine pathway has a potential to convert L-tryptophan into multiple medicinal molecules. This study aims to explore the biosynthetic potential of kynurenine pathway for the efficient production of actinocin, an antitumor precursor selected as a proof-of-concept target molecule. Kynurenine pathway is first constructed in Escherichia coli by testing various combinations of biosynthetic genes from four different organisms. Metabolic engineering strategies are next performed to improve the production by inhibiting a competing pathway, and enhancing intracellular supply of a cofactor S-adenosyl-L-methionine, and ultimately to produce actinocin from glucose. Metabolome analysis further suggests additional gene overexpression targets, which finally leads to the actinocin titer of 719 mg/L. E. coli strain engineered to produce actinocin is further successfully utilized to produce 350 mg/L of kynurenic acid, a neuroprotectant, and 1401 mg/L of 3-hydroxyanthranilic acid, an antioxidant, also from glucose. These competitive production titers demonstrate the biosynthetic potential of kynurenine pathway as a source of multiple medicinal molecules. The approach undertaken in this study can be useful for the sustainable production of molecules derived from kynurenine pathway, which are otherwise chemically synthesized.

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of ß-alanine.

ß-Alanine is an important ß-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of ß-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of ß-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of ß-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient ß-alanine biosynthesis was used to construct a ß-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative ß-alanine exporter was newly identified, and its overexpression further improved the ß-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of ß-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.

Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering.

BACKGROUND: Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-ß-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. RESULTS: Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. CONCLUSIONS: In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs.

Synthetic sRNAs allow knockdown of target genes at translational level, but have been restricted to a limited number of bacteria. Here, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. BHR-sRNA is tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria, with >50% of target gene knockdown achieved in 12 bacterial species. For medical applications, virulence factors in Staphylococcus epidermidis and Klebsiella pneumoniae are knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, high performance Corynebacterium glutamicum strains capable of producing valerolactam (bulk chemical) and methyl anthranilate (fine chemical) are developed by combinatorial knockdown of target genes. A genome-scale sRNA library covering 2959 C. glutamicum genes is constructed for high-throughput colorimetric screening of indigoidine (natural colorant) overproducers. The BHR-sRNA platform will expedite engineering of diverse bacteria of both industrial and medical interest.

Metabolic Engineering Strategies for the Enhanced Microalgal Production of Long-Chain Polyunsaturated Fatty Acids (LC-PUFAs).

Long-chain polyunsaturated fatty acids (LC-PUFAs), largely obtained from fish oil, serve as valuable dietary supplements with many health benefits, especially arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. In recent years, interest in the sustainable production of LC-PUFAs using heterologous production hosts has drastically increased because overfishing and polluted oceans have led to a gradual decline in the supply of LC-PUFAs from fish oil in the last few decades. Some species of microalgae have been considered to be an ideal producer of LC-PUFAs as they inherently accumulate large amounts of LC-PUFAs and utilize CO2 using light energy. Also, a growing number of genetic toolboxes have become available, and the microalgal cultivation process is amenable to a scale-up. In fact, tremendous progress has been achieved in the development of high-performance strains of microalgae through metabolic engineering that has led to the efficient production of fatty acids and various derivatives in the last decade. This review discusses metabolic engineering strategies that have contributed to the enhanced production of LC-PUFAs using microalgae, including optimizing fatty acid biosynthetic pathway and intracellular supply of precursors and cofactors, as well as engineering transcription factors.