Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids.

To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL--aidB+-ryfAM--gadAH-, producing 30.0 g L-1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

Pectin oligosaccharides from hawthorn (Crataegus pinnatifida Bunge. Var. major) inhibit the formation of advanced glycation end products in infant formula milk powder.

Pectin oligosaccharides (POSs) can not only be used as prebiotics to promote the growth of beneficial bacteria in the intestine but also can be used as natural food-borne antiglycation agents to inhibit the formation of advanced glycation end products (AGEs) in vitro, which is related to their structure, including molecular weight and galacturonic acid content. In this study, haw polysaccharides (HPSs) were isolated and purified, and POSs with high antiglycation activity in vitro were prepared. On this basis, the inhibitory effect of POSs on the formation of AGEs in infant formula milk powder was investigated. The results showed that no obvious inhibitory effect of POSs was found on the formation of AGEs in infant formula milk powder under accelerated storage at 25 °C and 45 °C. But, POSs showed a strong inhibitory effect on the formation of furosine, Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL) and the total AGEs in infant formula milk powder under accelerated storage at 65 °C. In addition, POSs also had a strong inhibitory effect on the formation of lipid oxidation products but did not affect the formation of protein degradation products. Simultaneously, the cytotoxicity experiments using human umbilical vein endothelial cells showed that the infant formula milk powder supplemented with POSs had the lowest cytotoxicity compared to the blank control (BC) and GOS/FOS supplementation milk powder under accelerated storage at 65 °C, which may be related to the inhibitory effect of POSs on the formation of AGEs in infant formula milk powder. Furthermore, the in vitro fermentation experiments showed that the antiglycation process did not affect the prebiotic activity of POSs in infant formula milk powder.

Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis.

BACKGROUND: Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear. RESULTS: In this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of L-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of L-leucine and L-valine, not only improved surfactin production but also increased the proportion of the C14 isoform. CONCLUSIONS: This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.

Pectin oligosaccharides from fruit of Actinidia arguta: Structure-activity relationship of prebiotic and antiglycation potentials.

Pectin oligosaccharides (POSs) have prebiotic and antiglycation activities in vitro, but the specific structure-activity relationship is unclear. In this study, POSs were obtained by enzymatic and ultrasound-assisted enzymatic degradation of pectin polysaccharide (PPS), respectively. Based on the chemical characterization, the antiglycation in vitro and prebiotic activities of POSs were compared and the structure-activity relationship was studied. The results showed that the antiglycation activity of POSs in vitro was proportional to the galacturonic acid content and GalA:Rha molar ratios except for the low molecular weight POSs (LM-POSs), and inversely proportional to its branching degree, such as Ara:Rha and Gal:Rha molar ratios. In addition, it was also found that the prebiotic activity of POSs was positively correlated with Ara:Rha and Gal:Rha molar ratios in molecule composition and the neutral sugar content, especially galactose and arabinose. The degree of esterification (DE) was less important for both antiglycation and prebiotic activity of POSs. These results provided an important theoretical basis for POSs application in food.

Pectin oligosaccharides from hawthorn (Crataegus pinnatifida Bunge. Var. major): Molecular characterization and potential antiglycation activities.

The crude polysaccharide was extracted from hawthorn (Crataegus pinnatifida Bunge. Var. major) and a salt-eluted polysaccharide (SPS-2) was fractionated by DEAE cellulose-52 chromatography. Monosaccharide composition and Fourier-transform infrared (FT-IR) analysis indicated that the SPS-2 contained 72.3% galacturonic acid and may be pectin. The results of antiglycation activity in vitro showed that the medium molecular weight pectin oligosaccharide (MM-POS) had the highest antiglycation activity among SPS-2 degradation products. Moreover, the MM-POS obtained by enzymatic degradation had stronger antiglycation activity than that by ultrasonic assisted enzymatic degradation. Composition analysis of MM-POS obtained from ultrasound-assisted enzymatic degradation showed that polygalacturonans content decreased from 85.1% to 61.9% but the arabinan and rhamnogalacturonans increased from 10.6% to 22.7% and from 3.5% to 13.2%, respectively, compared to the MM-POS obtained from enzymatic degradation. This study will provide a theoretical basis for the application of POSs in the food field, especially the exploitation of antiglycation agents.