A multi-layer genome mining and phylogenomic analysis to construct efficient and autonomous efflux system for medium chain fatty acids

Medium-chain fatty acids (MCFAs) are important components for food, pharmaceutical and fuel industries. Nevertheless, engineering microorganisms to produce MCFAs often induces toxicity and stresses towards host strains, which could be alleviated via accelerating the export of MCFAs from cells. However, current secretory systems are inefficient and require inducible promoters. Here, a multi-layer genome mining and phylogenomic analysis was developed to identify efficient efflux transporters. Firstly, based on the genomic mining of 397 strains throughout various representative species, the evolutionary history of efflux transporters was recapitulated, and further experimental analysis revealed that acrE from Citrobacter exhibited the best performance. Secondly, according to the further mining of 797 Citrobacter genomes and 1084 Escherichia genomes, a detailed phylogenomic analysis of efflux transporter-centric genomic vicinities was performed. This led to the identification of efficient efflux pump combination acrE and acrF. These efflux pumps were then combined with the quorum-sensing circuit from Enterococcus faecalis to regulate MCFA efflux in an autonomous manner, which achieved a 4.9-fold boost in MCFA production and firstly demonstrated the efficient and autonomous efflux pump specially for MCFAs. The integrative omics technologies described here are enabling the utilization of the increasingly large database and the effective mining of target gene diversities

A Novel Thermal-Activated β-Galactosidase from <i>Bacillus aryabhattai</i> GEL-09 for Lactose Hydrolysis in Milk

β-Galactosidase has been greatly used in the dairy industry. This study investigated a novel thermostable β-galactosidase (lacZBa) from Bacillus aryabhattai GEL-09 and evaluated the hydrolytic performance of this enzyme. Firstly, the lacZBa-encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). Phylogenetic analyses revealed that lacZBa belonged to the glycoside hydrolase family 42. Using SDS-PAGE, we determined that the molecular weight of lacZBa was ~75 kDa. Purified lacZBa exhibited a maximum activity at 45 °C, pH 6.0, and could be activated following incubation at 45 °C for several minutes. The half-life of lacZBa at 45 °C and 50 °C was 264 h and 36 h, respectively. While Co2+, Mn2+, Zn2+, Fe2+, Mg2+, and Ca2+ enhanced enzymatic activity, Cu2+ and ethylenediaminetetraacetic acid inhibited enzymatic activity. Moreover, lacZBa could hydrolyze lactose and oNPG with Km values of 85.09 and 14.38 mM. Molecular docking results revealed that lacZBa efficiently recognized and catalyzed lactose. Additionally, the hydrolysis of lactose by lacZBa was studied in lactose solution and commercial milk. Lactose was completely hydrolyzed within 4 h with 8 U/mL of lacZBa at 45 °C. These results suggested that lacZBa identified in this study has potential applications in the dairy industry

L-Tryptophan Production in Escherichia coli Improved by Weakening the Pta-AckA Pathway.

Acetate accumulation during the fermentation process of Escherichia coli FB-04, an L-tryptophan production strain, is detrimental to L-tryptophan production. In an initial attempt to reduce acetate formation, the phosphate acetyltransferase gene (pta) from E. coli FB-04 was deleted, forming strain FB-04(Δpta). Unfortunately, FB-04(Δpta) exhibited a growth defect. Therefore, pta was replaced with a pta variant (pta1) from E. coli CCTCC M 2016009, forming strain FB-04(pta1). Pta1 exhibits lower catalytic capacity and substrate affinity than Pta because of a single amino acid substitution (Pro69Leu). FB-04(pta1) lacked the growth defect of FB-04(Δpta) and showed improved fermentation performance. Strain FB-04(pta1) showed a 91% increase in L-tryptophan yield in flask fermentation experiments, while acetate production decreased by 35%, compared with its parent FB-04. Throughout the fed-batch fermentation process, acetate accumulation by FB-04(pta1) was slower than that by FB-04. The final L-tryptophan titer of FB-04(pta1) reached 44.0 g/L, representing a 15% increase over that of FB-04. Metabolomics analysis showed that the pta1 genomic substitution slightly decreased carbon flux through glycolysis and significantly increased carbon fluxes through the pentose phosphate and common aromatic pathways. These results indicate that this strategy enhances L-tryptophan production and decreases acetate accumulation during the L-tryptophan fermentation process

Improving the reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase through stabilizing a long loop in domain B.

The reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase were improved through site-directed mutagenesis. By using multiple sequence alignment and PoPMuSiC algorithm, Ser187 and Asn188, which located within a long loop in Domain B of Bacillus licheniformis α-amylase, were selected for mutation. In addition, Ala269, which is adjacent to Ser187 and Asn188, was also investigated. Seven mutants carrying the mutations S187D, N188T, N188S, A269K, A269K/S187D, S187D/N188T, and A269K/S187D/N188T were generated and characterized. The most thermostable mutant, A269K/S187D/N188T, exhibited a 9-fold improvement in half-life at 95°C and pH 5.5, compared with that of the wild-type enzyme. Mutant A269K/S187D/N188T also exhibited improved catalytic efficiency. The catalytic efficiency of mutant A269K/S187D/N188T reached 5.87×103±0.17 g·L-1·s-1 at pH 5.5, which is 1.84-fold larger than the corresponding value determined for the wild-type enzyme. Furthermore, the structure analysis showed that immobilization of the loop containing Ser187 and Asn188 plays a significant role in developing the properties of Bacillus licheniformis α-amylase

Efficient Expression of Maltohexaose-Forming α-Amylase from Bacillus stearothermophilus in Brevibacillus choshinensis SP3 and Its Use in Maltose Production

The maltohexaose-forming, Ca2+-independent α-amylase gene from Bacillus stearothermophilus (AmyMH) was efficiently expressed in Brevibacillus choshinensis SP3. To improve the production of AmyMH in B. choshinensis SP3, the temperature and initial pH of culture medium were optimized. In addition, single-factor and response surface methodologies were pursued to optimize culture medium. Addition of proline to the culture medium significantly improved the production of recombinant α-amylase in B. choshinensis SP3. This improvement may result from improved cellular integrity of recombinant B. choshinensis SP3 in existence of proline. Culture medium optimization resulted in an 8-fold improvement in α-amylase yield, which reached 1.72 × 104 U·mL−1. The recombinant α-amylase was applied to the production of maltose on a laboratory scale. A maltose content of 90.72%, which could be classified as an extremely high maltose syrup, could be achieved using 15% (m/v) corn starch as the substrate. This study demonstrated that the B. choshinensis SP3 expression system was able to produce substantial quantities of recombinant α-amylase that has potential application in the starch industry

L-Tryptophan Production in <i>Escherichia coli</i> Improved by Weakening the Pta-AckA Pathway

<div><p>Acetate accumulation during the fermentation process of <i>Escherichia coli</i> FB-04, an L-tryptophan production strain, is detrimental to L-tryptophan production. In an initial attempt to reduce acetate formation, the phosphate acetyltransferase gene (<i>pta</i>) from <i>E</i>. <i>coli</i> FB-04 was deleted, forming strain FB-04(<i>Δpta</i>). Unfortunately, FB-04(<i>Δpta</i>) exhibited a growth defect. Therefore, <i>pta</i> was replaced with a <i>pta</i> variant (<i>pta</i>1) from <i>E</i>. <i>coli</i> CCTCC M 2016009, forming strain FB-04(<i>pta</i>1). Pta1 exhibits lower catalytic capacity and substrate affinity than Pta because of a single amino acid substitution (Pro69Leu). FB-04(<i>pta</i>1) lacked the growth defect of FB-04(<i>Δpta</i>) and showed improved fermentation performance. Strain FB-04(<i>pta</i>1) showed a 91% increase in L-tryptophan yield in flask fermentation experiments, while acetate production decreased by 35%, compared with its parent FB-04. Throughout the fed-batch fermentation process, acetate accumulation by FB-04(<i>pta</i>1) was slower than that by FB-04. The final L-tryptophan titer of FB-04(<i>pta</i>1) reached 44.0 g/L, representing a 15% increase over that of FB-04. Metabolomics analysis showed that the <i>pta</i>1 genomic substitution slightly decreased carbon flux through glycolysis and significantly increased carbon fluxes through the pentose phosphate and common aromatic pathways. These results indicate that this strategy enhances L-tryptophan production and decreases acetate accumulation during the L-tryptophan fermentation process.</p></div

Flask cultivation of different strains.

<p>(a) Acetate levels; (b) L-tryptophan levels; (c) biomass levels; (d) glucose consumption levels. FB-04 (square); FB-04(<i>Δpta</i>) (circle); FB-04(<i>ΔackA</i>) <b>(</b>triangle); FB-04(<i>pta</i>1) (diamond).</p

Fed-batch fermentation of different strains.

<p>(a) Biomass levels; (b) Acetate levels; (c) L-tryptophan levels. FB-04 (square); FB-04(<i>Δpta</i>) (circle); FB-04(<i>ΔackA</i>) <b>(</b>triangle); FB-04(<i>pta</i>1) (diamond).</p

Strains, plasmids and primers used in this study.

<p>Strains, plasmids and primers used in this study.</p

SDS-PAGE analysis of enzymes.

<p>(a) SDS-PAGE analysis of Pta and Pta1 followed by the protein concentration. Lanes contain: cell lysates from expression cultures of <i>E</i>. <i>coli</i> BL21(DE3)/pET24a-<i>pta</i> (1.2 mg/mL) (lane 1) and <i>E</i>. <i>coli</i> BL21(DE3)/pET24a-<i>pta</i>1 (1.1 mg/mL) (lane 2); 50% saturated (NH₄)₂SO₄ precipitations of Pta (0.7 mg/mL) (lane 3) and Pta1(0.8 mg/mL) (lane 4); Purified enzymes: Pta (0.2 mg/mL) (lane 5) and (0.2 mg/mL)Pta1 (lane 6); and protein molecular weight markers (lane M).</p