Construction of a fimbriae absent Escherichia coli by deleting 64 genes and its application for efficient production of poly-3-hydroxybutyrate and L-threonine

Escherichia coli contains 12 chaperone-usher operons for biosynthesis and assembly of various fimbriae. In this study, each of the 12 operons was deleted in E. coli MG1655, and the resulting 12 deletion mutants all grew better than the wild type, especially in the nutrient-deficient M9 medium. When the plasmid pBHR68 containing the key genes for polyhydroxyalkanoate production was introduced into these 12 mutants, each mutant synthesized more polyhydroxyalkanoate than the wild type control. These results indicate that the fimbriae removal in E. coli benefits cell growth and polyhydroxyalkanoate production. Therefore, all the 12 chaperone-usher operons including 64 genes were deleted in MG1655, resulting the fimbriae absent strain WQM026. WQM026 grew better than MG1655, and no fimbriae structures were observed on the surface of WQM026 cells. Transcriptomic analysis showed that in WQM026 cells the genes related to glucose consumption, glycolysis, flagellar synthesis, and biosynthetic pathways of some key amino acids were up-regulated, while the TCA cycle related genes were down-regulated. When pBHR68 was introduced into WQM026, huge amounts of poly-3-hydroxybutyrate were produced; when the plasmid pFW01-thrA*BC-rhtC containing the key genes for L-threonine biosynthesis and transport was transferred into WQM026, more L-threonine was synthesized than the control. These results suggest that this fimbriae absent E. coli WQM026 is a good host for efficient production of polyhydroxyalkanoate and L-threonine, and has the potential to be developed into a valuable chassis microorganism. IMPORTANCE In this study, we investigated the interaction between the biosynthesis and assembly of fimbriae and intracellular metabolic network in E. coli. We found that eliminating fimbriae could effectively improve the production of polyhydroxyalkanoate and L-threonine in E. coli MG1655. These results contribute to understanding the necessity of fimbriae and the advantages of fimbriae removal for industrial microorganisms. The knowledge gathered from this study may be applied to the development of superior chassis microorganisms.

Resistance to serine in Bacillus subtilis: Identification of the serine transporter YbeC and of a metabolic network that links serine and threonine metabolism

SummaryThe Gram-positive bacterium Bacillus subtilis uses serine not only as building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, in excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.Originality-Significance StatementSerine is an important precursor for many biosynthetic reactions, and lack of this amino acid can induce biofilm formation in Bacillus subtilis. However, serine is toxic for the growth of B. subtilis. To understand the reason(s) for this toxicity and to identify the so far unknown serine transporter(s) of this bacterium, we performed exhaustive mutant screens to isolate serine-resistant mutants. This screen identified YbeC, the major serine transporter of B. subtilis. Moreover, we observed an intimate link between serine and threonine metabolism that is responsible for serine toxicity by inhibiting threonine biosynthesis.

Threonine in mammals: indispensability and transamination

As is known, amino acid threonine is not synthesized in the vertebrates when it does not come with food and the decomposition of threonine under the action of threonine dehydratase is irreversible process. Some facts point to the presence of insignificant threonine synthesis in animals. The question arises about the possibility of biosynthesis of threonine in animals in the absence of it in food, that is, its interchangeability. Research on this issue is important for compiling the diet of animals. The article shows that the threonine cannot be synthesized by reversibility of the reaction of its decomposition as well why threonine dehydrogenase in the tissues of mammals cannot be used in threonine biosynthesis. It is concluded that some quantity of threonine is involved in transamination.

Lysine and Threonine Biosynthesis from Aspartate Contributes to Staphylococcus aureus Growth in Calf Serum

ABSTRACTStaphylococcus aureusis a human pathogen, andS. aureusbacteremia can cause serious problems in humans. To identify the genes required for bacterial growth in calf serum (CS), a library ofS. aureusmutants with randomly inserted transposons were analyzed for growth in CS, and the aspartate semialdehyde dehydrogenase (asd)-inactivated mutant exhibited significantly reduced growth in CS compared with the wild type (WT). The mutant also exhibited significantly reduced growth in medium, mimicking the concentrations of amino acids and glucose in CS. Asd is an essential enzyme for the biosynthesis of lysine, methionine, and threonine from aspartate. We constructed inactivated mutants of the genes for lysine (lysA), methionine (metE), and threonine (thrC) biosynthesis and found that the inactivated mutants oflysAandthrCexhibited significantly lower growth in CS than the WT, but the growth of themetEmutant was similar to that of the WT. The reduced growth of theasdmutant was recovered by addition of 100 μg/ml lysine and threonine in CS. These results suggest thatS. aureusrequires lysine and threonine biosynthesis to grow in CS. On the other hand, theasd-,lysA-,metE-, andthrC-inactivated mutants exhibited significantly reduced growth in mouse serum compared with the WT. In mouse bacteremia experiments, theasd-,lysA-,metE-, andthrC-inactivated mutants exhibited attenuated virulence compared with WT infection. In conclusion, our results suggest that the biosynthesis ofde novoaspartate family amino acids, especially lysine and threonine, is important for staphylococcal bloodstream infection.IMPORTANCEStudying the growth of bacteria in blood is important for understanding its pathogenicity in the host.Staphylococcus aureussometimes causes bacteremia or sepsis. However, the factors responsible forS. aureusgrowth in the blood are not well understood. In this study, using a library of 2,914 transposon-insertional mutants in theS. aureusMW2 strain, we identified the factors responsible for bacterial growth in CS. We found that inactivation of the lysine and threonine biosynthesis genes led to deficient growth in CS. However, the inactivation of these genes did not affectS. aureusgrowth in general medium. Because the concentration of amino acids in CS is low compared to that in general bacterial medium, our results suggest that lysine and threonine biosynthesis is important for the growth ofS. aureusin CS. Our findings provide new insights forS. aureusadaptation in the host and for understanding the pathogenesis of bacteremia.

Production in Escherichia coli of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) with Differing Monomer Compositions from Unrelated Carbon Sources

ABSTRACTThe industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis inEscherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing thethrABCoperon inE. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.