1. Journal Club @ Ishii-Lab No. 1169
Recent Knowledge about PHA
~From Technological Aspect ~
Putative biosynthesis pathways of branched amino acids and endpoint feedback inhibition in R. eutropha. IlvBH, acetohydroxy acid synthase III; IlvC, ketol-acid reductoisomerase; IlvD, dihydroxy acid dehydratase; IlvE, branched-chain amino acid aminotransferase; LeuA, isopropylmalate synthase; LeuCD, isopropylmalate isomerase; LeuB, 3-isopropylmalate dehydrogenase. ⦵, inhibition; ⊕, relief of inhibition.
Masaharu Ishii, Prof.

2. Motivation for Today’s Journal Club
（１）I want to make it a reality that strain TH-1 should make useful compounds from carbon dioxide.
（２）Because TH-1 has a good ability to produce PHA, what I should think about must be how I can make the most of the biosynthetic ability.
（３）In terms of microbiological research on
PHA, those about Ralstonia eutropha must
be selected.
（４）Therefore, I selected a recent paper,
which I will present from now.

3. Recent Knowledge about PHA ~From Technological Aspect ~

4. Just for your information

5. Backgrounds
Because Leucine had a positive effect on the mol% increase of 3H4MV in PHB-4, authors isolated Leucine analog resistant mutant IF2 and analyzed the mutant together with its parent and KNK-DCD1 biochemically.

6. Structural similarity between Leucine and 3H4MV
Authors thought Leucine should be converted into 3H4MV.

7. Difference between IF2 and PHB-4
E. coli IlvH is known to be extremely sensitive to feedback inhibition by
valine. It was shown previously for the E. coli K-12 strain that mutagenesis of Ala-36 to Val (A36V) in IlvH resulted in the strain being less sensitive to feedback inhibition by valine. Probably, the same situation is happening on Ralstonia.

8. Putative biosynthesis pathways of branched amino acids and
endpoint feedback inhibition in R. eutropha.
Mutation in IlvH explains the escape of inhibition. But, there is LeuA, which can be inhibited by leucine.
Leu A activity is inhibited by Leu, but it can be recovered by Val. (Wiegel and Schelegel: 1977)
Putative biosynthesis pathways of branched amino acids and endpoint feedback inhibition in R. eutropha. IlvBH, acetohydroxy acid synthase III; IlvC, ketol-acid reductoisomerase; IlvD, dihydroxy acid dehydratase; IlvE, branched-chain amino acid aminotransferase; LeuA, isopropylmalate synthase; LeuCD, isopropylmalate isomerase; LeuB, 3-isopropylmalate dehydrogenase. ⦵, inhibition; ⊕, relief of inhibition.
Azusa Saika et al. J. Bacteriol. 2015;197:

9. Amino acids produced by R. eutropha strains PHB−4 and 1F2.
Amino acids produced by R. eutropha strains PHB−4 and 1F2. Cultures were grown in MS medium containing fructose (20 g/liter) at 30°C for 24 h. Concentrations of valine and leucine in culture supernatants were determined by HPLC. The results are the averages of data from three independent cultivations. ND, not detected.
Azusa Saika et al. J. Bacteriol. 2015;197:

10. Putative biosynthesis pathways of branched amino acids and
endpoint feedback inhibition in R. eutropha.
Putative biosynthesis pathways of branched amino acids and endpoint feedback inhibition in R. eutropha. IlvBH, acetohydroxy acid synthase III; IlvC, ketol-acid reductoisomerase; IlvD, dihydroxy acid dehydratase; IlvE, branched-chain amino acid aminotransferase; LeuA, isopropylmalate synthase; LeuCD, isopropylmalate isomerase; LeuB, 3-isopropylmalate dehydrogenase. ⦵, inhibition; ⊕, relief of inhibition.
It seems reasonable to think that IF2 made a success in escaping the feedback inhibition.
Azusa Saika et al. J. Bacteriol. 2015;197:

11. Expression of bktB, which encodes broad-substrate-specificity 3-ketothilase, showed a positive effect for the increase in average level of 3H4MV production. 3-Ketothilase catalyzes a condensation reaction between isobutyryl-CoA and acetyl-CoA.

12. Structural similarity between Leucine and 3H4MV
Authors thought Leucine should be converted into 3H4MV, which turned out not to be the case. Then, how 3H4MV is made?

13. Possible 3H4MV biosynthesis pathway from sugars in R
Possible 3H4MV biosynthesis pathway from sugars in R. eutropha strain 1F2.
Possible 3H4MV biosynthesis pathway from sugars in R. eutropha strain 1F2. IlvBH* has the A36T mutation in the IlvH subunit and is thought to be less sensitive to feedback inhibition by branched amino acids. BktB, 3-ketothiolase; PhaB, acetoacetyl-CoA reductase; PhaC, PHA synthase. See Fig. 1 for other enzymes.
Azusa Saika et al. J. Bacteriol. 2015;197:

14. PHA production by R. eutropha recombinants in the presence of fructose 　(20 g/liter) and various concentrations of l-leucine (0 to 10 g/liter).
Dry cell
PHB−4/pBBR1″C1PsAB
1F2/pBBR1″C1ABPtacBktB
PHA
PHA production by R. eutropha recombinants in the presence of fructose (20 g/liter) and various concentrations of l-leucine (0 to 10 g/liter). Recombinant strain PHB−4/pBBR1″C1PsABRe (A) and recombinant strain 1F2/pBBR1″C1ABPtacBktB (B) were cultured for 72 h at 30°C. Data for dry cell weight (triangles), PHA (circles), and the 3H4MV fraction in PHA (squares) are the averages of data from three independent cultivations.
3H4MV
When leucine concentration is low, leucine degradation does not occur. Therefore, isovaleryl-CoA is not formed. But, when leucine is supplied sufficiently, Leu A comes to be inhibited. Soon, 2-ketoisovalerate degradation will start. It gives a metabolic basis for the 3H4MV accumulation.
Azusa Saika et al. J. Bacteriol. 2015;197:

15. Concentrations of amino acids and residual fructose in culture supernatants of R. eutropha recombinants.
Fructose
PHB−4/pBBR1″C1PsAB
1F2/pBBR1″C1ABPtacBktB
Concentrations of amino acids and residual fructose in culture supernatants of R. eutropha recombinants. Recombinant strain PHB−4/pBBR1″C1PsABRe (A) and recombinant strain 1F2/pBBR1″C1ABPtacBktB (B) were cultured for 72 h at 30°C in the presence of fructose (20 g/liter) and various concentrations of l-leucine (0 to 10 g/liter). Data for residual fructose (circles), residual leucine (open triangles), and host-produced valine (closed triangles) are the averages of data from three independent cultivations.
Valine
When 3H4MV formation starts, there should be a metabolic “fighting” between valine formation and BktB. This result shows that Valine formation wins and that extracellular deposit of valine retards the total metabolism, which can be seen from the increase in residual fructose.
Azusa Saika et al. J. Bacteriol. 2015;197:

16. Possible 3H4MV biosynthesis pathway from sugars in R
Possible 3H4MV biosynthesis pathway from sugars in R. eutropha strain 1F2.
Possible 3H4MV biosynthesis pathway from sugars in R. eutropha strain 1F2. IlvBH* has the A36T mutation in the IlvH subunit and is thought to be less sensitive to feedback inhibition by branched amino acids. BktB, 3-ketothiolase; PhaB, acetoacetyl-CoA reductase; PhaC, PHA synthase. See Fig. 1 for other enzymes.
Azusa Saika et al. J. Bacteriol. 2015;197:

18. Leucine degradation
Label at C2 position can be retained within acetyl-CoA
Lower isotope peak by leucine should mean that acetyl-CoA is also supplied by using another route, probably via direct formation through glycolysis.

19. Am I satisfied with the results?
NO!

20. What should I (we) check?
（１）We know that TH-1 has three phbCs. Are there any enzymatic differences?
（２）Does TH-1 have bktB-related gene?
（３）Difference between phbC (PhbC) and bktB (BktB) in terms of gene sequence and enzymatic characteristics
（４） Is metabolic basis for the PHB accumulation in TH-1 similar to other microorganisms? In other words, is stringent response in TH-1 similar to other microorganisms?