This document discusses strategies for producing succinate in Lactococcus lactis. It explores knocking out genes in central metabolic pathways to redirect carbon flux toward succinate. Several key enzymes involved in the succinate pathway were investigated, including pyruvate carboxylase, fumarase, and fumarate reductase/succinate dehydrogenase. While some enzymes like fumarase showed improved activity when expressed in L. lactis, other efforts like cloning fumarate reductase were unsuccessful. Overall the document evaluates progress toward engineering L. lactis for succinate production and identifies remaining challenges like further testing enzyme activities and optimizing product export.

1. Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Production of succinate in Lactococcuslactis Michael GjevnøeJon Berg Poulsen S052811 s033225 Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives 1 of 21

2. Introduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Purpose of succinate production 1 of 12 valuable chemicals that can be produced biologically Potential market of $15 billion Organisms that are possible candidates Anaerobiospirillumsucciniciproducens Actinobacillussuccinogenes Corynebacteriumglutamicum Mannheimiasucciniciproducens Escherichia coli Lactococcuslactis GRAS, lactic acid bacteria (LAB) a group of gram positive, acid-tolerant, generally non-sporulating, non-respiring rod or cocci, lactic acid producing bacteria 2 of 21

3. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Goal: Succinate producing L. lactis Society has increased focus on the environment Symbiosis of large production plants Second generation feedstock Plant isolate to obtain natural utilisation of plant material 3 of 21

4. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives L. lactispathway EIIA, EIIB and EIIC are substrate specific Feedstock uptake by hierarchy Regulation of feedstock utilisation 4 of 21

5. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives L. lactispathway EIIA, EIIB and EIIC are substrate specific Feedstock uptake by hierarchy Regulation of feedstock utilisation General glycolysis in L. lactis 4 of 21

6. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives L. lactispathway EIIA, EIIB and EIIC are substrate specific Feedstock uptake by hierarchy Regulation of feedstock utilisation General glycolysis in L. lactis Oxidation of the 2 produced NADH to maintain redox balance 4 of 21

7. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives L. lactispathway EIIA, EIIB and EIIC are substrate specific Feedstock uptake by hierarchy Regulation of feedstock utilisation General glycolysis in L. lactis Oxidation of the 2 produced NADH to maintain redox balance General TCA cycle in other succinate producing organisms 4 of 21

9. Knockout of carbon competing products

10. Changed redox situation5 of 21

11. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Succinate producing pathway in L. lactis Redox balance accommodated by feedstock Symbiosis with biodiesel plant that delivers glycerol as a by-product and heavy CO2 emission 5 of 21

12. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Succinate producing pathway in L. lactis Glucose as feedstock PFL knockout to accommodate redox balance Use of PDH to yield extra NADH requires the gluoxylate shunt to incorporate acetyl-CoA in the succinate production 5 of 21

13. Bioproduction Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Potential enzymes to accommodate succinate production Glyoxylate shunt lacking ICL and MSYN PEPCK, PEPC, MDH, FUM and FRD also not present in L. lactis Possible knockout of enzymes not part of the strategy 6 of 21

14. Strategy Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Genes investigated in this project Genes most important to this research Original strategy for succinate production PC of pycAassayed from mutant strains and in purified form LDH/MDH homology investigated fumA cloned frdABCD cloned Excreated products of Dldh, Dadh and 2xpyk influenced by mutstions 7 of 21

15. Dldh/Dadh/2xpyk Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Mutant strains Mutant strains provided by Brian Koebmann Grown on SALN medium Analysed by HPLC Sampled at OD600 0.1 increase from 0.01 to stationary phase 8 of 21

17. Dadh no really change in production as expected

18. mix acid fermentation of DldhStrain produces also malate and acetoin9 of 21

20. Dadh no really change in production as expected

21. mix acid fermentation of DldhStrain produces also malate and acetoin

22. Amount of acetoin is so small that might not effluence succinate production

23. Malate is maybe produced by malic enzyme due to increased pyruvate pool

24. Or by LDH due to increased OAA pool created by PC with higher activity9 of 21

26. Substrate affinity determined by Gln90 in LDH and Arg90 in MDH

27. Important amino acids in LDHX and HICD is less conserved

28. LDH is not optimal for malate production because low pyruvate pool and high OAA pool is required10 of 21

29. Pyruvate carboxylase Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives PC assay PC assay on Dldh, Dadh and 2xpyk PC kinetic on purified PC 11 of 21

31. Activity unchanged, indicating MENZ activity for malate production

32. Background activity12 of 21

34. Negative control no activity

35. Can not distinguish between background activity and PC activity

36. Acetyl-CoA is precursor to many cellular building blocks

37. Alternative to convert OAA to malate and measure NADH consumption13 of 21

40. FUMB shows mainly anaerobic activity

41. FUMC shows mainly aerobic activity

42. Homology shows conservation of CP sequences in FUMA and FUMB that is often recognised in Fe-S binding proteins15 of 21

44. pCP34 with medium promoter strenght and pCP35 with stronger

45. Nisininduciable promoter16 of 21

47. Negative control of WT MG1363

48. Positive control of MC1000 expressing FUMA, FUMB and FUMC

49. MJ13A (pCP35+fumA) higher activity than MJ12A (pCP34+fumA)

50. Does not show activity standardised by protein amounts17 of 21

52. Parallel cloning strategy to FUMA18 of 21

54. Positive transformation control with no religations with pCP34 and pCP35

55. The colonies with frdABCDshowed no b-gal activity

56. Colony PCR was done several times with negative results

57. Plasmid purification and control digestion could have definitely determined presence of frdABCD19 of 21

59. Homology of 2 FRD and SDH from B. subtilis

60. Goodconservartion in FRDA and SDHA

62. Perspectives Introduction Bioproduction Strategy Dldh/Dadh/2xpyk PC FUMA FRD/SDH Perspectives Test of MENZ activity in the nonphysiological direction PC assay on purified PC to determine kinetics In vivo test of FUMA activity SDH instead of FRD with respect to greater chance of function in gram positive membrane Malate-, fumarate- and succinate excretion, potentially lack of transporter Investigate malate-, fumarate- and succinate tolerance level of L. lactis Multiple genes incorporated on chromosome without resistance markers 21 of 21