Microbial synthesis of high-value plant secondary products: Bioresource mining and engineering

Transcription

1 Microbial synthesis of high-value plant secondary products: Bioresource mining and engineering Bo Yu, Ph.D CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences

2 Biotechnology: old its underlying processes have been used by mankind for thousands of years. e.g. the production of wine and cheese. 2

3 Biotechnology: new Modern biotechnology uses enhanced micro-organism like yeast, bacteria as cell factories, as well as enzymes, to produce a variety of goods. Biomass Microorganism Products Downstream Bioprocess 3

4 Three Waves of Modern Biotechnology Red Biotechnology Medicines Green Biotechnology Transgenic plants White Biotechnology Industrial products 1980 s s s- Biotechnology has found its entry into medicine (red) and agriculture (green), and now a new wave of industry (white), also called industrial biotechnology. 4

5 Why Industrial Biotechnology? Demand People Health Technology Medical Biotechnol. Red Biotechnology Foods Agricultural Biotechnol. Green Biotechnology? Industrial Biotechnol. White Biotechnology 5

6 Fossil: the basis of modern civilization Refinery provides the essential chemicals and energy for modern society 6

7 The challenges for socio-economic People Environment pollution 7 Resource depletion Climate change China CO 2 emission 年 Environmental costs (white pollution, greenhouse, etc)

8 The solutions: Biotechnol. process -- from Petroleum to Biomass as materials Oil Coal Renewable biomass Corn Cassava industry exhaust Petrochemical Coal industries Biochemical Engineering Green & sustainable Rubber, fuel, nylon Plastics, chemicals Chemicals, pesticide, fine chemicals Antibiotics, Vitamins amino acids, enzymes Modern Industry 8

9 Definition of Industrial Biotechnology application of modern biotechnology for the industrial production of chemical substances and bio-energy, using living cells and their enzymes, resulting in inherently clean processes within minimum waste generation and energy use. Nutraceuticals Fine chemicals Bulk chemicals 9

10 Raw biomass Can biotechnology be competent? hydrolysis C6 sugar glucose 10 C5 sugar (xylose and arabinose hydrolysis ATP transport ADP C6 ~P ATP transport C3 aldehyde~p NAD + NADH ADP ATP C3 ~P ADP transform transform C5 ~P C7 ~P C4 ~P C3 Phenyl keto acids NADP + NADPH ~P H 2 O C3 keto C3 ADP ATP NADH NAD + H NADH NAD + 2 O C3 Acryloyl CoA C3 Hydroxy acids NAD + NADH ATP C3 acrylic acid NAD + ADP CO 2 C4 C3 diol CO 2 H 2 C1 NADH CO 2 acids C2 CoA C3 alcohol C3 C4 CoA NADH P i +O 2 CO 2 C6 ketone NAD + C4 CoA NADH NAD + C2 ~P NAD + NADH CO 2 ADP C2 aldehyde Polyketone C4 PHA ATP NADH NAD + CO 2 C5 C2 C2 keto acids Aromatic compounds alcohol acids alcohol Polymers Fuels C1-C6 Platform chemicals

11 Bio-products fit with current industries Gasoline Kerosine Diesel Fuel & Energy Bio-ethanol Bio-diesel Biogas, H 2 Petroleum Oxygenate Chiral chemicals Biomass Chemical industries HO OH OH H Hydrocarbons C n H 2n+2 11 Basic chemicals Fine chemicals Polymers Bulk chemicals Fine chemicals Bio-polymers OH OH O Carbohydrates C n (H 2 O) n

12 Bio-products fit with current industries Biomass 12 Carbonhydrates starch Hemicell ulose Cellulose Lignin Lipid Protein Thermochem Sugar glucose fructose xylose arabinose Microorganism Polymers SG C2 C3 C4 C5 C6 H 2 methane Ethanol, ethylene Lactic, glycerol acrylic acid 3-hydroxypropionic acid Fumaric acid Succinate Aspartate Malic acid Itaconic acid Levulinic acid Citric acid Gluconate Sorbitol Platform chem. Modern Chemical Indust.

13 I.B. reduces the processes and costs Chemical synthesis Organic substrate Bioprocess Pharmaceuticals Pesticides Fine chemicals Food additives Feed additives Cosmetic 1,3- PDO Traditional Chem. Biotechnology Fermentation Materials < 37% Energy < 30% CO 2 em. <63% 13

14 Case: 1,3-propanediol bio-production Glucose Native pathway in E. coli G3P Pyruvate TCA DHAP 3-PGA Gly 3-PHA 1,3-PDO Glycerol pathway from yeast 1,3-PDO pathway from Klebsiella pneumoniae 14 Modify >70 genes, 18 genes knockout or overexpressed Titer > 135 g/l, productivity >3.5 g/l/h First industrialization example of bio-based chemical

15 Biotech makes industry green Nutraceuticals Advanced Biocatalysts Fine chemicals Efficient Genetic Manipulation Rapid Microbial Evolution Protein Engineering & Screening System Metabolic Engineering Bulk chemicals Fuel 15

16 Plant secondary metabolites More than 2,000 kinds of plant natural products such as isoprenoids, alkaloids and flavonoids, which are all plant secondary metabolites, have been used by human as flavors, fragrances and medicines. (Chang et al., 2006)

17 Schematic overview of biosynthetic routes and precursors of the plant natural products (Marienhagen, et al., 2013)

18 Isoprenoids Isoprenoids comprise the largest class of the natural product products, encompassing more than 5,000 known compounds with an extremely diverse array of chemical structures, such as taxol, artemisinin, and carotenoids.

19 Alkaloids Alkaloids are nitrogen-containing, low-molecular-weight compounds and known for their medicinal use.

20 Glucosinolate Glucosinolates constitute a natural class of organic compounds that contain sulfur and nitrogen and are derived from glucose and an amino acid.

21 Natural diversity of glucosinolates They are synthesized from certain amino acids. About 132 different glucosinolates are known to occur naturally in plants. Aliphatic glucosinolates derived from mainly methionine, but also alanine, leucine, isoleucine, or valine. Aromatic glucosinolates include indolic glucosinolates, derived from tryptophan, phenylalanine and tyrosine.

22 Enzymatic activation The plants contain the endogenous thioglucosidases, called myrosinase, which, in the presence of water, cleaves off the glucose group from a glucosinolate. The remaining molecule then quickly converts to an isothiocyanate, a nitrile, or a thiocyanate; these are the active substances that serve as defense for the plant. To prevent damage to the plant itself, the myrosinase and glucosinolates are stored in separate compartments of the cell and come together mainly under conditions of physical injury.

23 Isothiocyanates Isothiocyanate was known for anti-carcinogenic activity, antimicrobial activity, and anti-inflammatory activity. Studies have suggested inverse relations between the intake of cruciferous vegetables, such as broccoli, and cancer incidence. The following compounds contribute to this function. Sulphoraphane (SFN) Benzyl Isothiocyanate (BITC) Phenylethyl Isothiocyanate (PEITC)

24 Natural diversity of isothiocyanates Its variation in the side group that is responsible for the variation in the biological activities of these plant compounds. Some glucosinolates: Sinigrin is the precursor to allyl isothiocyanate Glucotropaeolin is the precursor to benzyl isothiocyanate Gluconasturtiin is the precursor to phenethyl isothiocyanate Glucoraphanin is the precursor to sulforaphane

25 Shortcomings of natural extraction Natural diversity makes the extraction of separate pure compound high costly The enzymes of entire pathway in plants stored in separate compartments of the cell, make the synthesis inefficient Extraction process is complicated with high pollution

26 中国科学院微生物研究所 The comparison between plants and microorganisms as metabolic engineering platform plant E. coli Growing rate slow rapid Costs high low Yield low high Purity low high Extraction process difficult simple on an industrial scale 26

27 中国科学院微生物研究所 Microbial production of ITCs -- Benzyl Isothiocyanate as a case -- G ene m ining P rotein m odification E. coli P athw ay design F unctionally expressed enzym es 27 P henylalanine B enzylisothiocyanate

28 中国科学院微生物研究所 P450 enzymes myrosinase 28 Glucotropaeolin The original biosynthesis pathway in plant

29 中国科学院微生物研究所 The difficulties and solution strategies for P Translational incompatibility of the membrane signal modules 2. Absence of electron transfer machinery Modification of N-terminal membrane binding domain Fused with corresponding reductase via linker peptide ATR1: NADPH--cytochrome P450 reductase 1 29 ATR2:NADPH--cytochrome P450 reductase 2 This enzyme is required for electron transfer from NADP to cytochrome P450 in microsomes. Linker peptide linker peptide: (G 4 S) n

30 中国科学院微生物研究所 Functional expression of CYP79A2 The first eight modified amino acid from bovine-derived CYP17α was confirmed to be advantageous for anchorage to the membrane for E.coli amino acids were the functional domain amino acids were the functional domain without transmembrane sequence the 25 to 74 amino acids from sorghum derived CYP79A1 were proven to be effective for higher expression level 30

31 中国科学院微生物研究所 Functional expression of CYP83B1 ATR1: NADPH--cytochrome P450 reductase 1 ATR2:NADPH--cytochrome P450 reductase 2 31

32 中国科学院微生物研究所 The biosynthesis pathway of benzyl isothiocyanate 32

33 中国科学院微生物研究所 Design the pathway and reduce the step GSTF GGP1 Glutathione Pathway in plants Spontaneous reaction Cysteine 33

34 中国科学院微生物研究所 Selection and expression of C-S lyase C-S lyase was not redundant unlike the other enzymes in plant. SUR1 is the only C-S lyase for this pathway in plants. SUR1 In plants We tried to optimize the induction conditions but still failed to get the functional enzyme for inclusion body. 34

35 中国科学院微生物研究所 Selection and expression of C-S lyase The biosynthesis of methionine in E. coli involves cystathionine β- lyase which catalyzes a reaction similar to SUR1. 35

36 中国科学院微生物研究所 MetC 100 (β-lyase) was chosen for substituting SUR1 0 mau DAD1 C, Sig=210,4 Ref=off (D:\DATAS\LFX\LFX \ D) MetC min KDa 94 M mau min DAD1 C, Sig=210,4 Ref=off (D:\DATAS\LFX\LFX \ D) Pyruvic acid min 36

37 中国科学院微生物研究所 Condon-optimized UGT74B1 from Broccoli rapa was successfully expressed and purified which shows a high affinity for various types of thiohydroximate. UGT74B1 Marker UGT: UDP-glucose:thiohydroximate S-lucosyltransferase 37

38 中国科学院微生物研究所 SOT16 SOT18 both show catalyze activity on benzyl-derived glucosinolate. We failed to obtain the soluble SOT16 and select Arabidopsis thaliana ecotype Col-0 derived SOT18 which was confirmed to have higher V max compared to the other variants. SOT18 Marker 38 SOT: desulfoglucosinolate:paps sulfotransferase

39 中国科学院微生物研究所 Selection of suitable myrosinase Glycosylation modification was essential for plantderived myrosinases to form activity configuration. Myrosinase from Brevicoryne brassicae which need no secondary modification was selected to express in E.coli. 39

40 中国科学院微生物研究所 The activity of Myrosinase from Brevicoryne brassicae was confirmed 40

41 中国科学院微生物研究所 The modified biosynthesis pathway of benzyl isothiocyanate in E. coli CYP79A2 CYP83B1 O 2 NADPH CO 2 H 2 O NADP + O 2 NADPH CO 2 H 2 O NADP + All enzymes have been functionally expressed in E.coli PLP SOT18 UGT74B1 design 41 BMYR PAP PAPS UDP UDP-Dglucose Black indicate the original element Red indicate the modified in E.coli

42 中国科学院微生物研究所 42 As a proof of concept, successful biosynthesis of BITC in vitro by functionally expressed enzymes from different sources was confirmed.

43 中国科学院微生物研究所 Conclusion 43 ACS Synth. Biol. (2016)

44 中国科学院微生物研究所 Next Functionalizing the synthesis in vivo Cofactor supplement: cysteine and PAPS Coenzyme balance and supplement: NADPH Remove susbtrate feedback inhibition and degadation: cysteine 44

45 中国科学院微生物研究所 Cofactor supplement ATP regenaration ATP AMP Regeneration Remove feedback inhibition cysq 45 SAT-m from Arabidopsis Remove product inhibition tnaa Pyruvate + NH 3 + H 2 S Knock-out degradation path.

46 中国科学院微生物研究所 NADPH balance (P450 enzymes needs NADPH) Expression of zwf increases PPP pathway for NADPH supplement 46

47 中国科学院微生物研究所 Schematic map of BITC synthesis in vivo zwf Glucose-6-P Glucose sulfide O-Acetylserine SAT-m serine 47 PAPS regeneration

48 中国科学院微生物研究所 Acknowledgement 48 Feixia Liu

49 Thanks for your attention! Bo Yu 49