Dr. G. S. Prasad Director TIE-U

Coordinator Research and Development

University of Hyderabad

Hyderabad, INDIA

Exploiting Yeasts Diversity for

Novel Industrial Applications

0

200

400

600

800

1000

1200 N

um

ber

of

Yeast

Sp

ecie

s

Year

Number of Yeast Species Since 1952

Number of Described Yeast Species

Histogram showing the number of yeast species discovered and

characterized since 1952. (modified from Daniel et al., 2006)

Exploration sites

19%

81%

Distribution of Soil Yeast isolates between Ascomycetes and Basidiomycetes

Ascomycetous yeasts Basidiomycetous yeasts

0

5

10

15

20

25

30

Nu

um

be

r o

f Is

ola

tes

in E

ac

h G

en

us

Name of The Genus

Genus Diversity of Soil Yeast Isolates

Ascomycetous Yeasts Zygomycetous YeastBasidiomycetous Yeasts

Pie chart of the distribution of soil yeast

isolates between ascomycetous and

basidiomycetous yeasts.

0

5

10

15

20

25

Num

ber o

f Yea

st Is

olat

es am

ong

Diffe

rent

Seq

unec

e Typ

es

Name of the Nearest Yeast Species

Species Diversity among Soil Yeast Isolates

ST-10

ST-9

ST-8

ST-7

ST-6

ST-5

ST-4

ST-3

ST-2

ST-1

Basidiomycetous yeast species Ascomycetous yeast species Zygomycetous yeast species

Bar graph showing the species diversity among soil yeast isolates.

X-axis represents the name of the species to which the isolates are closely related.

Y-axis represents number of yeast isolates related to a species. Color coding represents number of sequence types related to type strain of the species.

0

2

4

6

8

10

12

14

16

Distribution of Yeast Isolates Related to Pseudozyma spp. among Flower Samples

Ustilago sparsa Ustilago esculenta Ustilago trichophora

Sporiosporium chrysopogonis Sporiosorium sorghi Pseudozyma aphidis2

Pseudozyma hubiensis Trichosporonoides maida Pseudozyma spp.

Cryptococcus rajasthanensis sp. nov. MTCC 7075T

Debaryomyces singareniensis sp. nov. MTCC 7061T Published

Candida ruelliae sp. nov. MTCC 7739T

Cryptococcus andhrapradeshensis sp. nov. MTCC 9220T

Rhodosporidium singareniensis sp. nov. MTCC 8849T Very rare

Sympodiomycopsis ganganagarensis sp. nov. MTCC 7062T

Sympodiomycopsis indica sp. nov. MTCC 9313T

Candida cucurbitacearum sp. nov. MTCC 9314T

Candida imtechensis sp. nov. MTCC 8174T

Candida sesame sp. nov. MTCC 7738T

Cryptococcus solicola sp. nov. MTCC 9239T

Cryptococcus floricola sp. nov. MTCC 7076T

Cryptococcus khammamensis sp. nov. MTCC 9221T

Rhodotorula shivajii sp. nov. MTCC 9223T

Rhodotorula tapanii sp. nov. MTCC 9222T

Starmerella dubei sp. nov. MTCC 7848T

Torulaspora soli sp.nov. MTCC 8805T

Wickerhamiella lachancei sp. nov. MTCC 8068T

Several other interesting yeasts were isoalted from flowers, insects, tree barks etc., and they

are being screend for production of pullulan, lipase, xylitol, erythritol etc.,

List of some novel yeast species isolated in this study

MTCC9314

MTCC 8068

MTCC 7739

Fellomyces polyborus CBS6072T (AF189859)

Tremella encephala CBS 6968 (AF189867)

Bullermyces albus CBS 501 T (AF075500)

Bullera unica CBS 8290 T (AF075524)

Cr. andhrapradeshensis 864T MTCC 9220T (FM178287)

Cryptococcus rajasthanensis S15L T MTCC7075 T (AM262324)

Cryptococcus sp APSS 823 Cr. solicola 862T MTCC 9239T (FM178286)

Cryptococcus sp APSS 830 Cryptococcus sp APSS 850

Cryptococcus sp APSS 851 Cryptococcus sp APSS 854

Cryptococcus laurentii CBS 139 T (AF075469)

Cryptococcus sp RBSS 307 Cryptococcus sp. RBSS 321 Cryptococcus sp. APSS 333 Cryptococcus laurentii CBS 7140 (AY315663)

100

86

Cryptococcus cellulolyticus CBS 8294 T (AF075525)

Bullera psuedoalba CBS 7227 T (AF075504)

Cryptococcus flavescens CBS 942 T (AB035042)

Cryptococcus aureus CBS 318 T (AB035041)

Cryptococcus taeanensis KCTC17149 T (AY422719)

Bullera japonica CBS 2013 T (AF444760 )

84

Cryptococcus perniciosus VKM Y-2905 T (AF472624)

Cryptococcus nemorosus VKM Y-2906 T (AF472625)

99

100

75

99

0.02

Neighbor-joining tree of Cryptococcus solicola, Cr. andhrapradeshensis and related species based on

the sequences of D1/D2 domains. Evolutionary distances were calculated according to Kimura (1980)

Numbers at nodes represent 100 replicate bootstrap samplings. Bootstrap values less than 70% are not

shown. Fellomyces polyborus was used as outgroup. Bar indicates 2 % variation.

Candida bombicola Biosurfactant Producers

Predicting Functionality from Phylogeny

Strain designation Diameter of the drop in mm

PDB YGPM

Fresh culture medium 0.5 0.5

Starmerella sp.(16S1 ) 0.6 0.75

P. flocculosa 0.8 0.9

P. antarctica

0.9 0.8

H.polymorpha 0.6 0.5

Diameter of the droplet (50 μl of culture supernatant) on the hydrophobic surface of Para film M

16S1 (putative new sp.

Biosurfactant Producers

Predicting Functionality from Phylogeny

P. antarctica, U.maydis

3E4,9D2,9E4 3F1,3G2,6P1

Present Study

Strain designation Diameter of the drop in mm

PDB YGPM

Fresh culture

medium 0.5 0.5

3E4 0.5 0.85

3F1 0.85 0.75

3G2 0.5 0.8

6P1 0.85 0.6

9D2 0.54 0.85

9E4 0. 5 0.89

15E1 0.85 0.6

16P2 0.75 0.5

16R1 0.81 0.5

MTCC 225 T 0.6 0.5

MTCC 2658 T 0.8 0.8

MTCC 2706 T 0.8 0.8

Predicting Functionality from Phylogeny

Wicherhamiella sp.

9G3 and 9A2 (putative new sp. Present study)

Producers(Lipase/Esterase)

Commercial Lipase was used as a positive control

1. Biopolymer production using yeast like fungus Aureobasidium pullulans 2. Characterization of uricases of yeasts with special emphasis on Kluyveromyces lactis 3. Application of purine degrading enzymes in lowering the purine content in beverages

1. Biopolymer production using yeast like fungus

Aureobasidium pullulans

Pullulan production by an osmotolerant Aureaobasidium pullulans

Pullulan a biopolymer synthesized by yeast-like fungal species Aureobasidium pullulans, is a linear α-D-glucan built of maltotriose subunits, connected by α-1,6-D-glucosidic and α-1,4-D-glucosidic linkages in 2:1 ratio to produce a linear glucan. This typical feature is responsible for structural flexibility and superior solubility of pullulan, and confers it with certain unique physical and chemical properties •film- and fibre-forming capability (compession moldings, acoating and sizing agent)

•impermeablity to oxygen •non-reducing •biodegradable nature It is aslo •non-mutagenic •non-toxic •tasteless •Odorless •Edible being used as a starch replacer in low-calorie food formulations, in cosmetics, lotions and shampoos

Pullulan in cosmetics

Pullulan has wide application as additive and thickener in cosmetic formulation

by providing smoothness due to its stable viscous nature in wide range of pH, to

heating, in most metal ion including sodium chloride.

Pullulan has film forming ability. Also it has characteristics like water solubility,

dispersibility, moisture absorptivity, tackiness and toxicity.

These properties of Pullulan helps to be used in various cosmetic formulations

like lotion, shampoo, face pack, eyeliner, lipstick, powder, gel, mascara, shrub

etc.

The adherence nature of Pullulan helps considerably in tightening of the skin

resulting in exhibiting strong anti-aging effect.

Being a biopolymer it also forms sheer film improving skin texture.

Being viscous, oil resistance and water soluble it can be easily washed away

from the skin and maintaining freshness of the skin.

In pharmaceutical industry Pullulan’s film forming ability is exploited for

making oral strip where colours flavour and functional ingredient can

be entrapped in the film matrix and effectively stabilized.

Further this film forming ability is also utilized for making of capsule

and soft gel capsule.

Pullulan can also be used as bulking agent in tablets.

Pullulan coating of dietary supplement tablets and soft gel helps to

maintain the stability of active ingredient which is prone to oxidation.

Pullulan’s oxygen impermeability is 9 times better then gelatin capsule

and 300 times better then HPMC capsule. Being fermentation based

product it is considered VEG.

The capsule from Pullulan can be considered as vegetal, non starch,

transparent, Gluten free, preservative free and non-GMO.

Pullulan in Pharmaceuticals

Pullulan is being used extensively in the food industry as a food ingredient for

over 20 years in Japan, and has Generally Regarded As Safe (GRAS) status

in the USA (USFDA 2002).

Pullulan which was earlier considered as an indigestible polymer was shown

to be slowly digestible and found application as a low-calorie food additive

providing bulk and texture (Wolf, 2005).

Recently pullulan is also being investigated for its biomedical applications in

various aspects like targeted drug and gene delivery, tissue engineering,

wound healing and in diagnostic imaging using quantum dots (Rekha &

Sharma 2007).

Despite the large number of uses, some of the problems associated with

fermentative production of pullulan are

(i)the formation of a melanin pigment;

(ii)the inhibitory effects caused by high sugar concentrations in the medium

(iii) the high cost associated with pullulan precipitation and recovery

Major bottlenecks

• The major bottleneck for its commercial viability is the price of the final product. Pullulan is 3 times or more costly as compared to other microbial polysaccharides like xanthum gums etc.

• Hence, it is important to develop a process for production of Pullulan which will be cost effective and economically viable.

• The first step of developing such processes is to find out a microbe having high pullulan producing capacity.

Screening

• Total 10 strains isolated from soil and flower samples were selected.

• Screened on the basis of higher exo-polysaccharide (EPS) production and less pigment formation.

EPS production by different strains

0

2

4

6

8

10

12

RBSS-125 RBSS-302 RBSS-303 APSS-865 3B2 4A3 8B1 6C1 17A2 17BR13

24h 48h 72h 96h 120h

Soil isolates_________ _________Flower isolates____________

S.No. Strain Designation Colony colour Source of Isolation

1. RBF-3B2 Cream Flowers of Andropogonis

echioides

2. RBF-4A3 Cream Flowers of Caesulia axillaris

3. RBF-8B1 Cream to pink Flowers of wild plant

4. RBF-17A2 Cream to pink Flowers of a Fabaceae family

plant

5. RBF-17BR13 Cream to pink Flowers of a Malavaceae family

plant

Details of Aureobasidium pullulans isolates used in furtjer study

Five flower isolates were selected for further studies

0

5

10

15

20

25

30

24 48 72 96 120 144

Fermentation Time (hrs.)

EPS

(gl-1

)

RBF-4A3 RBF-3B2 RBF-17A2 RBF-17BR13 RBF-8B1

These strains were further studied for pullulan production using

5% (w/v) concentration of glucose

Selected 4A3 for further studies

0

10

20

30

40

50

60

70

80

5% 10% 15% 20% 25%

Glucose Concentration

EPS

and

Bio

mas

s gl

-1

EPS

Biomass

Effect of different concentrations of glucose on exopolysaccharide and biomass production by A. pullulans 4A3

• As the physiological characterization of isolate A. pullulans RBF-4A3 showed that it is

able to grow at 50% glucose concentration, the ability of this isolate to produce EPS at

higher concentrations of glucose was examined.

• Glucose concentrations ranging from 5 to 25% w/v were used for examining the EPS

production, biomass, change in pH, consumption of glucose.

66.79 gl-1 EPS in 96 h

Biomass 34.94 gl-1

Cell morphology in 5% (w/v) glucsoe Cell morphology in 15% (w/v) glucsoe

Assignment Sigma Pullulan

wave number (cm-1)

EPS of RBF-4A3

wave number (cm-1)

O-H Str 3399.4 3397.9

C-H str 2924.7 2927.9

O-C-O str 1651.9 1653.1

C-O-H bend 1418.6 1418.4

C-O-C str 1156.0 1154.7

α-1,6- glucosidic bonds 1022.1 1018.7

α-D-glucopyranoside 852.8 847.5

α-1,4-D-glucosidic bonds 756.0 755.3

Comparison of FTIR data from Sigma pullulan and EPS produced by A. pullulans RBF-4A3

Comparison of production at different scales.. Just initiated.. Yet to be standardized

Age (hrs.)

Shake flask ( 50ml/250 ml)

20 L fermenter ( 12L/20L)

1500 L fermenter (560L/1500L)

Reducing

sugar (%)

Biomass

(g/L)

EPS

(g/L)

Reducing

sugar (%)

Biomass

(g/L)

EPS

(g/L)

Reducing

sugar (%)

Biomass

(g/L)

EPS

(g/L)

24 14.5 17.4 22.5 13.9 18 14.3 14 14.1 18.1

32 11.5 22.6 34.7 10.7 20 27 11.2 16 24.5

48 9.1 27.5 45.5 9.5 24.2 34.8 7.9 19.5 38.2

56 6.2 29 52.3 7.2 28.1 41.5 6.5 23.2 46

72 3.8 35.6 60.5 2.89 35.35 55.7 4.2 29 52.2

2. Characterization of uricases of yeasts with special emphasis on Kluyveromyces lactis

43

(from So & Thorens, 2010)

Novel uriase mutants – (European patent granted)

•Rasburicase: a recombinant uricase from A.

flavus, was approved by EMEA in 2001

(Fasturtec®) and the FDA in 2002 (Elitek®) for

tumour lysis syndrome.

•It costs $387 per 1.5 mg vial

•Day's dose for a 70 kg adult costs $3870.

•Krystexxa®: a PEGylated recombinant porcine

uricase, with a baboon C-terminal sequence

Approved by FDA in October 2010 for the

treatment of patients with chronic gout,

refractory or intolerant to conventional therapy.

• given as 8 mg dose every 2-4 weeks.

• It costs $5390 per 8 mg/ml vial.

Microbial Uricases

Aspergillus flavus

A. nidulans

Candida utilis

Bacillus fastidiosus

Arthrobacter globiformis

Cellulomonas flavigena

Klebsiella pneumoniae

Pseudomonas aeruginosa

Rasburicase

Phase III trials

45

Screening of uricase producing yeast isolates

36 isolates were screened on uric acid medium

46

Screening of yeasts on uric acid plates

47

S.No. Scientific name MTCC Number/ Strain designation Growth on UA Plate

1 Candida utitlis1 MTCC 185 +++

2 S. cerevisiae2 MTCC 170 -

3 Aureobasidium pullulans 4A3 -

4 Candida imtechenis 1A1 -

5 Clavispora lusitaniae MTCC 1648 ++

6 Cryprococcus sp. CDG-69

7 Cryptococcus floricola S1C -

8 Cryptococcus magnus CDG-2 -

9 Cryptococcus (Filobasidiella)

neoformans

MTCC 1346 ++

10 Cryptococcus rajasthanensis 3C1 -

11 Kluyveromyces lactis var. lactis MTCC 117 ++

12 Kluyveromyces lactis var. lactis MTCC 458 ++++

13 Kluyveromyces marxianus MTCC 3774 ++

14 Lachancea thermotolerans MTCC 33 +++

15 Lodderomyces elongisporus MTCC 94 ++

48

S.No. Scientific name MTCC Number/ Strain designation Growth on UA Plate

16 Metschinikowia koreens 16A -

17 Metschinikowia koreens 9G1 -

18 Meyerozyma guilliermondii MTCC 1311 ++

19 Pichia segobiensis MTCC 9716 +++

20 Pichia stipitis MTCC 9718 ++++

21 Pseudozyma aphids 9D2 -

22 Pseudozyma aphids 9E4 -

23 Pseudozyma hubeiensis 16P1 -

24 Pseudozymeantarctica MTCC 2706 -

25 Rhodosporidium kratochvilova RBSS 911 -

26 Sporisorium chrysopogonis 6P3 -

27 Sporisorium sorghii 4C3 -

28 Sporisorium sp. S1E -

29 Starmerelladuebi 16S1 -

30 Sympodiomycopsis ganganagarensis 1A1 -

31 Sympodiomycopsis indica 8A1 -

32 Ustilago sparsa 15B1 -

33 Ustilago sparsa 15P1 -

34 Ustilago trchospora 14S2 -

35 Wickerhamiella cucurbitacearum 9A2 -

36 Wickerhamiella sp. 9G3 -

37 Yarrowia lipolytica MTCC 35 +++

38 Zygosaccharomyces rouxii MTCC 2635 ++

49

5 isolates were selected for further studies

Kluyveromyces lactis

Lachancea thermotolerans

Pichia segobiensis

Pichia stipitis

Yarrowia lipolytica

Genome sequences of all the above yeasts except P. segobiensis

are available

50

Cloned uricase gene from 5 yeast species in E. coli and Over expressed

Biochemical Characterization of all the cloned enzymes was done

Optimum pH Optimum temperature pH stability Temperature stability Kinetics

Structural studies of enzyme were done

51

Purification of uricase from different yeast species

P. segobiensis K. lactis

L. thermotolerans P. stipitis

Y. lipolytica

1- Marker

2 – Crude

3 – FT

4 – Wash

5 – 9 - Elutions

37 kDa

Crystal of Uricase enzyme

Diffraction up to 2.6 ?

Crystallization of Uricase

Top view Side view of tetramer

TETRAMER

1 2

3

4

Kinetics studies of uricases

S. No. Organism KM (µM) kcat (min-1) kcat/KM (µM-1 min-1)

1 A. flavus 96.2 4.3 0.044

2 C. utilis 38.5 ± 5.16 12.16 0.315

3 A. globiformis 215±2.16 0.87 0.004

4 KlUOX 42.2 ± 2.90 12.16 0.288

6 LtUOX 48 ± 4.75 10.12 0.21

7 PseUOX 106±7.12 4.2 0.039

8 PstUOX 84±2.20 5.5 0.065

9 YlUOX 98±4.79 6.5 0.066

C. utilis 38.5 ± 5.16 12.16 0.315

ELECTRON DENSITY AT ACTIVE SITE

62 Thr - B

162 Phe - A

184 Arg - A

240 Gln - A 266 Asn - A

Kluyveromyces lactis

56

Site Directed Mutations

Using the crystal structure information 5 residues at the active site

were found to be close to the substrate

62 Threonine alanine, serine

167 Phenylalanine alanine, tryptophan, tyrosine

184 Arginine alanine, lysine, histidine

240 Glutamine alanine, aspargine,

266 Aspargine alanine, glutamine

All these mutants were cloned and over expressed in E. coli

S. No Mutant Km (μM) Kcat (min-1) Kcat/Km (μM-1 min-1)

1 Candida utilis 38.5 12.16 0.315

2 K. l (wild type) 40.8 12.16 0.29

3 T62A 68.3 2.97 0.04

4 T62S 16.2 14.32 0.88

5 F167A 76 1.35 0.017

6 F167W 28.07 14.5 0.516

7 F167Y 35.6 11.08 0.311

8 R184A 79.7 0.81 0.01

9 KL R184K 57.9 2.97 0.051

10 KL R184H 118.5 1.08 0.009

11 KL Q240A 44.0 1.62 0.03

12 KL Q240N 57.9 5.13 0.08

13 KL N266A 62.7 2.97 0.047

14 KL N266Q 62.9 3.7 0.058

Three mutations seems to have better catalytic efficiency

(from So & Thorens, 2010)

Novel uriase mutants – (European patent granted)

•Rasburicase: a recombinant uricase from A.

flavus, was approved by EMEA in 2001

(Fasturtec®) and the FDA in 2002 (Elitek®) for

tumour lysis syndrome.

•It costs $387 per 1.5 mg vial

•Day's dose for a 70 kg adult costs $3870.

•Krystexxa®: a PEGylated recombinant porcine

uricase, with a baboon C-terminal sequence

Approved by FDA in October 2010 for the

treatment of patients with chronic gout,

refractory or intolerant to conventional therapy.

• given as 8 mg dose every 2-4 weeks.

• It costs $5390 per 8 mg/ml vial.

3. Application of purine degrading enzymes in lowering the purine content in beverages

Selection of sample

Alcohol gives you ????

Kick

Any type of alcohol (beer, wine and spirit) increases the risk of gout attacks

Even in moderate level can also increases the risk of hyperuricemia and gout

Among the alcoholic beverages, beer has greater effect on hyperuricemia contains high amount of purines contains nucleosides that are easily absorbable

Globally beer consumption is 187.37

million kilolitres in 2012

Beer is the 3rd largest drink in the world after water and tea. Globally beer consumption is

187.37 million kilolitres in 2012

Among the world’s 25 largest beer-consuming countries, Thailand and India

achieved the highest annual growth rates of 13.2% and 12.4%

IMTECH

(Kirin Beer university report, 2014)

HPLC chromatogram of standards

Experimental parameters Solvent system : Sodium phosphate buffer (pH 2.3) / 10 % methanol Temperature : 35 ºC Flow rate : 0.5 mL/min

Standards

1 Adenine

2 Guanine

3 Uric acid

4 Hypoxanthine

5 Xanthine

6 Adenosine

7 Inosine

8 Allantoin

9 Xanthosine

IMTECH

Purine content in different beers

Beer brand Alcohol

content

Adenine

(µMol/l)

Guanine

(µMol/l)

Hypoxanthin

e

(µMol/l)

Xanthin

e

(µMol/l)

Uric acid

(µMol/l)

Adenosin

e

(µMol/l)

Inosine

(µMol/l)

Guanosi

ne

(µMol/l)

Xanthosi

ne

(µMol/l)

Total

purine

content

Beer-KP 5.25% 12.6 - 13.2 - - - 29.8 - - 55.6

Beer-KS <8% 43.0 70.0 82.1 51.1 - - 93.0 - - 339.2

Beer-KUL <5% 21.0 - 39.6 - - - 65.2 - 4.1 130.0

Beer-KUM <8% 29.8 - 46.5 - - - 85.9 - 8.3 170.5

Beer-BW <5% 26.7 - 48.1 - - - - 69.5 5.8 150.1

Beer-CB <5% - 11.4 49.8 - - - - 56.2 6.1 123.5

Beer-CR <5% 27.9 - 19.3 22.2 - 12.7 - 6.7 5.7 94.5

Beer-HK <5% 83.6 - 20.9 - - - - - - 104.5

Beer-TG <5% 23.2 34.8 41.2 32.1 - 18.4 - - - 149.7

Beer-TB <8% 32.9 59.8 62.3 30.6 - - - 62.0 8.2 255.8

Beer-FL <5% 26.5 2.5 81.7 - - - - 65.4 - 176.1

Beer-FG <8% 32.3 3.9 39.5 2.3 - - - 73.9 - 151.9

Beer-MHL <5% 22.4 - 30.9 - - - - 58.2 2.6 114.1

Beer-MA <8% 26.2 - 43.2 6.5 - - - 15.6 - 91.5

Beer-KD <5% 16.2 2.5 24.8 10.0 - - 28.6 40.5 - 122.6

Beer-HW <8% 28.2 5.4 14.3 2.18 8.7 - - 39.8 - 98.58

Quantification of different beer available in Indian market

Purine content analysis in Beer-KS

Purine

1 Adenine

2 Guanine

3 Hypoxanthine

4 Xanthine

5 Inosine

Purine degradation by enzyme cocktail from E. coli and A. adeninivorans

Cocktail of enzymes (ADA, GDA, PNP, XanA, and Uricase) is capable of reducing the overall purine content in beer by 50% and converted into more

soluble form, allantoin.

E. coli A. adeninivorans

First time, quantification of purine content in 16 different

beers from Indian market and found that adenine, guanine, hypoxanthine, xanthine, and inosine are major purine contents in beer whereas some beers also contained guanosine, adenosine, and xanthosine.

Beer treated with individual enzyme and found that each enzyme was able to reduce the respective purine content in beer.

The cocktail of enzymes was able to reduce the overall purine content of beer. Hence, demonstrated the enzymatic approach for lowering the purine content in beverage.

Publication

Functional and structural characterization of Kluyveromyces lactis Purine Nucleoside Phosphorylase and its potential applications in reducing purine content in food. Durga Mahor1, Anu Priyanka2, Gandham S Prasad1* and Krishan Gopal Thakur2* PLoS One. http://dx.doi.org/10.1371/journal.pone.0164279

Thank you for your kind attention