Xylitol Production from Lignocellulosic Pentosans: A Rational Strain

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Biotechnology and Biological Transformations

Xylitol production from lignocellulosic pentosans: a rational strain engineering approach towards a multiproduct biorefinery Dr. Diptarka Dasgupta, Vivek Jhungare, Abhilek Kumar Nautiyal, Arijit Jana, Saugata Hazra, and Debashish Ghosh J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05509 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Journal of Agricultural and Food Chemistry Xylitol fermentation with engineered yeast

Xylitol production from lignocellulosic pentosans: a rational strain engineering approach towards a multiproduct biorefinery

1 2 3 4 5 6 7 8 9 10 11 12 13

Abstract: Kluyveromyces marxianus IIPE453 can utilize biomass-derived fermentable sugars

14

for xylitol and ethanol fermentation. In this study, the xylitol production in the native strain

15

was improved by overexpression of endogenous

16

expression cassette harboring the gene of interest was constructed and incorporated in the

17

native yeast. qPCR analysis demonstrated the 2.1-fold enhancement in D-xylose reductase

18

transcript levels in the modified strain with 1.62-fold enhancement in overall xylitol yield

19

without affecting its ethanol fermenting capacity. Material balance analysis on 2 kg of

20

sugarcane bagasse-derived fermentable sugars illustrated an excess of 58.62 ± 0.15 g xylitol

21

production by transformed strain in comparison to the wild variety with similar ethanol yield.

22

The modified strain can be suitably used as a single biocatalyst for multiproduct biorefinery

23

application.

24 25 26 27 28

Diptarka Dasgupta1*, Vivek Jhungare2, Abhilek K Nautiyal1, Arijit Jana1, Saugata Hazra2, Debashish Ghosh1* 1Biotechnology

Conversion Area, Bio Fuels Division, CSIR-Indian Institute of Petroleum, Dehradun, Uttarakhand – 248 005, 2 Department of Biotechnology and 3Centre for Nanotechnology, Indian Institute of Technology Roorkee (IIT-R), Uttarakhand- 247667 *corresponding authors: [email protected], [email protected]

Keywords:

D-xylose

reductase gene. A suitable

D-Xylose

reductase; Kluyveromyces; biorefinery; xylitol; lignocellulosic biomass; ethanol

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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Xylitol fermentation with engineered yeast

30 31 32

Nomenclature 2µ

Saccharomyces origin of replication

33

ARSSac

Saccharomyces autonomous replicating sequence

34

Cre-LoxP

recombinase mediated LoxP

35

DO2

dissolved oxygen

36

fs

femtosecond

37

GAPDHKS

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of Kluyveromyces marxianus IIPE453

38 39

HMF

hydroxymethyl furfural

40

HXT

Hexose transporter

41

KSIIP453_Ura3-

Ura3 mutant of KSIIPE453

42

KSIIPE453

Kluyveromyces marxianus IIPE453 (MTCC 5314)

43

KSIIPE453t

KSIIPE453 with overexpressed XRKS

44

loxP–KanMX–loxP

LoxP-Kanamycin cassette for gene disruption

45

metric tons

MT

46

ns

nano second

47

ori_S11

Kluyveromyces origin of replication S11

48

pDrive_Ura3KS

pDrive vector carrying Ura3KS gene

49

pDrive_Ura3KS_KanMX

pDrive carrying partial Ura3KS gene and Kanamycin disruption cassette

50 51

PPP

pentose phosphate pathway

52

pYES2_XRKS

pYES2 expression vector carrying XRKS gene

53

pYES2_XRKS_S11

pYES2_ XRKS with ori_S11

54

productivityc

calculated productivity from literature data 2


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55

ps

pico second

56

qRT-PCR

quantitative real time PCR

57

SCADA

supervisory control and data acquisition

58

SCB

sugarcane bagasse

59

URA3KS

Orotidine 5'-phosphate decarboxylase (Ura3) gene of KSIIPE453

60 61

Ura3Sac

S. cerevisae Ura3

62

vvm

volume of air/volume of medium

63

XRKS

homologous D-xylose reductase of KSIIPE453

64

YEV

Yeast expression vector

65

YNBD

Yeast nitrogen base supplemented with dextrose and amino acid mix without uracil

66 67

YNB-5 FOA

Yeast nitrogen base with 5-fluoro orotic acid

68

YPX

yeast extract peptone D-xylose

69 70 71 72

Kinetic parameters YX/S

Cell biomass yield on D-xylose (g/g)

73

YP/SX

Xylitol yield on consumed sugar (g/g)

74

QPX

volumetric xylitol productivity (g/L.h-1)

75

YP/SE

ethanol yield on consumed sugar (g/g)

76

QPE

volumetric ethanol productivity (g/L.h-1)

77 78 79 80 81

3



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83 84 85

1.

86

food and beverage industries as a low-calorie sweetener with a stable and gradually rising

87

market demand of 250,000 metric tons (MT) by 2020.1 Commercially, xylitol is produced by

88

catalytic hydrogenation of ultrapure D-xylose using Raney nickel catalyst which comes with

89

certain disadvantages like severe reaction conditions, high hydrogen requirement and low

90

catalyst selectivity resulting in multiple byproducts which necessitate extensive purification

91

for downstream product recovery.2,3 Biocatalytic production of xylitol by single step

92

reduction of the hemicellulosic fraction has been considered as one of the lucrative options

93

which can be more green and sustainable than their chemical counterparts with fewer steps

94

for recovery due to xylitol production with higher selectivity.4 D-xylose accounts for nearly

95

40% of the total carbohydrates and is the second most abundant fermentable sugar in

96

lignocellulosic biomass available in the hemicellulosic form.5,6 The escalating demand for

97

renewable biofuel and success of first-generation ethanol production by fermentation route

98

has led to the focus on lignocellulosic fermentable sugars for the production of second-

99

generation biofuels.7 Use of cellulose derived hexosans for ethanol production was achieved,8

100

but utilization of pentose fraction till date remained industrially non-viable. Genetically,

101

yeasts have been engineered to ferment pentose fraction into ethanol,9 but their performance

102

in terms of titer and productivity were significantly low. Instead of conceptualizing ethanol as

103

the sole energy product in a lignocellulosic biorefinery, use of hemicellulosic fraction for a

104

non-energy specialty chemical such as xylitol could be considered as an option for multi-

105

product biorefinery scheme.10,11

106 107

Introduction

Xylitol is a 5-carbon sugar alcohol, that is widely used in pharmaceuticals, nutraceuticals,

Biocatalytic conversion of lignocellulosic pentosans into xylitol is limited by issues such as non-availability of

D-xylose

fermenting industrial strain.12 Out of few naturally 4


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available D-xylose fermenting yeasts, Candida sp. has been so far reported to be the best

109

producer of xylitol, but its pathogenic nature was the hindrance for its commercial

110

application.13,14 Other yeasts like Pichia sps., Hansunela sps. are capable of xylitol

111

production, but, have low titer and productivity.15 Saccharomyces cerevisiae, widely used in

112

industrial ethanol fermentation do not possess a native D-xylose assimilation pathway and

113

require extensive genetic modification for xylitol production1,16 from biomass-derived sugars.

114

Dairy yeast Kluyveromyces is nowadays being explored, due to its generally regarded as safe

115

(GRAS) status, established ethanol fermentation, natural pentose assimilation, high

116

temperature, and high gravity fermentation and gradual increasing knowledge about its

117

genetic infrastructure.17,18 With such advantages, it is now being regarded as a promising

118

yeast for industrial application.

119

The present study is focussed on improving xylitol titer by overexpressing

120

homologous xylose reductase (XR) in Kluyeveromyces marxianus IIPE453 (KSIIPE453)

121

from lignocellulosic pentosans and at the same time, recycling of the yeast for ethanol

122

fermentation without affecting its titer and productivity. A rational strain engineering

123

approach was devised based on the consideration that xylitol dehydrogenase (XDH) gene

124

deletion would help in accumulating xylitol,19 but the yeast’s inherent capability of cell

125

biomass generation from pentose stream would be lost (Table 1). Instead, hexose sugar

126

would be necessary for cell biomass generation thereby reducing the overall ethanol titer.

127

Considering the improvement of overall yield of xylitol and ethanol from sugarcane bagasse,

128

this engineered strain may be a promising catalyst for biorefinery using the lignocellulosic

129

resource.

130 131 132

2.

Experimental

2.1

Strains, plasmids, primers and chemicals 5



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Kluyveromyces marxianus IIPE453 (KSIIPE453) (MTCC 5314), a thermophilic yeast, was

134

used as an experimental host for homologous overexpression. Escherichia coli TOP10

135

(Invitrogen, USA) was used as a host for plasmids amplification and was cultured in Luria-

136

Bertani (LB) medium. Yeast extract/peptone D-xylose (YPX) medium (composition in g/L;

137

peptone, 20; yeast extract, 10; D-xylose, 20) was used to culture the KSIIPE453 aerobically.

138

YNB-5 FOA (composition in g/L; YNB, 6.7; dextrose, 20; amino acid mix without uracil,

139

1.3; uracil, 0.1; 5-fluoroorotic acid, 0.8) and synthetic dropout medium YNBD (composition

140

in g/L; YNB devoid of amino acids, 6.7; dextrose, 20; amino acid mix without uracil, 1.3)

141

were used for mutant screening and transformant selection. Strains, plasmids, and primers,

142

used in this study were summarized in Tables 2, 3 & 4. Sugars, salts, ready-to-use media

143

were of the analytical (Sigma-Aldrich, USA) or commercial (Himedia, India) grade.

144

Molecular biology kits, reagents, enzymes were procured from Fermentas, Germany unless

145

specified otherwise.

146

2.2

147

The strain engineering strategy is schematically depicted in Fig. 1. Orotidine 5'-phosphate

148

decarboxylase (Ura3KS, NCBI accession number KX453285) was amplified from KSIIPE453

149

gDNA and cloned to construct pDrive_Ura3KS. Fragment with a major portion of Ura3KS open

150

reading frame (ORF) was substituted by 1.6 kb loxP-KanMX-loxP from pUG6 to form

151

pDrive_Ura3KS_KanMX (Fig. 1, 3a). It was further digested with EcoRI and transformed in

152

KSIIPE453 to disrupt Ura3KS by homologous recombination. The auxotrophic mutant

153

(Ura3KS disrupted KSIIPE453) was selected on YNB-5 FOA plate. XRKS was amplified from

154

gDNA of KSIIPE453, and the amplicon and pYES2/CT/lacZ were double digested to insert

155

XRKS into pYES2/CT/lacZ following gel extraction and ligation to form pYES2_ XRKS. The

Expression cassette construction

6


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156

expression cassette pYES2_XRKS_S11 was constructed by inserting PCR-amplified ori_S11

157

from pSPGK1within the unique restriction sites (KspAI and Eco105I) in pYES2_ XRKS.

158

2.3

Yeast transformation and expression analysis in the engineered strain

159

The

plasmid

160

KSIIPE453_Ura3- by lithium acetate method.25 Transformants (KSIIPE453t) were selected

161

on YNBD plates after 3-4 days of incubation. KSIIPE453 and KSIIPE453t were cultivated in

162

YPX medium. Cultures were washed with distilled water and subsequently transferred into

163

YNB medium (supplemented with 2% D-xylose as the carbon source with 1% D-galactose as

164

an inducer for 48 h of incubation). RNAs were isolated from 24 and 48 h aliquots. cDNA was

165

synthesized from isolated total RNA as per standard protocol. Expression level analyses were

166

confirmed with quantitative real-time PCR (qRT-PCR) against XRKS gene in KSIIPE453 as

167

well as KSIIPE453t with Glyceraldehyde 3-phosphate dehydrogenase (GAPDHKS, NCBI

168

accession number KX453284) as an internal control. Relative transcription levels of XRKS in

169

KSIIPE453 and KSIIPE453t were normalized to GAPDHKS signal and calculated as 2-∆∆CT.26

pYES2_XRKS_S11

(expression

cassette)

was

transformed

into

170

The selected transformant was further analysed for the retention of the plasmid for 5

171

successive generations. The culture was initially grown on YPX and further cultivated on

172

YNBD plates at 45°C. Colonies were collected at each generation from subsequent plates and

173

grown in liquid YNBD medium. Plasmid isolation was carried out from the liquid culture

174

(from individual generations) using plasmid extraction kit (Zymoprep yeast plasmid

175

miniprep, USA). The plasmid was further digested with PagI to confirm the isolated vector as

176

pYES2_XRKS_S11 based on its band size. Additionally, PCR amplification of the plasmid

177

(from individual generations) with XRKS primers was carried out to confirm the presence of

178

the XRKS gene in the isolated expression cassette.

179

2.4

Enzyme activity, 3D modelling and MD simulation 7



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XRKS purification and activity analysis in both KSIIPE453 and KSIIPE453t was carried out as

181

previously described.23 Purified XRKS from both KSIIPE453 and KSIIPE453t was assayed for

182

reductase activity and evaluated on native PAGE for activity staining. For a molecular level

183

understanding of the enzyme, a 3D homology model of XRKS was constructed. Candida

184

tenuis D-xylose reductase (CtXR, PDB ID 1JEZ, 2.20 Å structural resolution) was selected as

185

the template (based on BLAST analysis with XRKS protein sequence; NCBI accession

186

number AHY04295) for model building. The model was energy minimized (GROMACS;

187

v5.1.2) with AMBER99SB-ILDN force field.27,28 Docking simulation of NADPH and NADH

188

was performed using Autodock Vina 29 with CtXR in complex with either NADPH (PDB ID:

189

1K8C, resolution 2.1 Å) or NADH (PDB ID: 1MI3, resolution 1.8 Å) as template. The

190

binding grid of cofactors from the reference structures was used to develop a similar grid map

191

in XRKS and enabled docking of the cofactors into the latter using a knowledge-based

192

approach. The refinement of the active site residues in the docked complex was carried out

193

using Crystallographic Object-Oriented Toolkit (COOT).30 PROCHECK31 was used to

194

validate modelled XRKS enzyme in complex with NADH, NADPH and the patterns of

195

nonbonded atomic interactions within the protein and the co-substrate were verified using the

196

ERRAT 32, PyMol 33 and Chimera 34 tool.

197

Molecular dynamics (MD) simulation studies were performed to understand the

198

structure and dynamic behaviour of the modeled XRKS protein both in the apo and substrate

199

(D-xylose) bound form. Simulations were performed using the open-source software

200

GROMACS 5.1.2 and AMBER99SB force field. The systems were solvated in a cubic box

201

with TIP3P water molecules. Water molecules were replaced by Na+ or Cl- to neutralize the

202

net charge of the system for each complex system with protein atoms maintained at a distance

203

of 1.0 nm from the box edges. Before sampling simulations, the solvated systems were 8


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energy minimized by steepest descent method (3000 steps) and then equilibrated for 100 ps at

205

300K temperature by position restrained MD simulation. Following equilibration, the systems

206

were subjected to final MD simulations for 50 nanoseconds at 300K. Periodic boundary

207

conditions were applied as isothermal and isobaric using Berendsen Coupling algorithm with

208

relaxation times of 0.1 and 0.2 picoseconds respectively. The LINCS algorithm was used to

209

constrain bond lengths using a time step of 2 femtoseconds for both systems. Electrostatic

210

interactions were calculated using the Particle Mesh Ewald method, van der Waals and

211

coulombic interactions were calculated with a cut-off at 1.0 nm. The different MD

212

trajectories were analysed with tools provided by GROMACS program package. The root-

213

mean-square deviations (RMSD) from the initial structure and the root-mean-square

214

fluctuations (RMSF) were calculated during all MD simulations. Also, in case of complex

215

structure, the distance between the active site and D-xylose atoms has been calculated in each

216

frame.

217

2.5

218

Fermentation was carried out in a 5 L in-situ sterilizable stir tank reactor (BioSac, India)

219

equipped with supervisory control and data acquisition (SCADA). Sugarcane bagasse (SCB)

220

hydrolysate (pentose sugar-rich stream) was used for yeast cell biomass generation, and

221

xylitol production and saccharified broth (hexose rich stream) were used for ethanol

222

fermentation. Sugarcane bagasse hydrolysate and saccharified broth were generated as

223

described earlier.35,36 Yeast cultivation and fermentation were carried out at 45°C and pH 4.5.

224

Cultivation and fermentation were terminated after consumption of ~ 90% sugar. KSIIPE453t

225

was compared with KSIIPE453 based on its xylitol and ethanol fermentation capability from

226

biomass-derived pentose & hexose sugars. A complete material balance for alcohols

227

production with cell biomass as intermediates was carried out to visualize the process with

Fermentation study with yeast recycling

9



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improved xylitol yield that could be integrated into lignocellulosic biorefinery concept. The

229

multiproduct refinery process was estimated with 2 kg of sugarcane bagasse (after maximum

230

recovery of fermentable sugars as reported earlier) as a feed basis.

231

2.6

232

The concentrations of D-xylose, D-glucose, xylitol, ethanol, furfural, and acetic acid were

233

quantitatively analyzed by HPLC.20 Briefly, a refractive index (RI) detector and Hiplex-H

234

acid 8 μm column (100 mm × 7.7 mm diameter column, PL Polymer Laboratory, U.K.) were

235

used for analyses. The column was eluted with 1 mM sulfuric acid as the mobile phase (flow

236

rate being 0.7 mL/min), and the oven temperature was maintained at 70°C. Dry cell mass

237

(DCM) was determined with a known quantity of cellular broth (1 mL) by hot air drying of

238

cell pellets in microfuges. Average of triplicate data was considered for DCM determination.

239

DNA and RNA concentrations were estimated spectrophotometrically by measuring

240

absorbance (Biophotomerter plus, Eppendorf) at 260 and 230 nm respectively. Threshhold

241

cycle (CT) values in real time PCR was reported with statistical mean and standard deviation

242

(SD). Following calculations were considered.

243

Cell biomass yield = generated yeast biomass / consumed substrate (g/g)

244

Yield (xylitol or ethanol) = product / consumed substrate (g/g)

245

Productivity (xylitol or ethanol) = titre / time (g/L.h-1)

246 247 248

3.

Results and discussion

3.1

Strain engineering for XRKS overexpression

249

An auxotrophic mutant of KSIIPE453 was constructed (Fig 1) by disrupting Ura3KS gene.

250

The Cre loxP–KanMX–loxP gene from pUG6 was used to disrupt Ura3KS for Cre-mediated

251

recombination between the two loxP sites37-39 (Fig. 2a). The Ura3KS gene disruption cassette

252

was constructed with a threshold flanking homologous sequences of ~400 bp on either side to

Analytical techniques and calculations

10


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253

improve in selective gene deletion efficiency and avoid non-homologous end joining

254

recombination.22, 40-41

255

Ura3KSF and Ura3KSR, with homology to both Ura3 gene and disruption cassette, led

256

to amplification of 0.86 kb (partial nucleotide sequence) of Ura3KS ORF in wild-type allele

257

and a lone 2.3 kb fragment (Fig. 2b, c) for the correctly integrated disruption cassette.

258

Further, the 2.2 kb amplification band and KSIIPE453 genome sequence analysis (NCBI

259

accession number LDJA00000000; scaffold 1) confirmed the presence of a single copy of

260

Ura3 gene (haploid hemiascomycetes) as reported in other Kluyveromyces.42,43 The growth

261

rates of KSIIPE453 and KSIIPE453_Ura3- evaluated in YNB medium devoid of uracil

262

illustrated a 2 h doubling time for KSIIPE453 and no growth of KSIIPE453_Ura3-

263

respectively. Growth kinetics were similar for both strains with uracil complementation;

264

however, the final biomass yield in the mutant (YX/S = 0.215) was slightly lower than that of

265

the KSIIPE453 (YX/S = 0.223).

266

The XRKS gene (0.99 kb) was PCR amplified from the KSIIPE453 gDNA and cloned

267

within the restriction sites in pYES2/CT/lacZ resulting in the modified yeast expression

268

vector (YEV) termed as pYES2_XRKS. Restriction digestion with NdeI and SalI yielded two

269

fragments corresponding to 3.15 kb and 3.73 kb (Fig. 2e), whereas native pYES2/CT/lacZ

270

produced a linear fragment of 8.97 kb, indicating the absence of SalI cut site. Bidirectional

271

sequencing of pYES2_XRKS (with primers Gal1F and CYC1R) generated a 1.41 kb fragment

272

spanning the entire promoter to terminator region including XRKS gene. BLAST alignment of

273

sequenced fragment with database deposited XRKS nucleotide data (NCBI accession number

274

KJ563917) showed 100% identity, with SalI restriction site found 483 bp downstream to the

275

translation start codon and affirmed the XRKS inclusion in the yeast expression vector (YEV).

276

A putative TATA box (TATATAAA) was found in the promoter region at position 205 bp 11



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277

upstream to the CDS. The presence of Ura3Sac in YEV was considered to complement the

278

deficiency of Ura3KS in KSIIPE453_Ura3-, as the former has been reported to function in

279

uracil deficient Kluyveromyces.44 Saccharomyces autonomous replicating sequence (ARSSac;

280

2µ origin of replication) originally present in YEV was non-functional in Kluyveromyces due

281

to the absence of a required conserved sequence in the plasmid.45 Thus, to facilitate the

282

replication of expression cassette in transformed Kluyveromyces, 0.9 kb (Fig. 2f) S11

283

fragment (ARSKS) from plasmid pSPGK1 was introduced into pre-existing pYES2_XRKS to

284

construct 7.1 kb pYES2_ XRKS _S11 (Fig. 3b, c). Improvement of xylitol production by

285

homologous overexpression of XRKS in Kluyveromyces was not reported earlier, due to

286

unavailability of suitable expression system. The vectors reported for recombinant

287

Kluyveromyces protein production were derived from K. lactis vector pKD1, with a single

288

cloning site EcoRI.46,47 As XRKS possessed internal EcoRI cut site, several alterations in the

289

nucleotide sequence were required for its incorporation in such vectors. Hence, expression

290

cassette was constructed with pYES2/CT/lacZ backbone having multiple cloning sites for

291

several restriction enzymes and the ability to replicate in transformed KSIIPE453.

292

3.2

293

pYES2_XRKS_S11 was transformed into KSIIIPE453_Ura3- and XRKS expression levels

294

were analyzed. Recombinants were selected by growth in the absence of uracil and

295

transformation efficiency was estimated as ~300 transformants/ µg of plasmid. A few

296

transformants were selected from the plates and were subjected to colony PCR using

297

Ura3_pYES2 primers. A single 0.8 kb DNA fragment corresponding to Ura3 of

298

Saccharomyces (Ura3Sac, selection marker in the YEV) was amplified without any non-

299

specific bands. This confirmed the incorporation of the expression cassette (Fig. 2 g1-g7, g9-

300

g16) within the transformed cells. Amplification bands were absent in KSIIPE453_Ura3-.

Transformation and gene expression

12


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301

XRKS expression levels in one selected clone was estimated based on colony PCR band

302

intensity and shake flask fermentation studies (data not shown). RNA yield of

303

KSIIIPE453_transformed cells was up to ~100 ng/µL with high quality (A260/280 = 2.01).

304

XRKS expression by qRT-PCR (GAPDHKS normalized) after 24 h, illustrated ~2.1-fold

305

improvement in transcript levels of KSIIPE453t over KSIIPE453. The transcription levels of

306

XRKS at different time intervals and the corresponding xylitol yields are illustrated in Table

307

5. High CT values (~16.97) indicated that XRKS expression was strongly enhanced by D-

308

galactose induction and was similar to constitutive gene expression. Improved XRKS

309

expression was also reflected in enhanced D-xylose consumption and xylitol yield. D-Xylose

310

was completely depleted after 48 h yielding 6.89 g/L xylitol. The expression folds were

311

higher in comparison with reported heterologous gene expression in K. marxianus from N.

312

crassa (1.49)48, and lower with P. stipitis (2.70) XR.49 Similar improvements in expression

313

levels have been reported in mutant K. marxianus 36907-FMEL1 by Kim et al. using random

314

XR mutagenesis.50 KSIIPE453t retained the expression vector even after 5 generations.

315

Restriction digestion of the vector (isolated from KSIIPE453t) with PagI generated a single

316

diagnostic band of size 7.1 kb (Fig. 2 h) which confirmed the vector as pYES2_XRKS_S11.

317

PCR amplification of the isolated plasmid (from each of the 5 generations) resulted in intense

318

XRKS bands corresponding to the size of 0.99 kb (Fig. 2 i1-i5) as in the wild type (Fig. 2 d).

319

This confirmed that the expression cassette harbouring the XRKS gene was retained in

320

KSIIPE453t for the successive generations.

321 322 323

3.3

324

as cofactor which was ~ 1.7-fold higher compared to KSIIPE453. Zymogram analysis

325

corroborated the activity assay results (Fig. 4), where purified XRKS activity bands were

XRKS activity and co-factor specificity

KSIIPE453t demonstrated an overall XRKS activity of 1.82 ± 0.03 IU/mg with only NADPH

13



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326

significantly more intense in KSIIPE453t compared to KSIIPE453. The assay results were in

327

accordance with the qRT-PCR data and correlated to overall xylitol yield improvement in the

328

transformed variety.

329

3D model of XRKS (Fig. 5) illustrated that the active site interactions would vary

330

significantly depending on the cofactor binding. Both NADPH and NADH showed

331

interactions with XRKS amino acid residues within the active site (Table 6, Fig. 6). The

332

important conformations of Glu227, Lys 275, Ser175 were conserved in XRKS similar to

333

CtXR in complex with either NADH or NADPH. In CtXR NADPH specificity was imparted

334

by residues Lys274, Ser275 and Asn276 while in XRKS an interesting difference was

335

observed where unlike CtXR, Asn276 was replaced by Ser280 (Fig. 7a). Although a triad of

336

residues Tyr221, Ser280 and Glu287 were involved in interacting with NADPH moiety in

337

XRKS, Ser280 was believed to be the principal governing factor. Ser280 in the active site did

338

not show interaction with NADH bound form which may be attributed to the loss of

339

phosphate interactions observed with the NADPH (Table 6, Fig. 7c) that promote the

340

conformation flexibility of the adenosine molecule.51 NADH moiety was at a distance of

341

~4Ao (Fig. 7b) from the amino acid residue which was above the threshold for favourable

342

non-bonded interaction. This demonstrated as to why the binding of NADH in XRKS was less

343

favourable compared to NADPH.

344

3.4

345

The MD simulations for both apoenzyme and the enzyme-substrate complex generated stable

346

trajectories indicating that the systems were well equilibrated. The RMSD of the enzyme

347

complex showed better stability (0.2-0.22 nm) in comparison to the apo form (0.2-0.29 nm)

348

(Fig. 8) which clearly indicated that D-xylose binding confers stability to the XRKS enzyme

349

system.

Molecular Dynamics Simulations of XRKS

D-xylose

was preferentially non-bonded in the active site with the amino acid 14


Page 15 of 50


350

residues Trp 24(NE1), Asp 51(OD2), Tyr 52(OH), His 114(NE2), Asn 315(OD1). The RMSF

351

variations for the residues Asp 51 and Tyr 52 were mostly stable (~ 0.5 nm) in the enzyme-

352

substrate complex while Trp 24 and Asn 315 demonstrated RMSF fluctuation of ~1 and 1.5

353

nm in certain instants of trajectory. Trp24, in particular, showed a high spike variation (in

354

RMSF) in later stages demonstrating flexible motion. The fluctuations in His 114 ranges

355

differently in case of the complex structure which is ~ 0.5 nm in more than 80% of the frames

356

while the apo structure illustrates RMSF values near to 1. This represents the stability of the

357

Histidine residues with D-xylose due to interaction with substrate atoms. Active site analysis

358

revealed that D-xylose oxygen, O2, is involved in interacting with Nitrogen NE2 of His 114

359

and Oxygen OD2 of Asp 51 with their interactions ranging from 2.8-13.0Å and 2.6-13.5Å

360

respectively (Fig. 9). The motion of these three atoms generates a stable pattern of triangular

361

formation which is accordance with the constant RMSF values of these residues (within the

362

complex). Further, the Oxygen at O3 of D-xylose interacts with oxygen (OH) of Tyr52 and

363

nitrogen (NE2) of His 114 with dynamic variation in distance among pairs ranging from 2.5-

364

11.7Å and 2.7-13.1 Å respectively. The D-xylose has more affinity towards oxygen of Tyr 52

365

instead of nitrogen of His 114 because of higher electronegativity of OH in Tyr52 and

366

Nitrogen of His114 has a partial pull towards O2 of the substrate. The nitrogen of Trp24 and

367

oxygen of D-xylose displayed stable states by attending lower distances at various frames and

368

it ranges from 2.8-10.2 Å. The interaction of oxygen of Asn315 and D-xylose showed the

369

variation in between 2.5-12 Å having mixed variant fluctuation as similar to its RMSF values.

370

MD analysis enabled us understanding the dynamics of important catalytic residues as well as

371

their enhanced stabilization through substrate binding in the active site.

372

3.5

Xylitol and ethanol fermentation by KSIIPE453t

15



Page 16 of 50


373

Overexpression of XRKS in KSIIPE453t resulted in improved xylitol yield with unaltered

374

ethanol titer. The yeast was grown in pentose sugars to generate optimum cell biomass for

375

either xylitol or ethanol fermentation and the cells were further recycled to undertake the next

376

fermentation batches as per the process requisite. Each fermentation process has been

377

segregated into five stages. First was the cell biomass generation stage (G). In the second

378

stage (S1), the yeast cell biomass was allowed to settle for 4 h, and the liquid broth was

379

replaced by pumping fresh biomass hydrolysate for fermentation (F1). The process was

380

repeated with another cell settling (S2), followed by fermentation (F2) with one-time cell

381

recycling.

382

3.5.1

383

KSIIPE453t produced xylitol from pentose during cell biomass generation under aerobic

384

condition due to its inherent crabtree positive nature.52 A cell biomass concentration of 4.52 ±

385

0.18 g/L was obtained from the SCB hydrolysate with a xylitol accumulation of 3.19 ± 0.1

386

g/L. D-Xylose consumption by KSIIPE453t during growth phase (0.41 ± 0.01 g/L.h-1) showed

387

little variation with respect to KSIIPE453 and xylitol yields were nearly similar (0.186 ±

388

0.003 g/g). Fig. 10 (a, b) depicted the time course profile of xylitol production. KSIIPE453t

389

produced higher amounts of xylitol (with D-galactose induction) compared to KSIIPE453

390

with a maximum xylitol concentration of 17.45 ± 0.1 g/L. D-Xylose consumption rates in the

391

first 24 h were very high (0.79 ± 0.05), with high xylitol YP/SX (0.61 ± 0.05 g/g). This

392

indicated that XRKS overexpression in turn increased the D-xylose concentration gradients

393

across the cell leading to faster uptake rates and conversion. Overall xylitol yield (YP/SX) and

394

volumetric productivity (QPX) of the recombinant strain in SCB hydrolysate were 0.51 ± 0.02

395

g/g and 0.335 ± 0.01 g/L/h respectively, with a 1.62-fold increase in xylitol production

396

compared to KSIIPE453 (0.315 ± 0.01 g/g). Cell recycling improved the overall xylitol

Yeast biomass generation and xylitol fermentation with cell recycling

16


Page 17 of 50


397

productivity (combining F1 and F2) since there was no lag phase (for the cells) in F2

398

compared to F1. However, the volumetric productivity in SCB hydrolysate was slightly lower

399

compared to pure sugar (0.42 ± 0.01 g/L/h), which might be attributed to differential levels of

400

expression (in biomass hydrolysate and pure sugar) of the moderate affinity

401

transport gene HXT1 elucidated from the genome sequence data. Control of oxygen

402

availability was the major factor governing xylitol fermentation and very low dissolved

403

oxygen (DO2) levels (< 2%) reduced XR activity owing to limited intracellular cofactor

404

concentrations.53 DO2 was maintained at 7% saturation through cascading, provided the

405

suitable micro-aerobic condition for cofactor regeneration (NADPH) in vivo via the pentose

406

phosphate pathway.54 Xylitol could be accumulated without the need for any additional co-

407

substrate such as glycerol or glucose as a slight amount of D-xylose was utilized for the

408

purpose. This explained the little reduction in xylitol yield (from initial 0.61 ± 0.05 g/g to

409

0.51 ± 0.02 g/g final) over the entire cycle. The xylitol titer and productivities were either

410

higher or at par in comparison to reported heterologous XR gene expression from N. crassa

411

and P. stipitis in K. marxianus YZJ009 (xylitol titer; 16.86 ± 0.07 g/L and productivity; 0.16

412

± 0.01 g/L/h) and K. marxianus YZB014 (xylitol titer; 11.32 g/L and productivityc; 0.35

413

g/L/h) respectively.48,49 Similar improvements in xylitol yield have been reported in mutant

414

K. marxianus 36907-FMEL1 by Kim et al. using random XR mutagenesis.50 YP/SX and QPX

415

for xylitol production in hemicellulosic hydrolysate were in fact, higher than the ones

416

reported for few Candida sps., which are known xylitol producers. Mateo et al. reported

417

xylitol yields varying from 0.12 g.g-1 to 0.23 g.g-1 from acid hydrolysate using C. tropicalis

418

NBRC 0618.55 Cheng et al. fermented corncob hydrolysate to produce xylitol with a

419

maximum titer of 17.1 g/L and YP/SX ranging between 0.32-0.44 g.g-1 respectively.56

420

KSIIPE453t did not require any co-substrate for biomass growth or cofactor generation and

D-xylose

17



Page 18 of 50


421

was advantageous for overall bioprocess development. The fermentation data (Table 7)

422

clearly illustrated that homologous XRKS gene overexpression in KSIIPE453t enhanced D-

423

xylose conversion rates and xylitol production compared to the native KSIIPE453 without

424

any requirement of the additional cofactor.

425

3.5.2

426

Ethanol fermentation by KSIIPE453t showed no variation in terms of yield, YP/SE (0.444 ±

427

0.001) compared to wild variety. The growth pattern on acid hydrolysate in the second cycle

428

of biomass generation was similar with xylitol accumulation (3.1 ± 0.12 g/L) (Fig. 10 c, d).

429

During fermentation cycle F1, entire hexose was consumed in ~24 h of fermentation cycle

430

from the saccharified broth with consumption rates of 1.85 ± 0.05 g/L.h-1, slightly lower

431

(~2.01 ± 0.05 g/L) in comparison to pure sugar. The second fermentation cycle with cell

432

recycling was even faster and accumulated ethanol with an overall titer of 21.1 ± 0.2 g/L. The

433

ethanol volumetric productivity (QPE) in the KSIIPE453t was 0.81 ± 0.05 g/L/h in biomass

434

hydrolysate over the entire course of the cycle which was near identical to wild-type

435

KSIIPE453 (0.83 ± 0.08g/L/h) (Table 7). YP/SE and QPE were significantly higher in

436

comparison to ethanol fermentation by industrial S. cerevisiae strain UFPEDA 1238 from

437

non-delignified SCB.57

438

3.6

439

A complete material balance for alcohols production from biomass-derived sugars (obtained

440

from 2 kg SCB hydrolysis) for KSIIPE453t in comparison with KSIIPE453 is illustrated in

441

Table 8. Alcohol production with KSIIPE453t resulted in an overall 184.43 ± 0.3 g of xylitol,

442

combining growth and fermentation cycles. Altogether, an excess yield of 58.62 ± 0.15 g

443

xylitol was achieved by KSIIPE453t over KSIIPE453, without significant alteration in

444

ethanol titer (~ 287.64 ± 0.25 g) from total fermentable sugars. Pentose value addition is one

Ethanol fermentation from saccharified broth with cell recycling

Sugar management by KSIIPE453t for SCB based bio refinery

18


Page 19 of 50


445

of the major process constraints for SCB based biorefinery which accounts for ∼ 25-30% of

446

total fermentable sugars. Xylitol yield enhancement by 1.62 fold would significantly benefit

447

the process in terms of overall economics owing to higher xylitol costs. Existing pentose

448

fermenting strains such as Pichia stipitis or genetically modified S. cerevisae had been

449

considered for D-xylose fermentation into ethanol, but were not economically viable, owing

450

to low ethanol titer and productivity.58-60 In this regard, KSIIPE453t is advantageous as a

451

single biocatalyst that can completely valorize biomass-derived pentose and hexose into

452

specialty chemical xylitol and fuel ethanol in a multiproduct biorefinery scheme.

453 454 455

Authors’ contributions

456

on experiments with data collection along with the drafting of the manuscript; VJ carried out

457

the bioinformatics analysis of the entire research work. DG and SH had the primary

458

responsibility for data representation and final shaping of the manuscript; AKN carried out

459

cell culture maintenance, inoculum preparation and analytical data interpretation with DDG.

460

DG supervised overall work and participated in result interpretation with DDG and SH; DDG

461

and DG are the corresponding authors. All authors read and approved the final manuscript.

462

Acknowledgement: Authors thankfully acknowledge Dr. Anjan Ray, Director CSIR-IIP, for

463

his constant motivation and support to this study, and for facilitating necessary

464

infrastructure. The authors are also grateful to Mr. Sunil Kumar Suman and Mr. Deepchand

465

(CSIR-IIP) for the scientific and technical support extended during the experimental studies

466

Funding sources: CSIR Mission Mode project No. HCP0009

467

Conflict of interest: The authors declare that they have no competing interests

DDG, AKN and AJ designed the overall research plan, study oversight and conducted hands-

19



Page 20 of 50


469 470 471

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633

55.

Mateo, S.; Puentes, J. G.; Moya, A. J.; Sánchez, S. Ethanol and xylitol production by

634

fermentation of acid hydrolysate from olive pruning with Candida tropicalis NBRC

635

0618. Bioresour. Technol. 2015, 190, 1-6.

636

56.

Cheng, K.K.; Wu, J.; Lin, Z.N.; Zhang, J. Aerobic and sequential anaerobic

637

fermentation to produce xylitol and ethanol using non-detoxified acid pretreated

638

corncob. Biotechnol. Biofuel. 2014, 7, 166-184.

639

57.

de Albuquerque, W. M. C.; Martin, C.; de Moraes, R. G. J.; Gouveia, E. R. Increase

640

in ethanol production from sugarcane bagasse based on combined pretreatments and

641

fed-batch enzymatic hydrolysis. Bioresour. Technol. 2013, 128, 448-453.

642

58.

Silva, J. P. A.; Mussatto, S. I.; Roberto, I. C.; Texeira, J. A. Ethanol production from

643

xylose by Pichia stipitis NRRL Y-7124 in a stirred tank bioreactor. Braz. J. Chem.

644

Eng. 2011, 28,151−156.

645

59.

Ha, S. J.; Galazka, J. M.; Kim, S. R.; Choi, J. H.; Yang, X.; Seo, J. H.; Glass, N. L.;

646

Caté, J. H.; Jin, Y. S. Engineered Saccharomyces cerevisiae capable of simultaneous

647

cellobiose and xylose fermentation. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 504−509.

648

60.

Bettiga, M.; Bengtsson, O.; Hahn-Hägerdal, B.; Gorwa-Grauslund, M. F. Arabinose

649

and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a

650

fungal pentose utilization pathway. Microb. Cell. Fact. 2009, 8, 40−51.

27



Page 28 of 50


652 653

Figure legends

654

Fig. 1

Strain engineering scheme

655

Fig. 2

PCR amplification and restriction digestion during over expression process;

656

(L) Ladder (GeneRuler 1kb); a, loxP-KanMX-loxP cassette (1.62 kb); b,

657

KSIIPE453_Ura3- with integrated disruption cassette (2.3 kb); c, Ura3KS in

658

KSIIPE453 (0.86 kb); d, XRKS in KSIIPE453 (0.99 kb); e, restriction digestion

659

of pYES2_XRKS with NdeI and SalI (3.6 Kb and 3.2 kb); f, S11 amplification

660

from pSPGK1 (0.9 kb); g1 - g7 & g9 - g16, validation of pYES2_XRKS_S11

661

in KSIIPE453t (0.8 kb); g8, false positive colony without amplification; h,

662

restriction digestion of the isolated pYES2_XRKS_S11 with PagI (7.1 kb);

663

i1-i5, XRKS (0.99 kb) amplified from PYES2_XRKS_S11 isolated from 5

664

successive generations of KSIIPE453t

665

Fig. 3

pYES2_XRKS_S11

666 667

Constructed vectors; (a) pDrive_Ura3KS_KanMX; (b) pYES2_XRKS; (c)

Fig. 4

Zymogram of purified XRKS from KSIIPE453 and KSIIPE453t; (Lane L:

668

Native PAGE ladder; W24, W48 and T24, T48; purified XRKS from KSIIPE453

669

and KSIIPE453t during 24 and 48 h of fermentation from SCB acid

670

hydrolysate.

671

Fig. 5

potential surface of the enzyme

672 673

Fig. 6

676

Electrostatic potential of NADH and NADPH binding groove in XRKS (A, C); Molecular interaction of NADH and NADPH with the enzyme (B, D).

674 675

(a) Cartoon representation for the modelled structure of XRKS; b) Electrostatic

Fig. 7

a) Multiple sequence alignment of XR from different hemiascomycetes yeast. The numbering has been done using template sequence as Candida tenuis. The 28


Page 29 of 50


677

sequence in red highlights XRKS. protein sequence. The blue box represents

678

Ser280 position. b) Interaction of Ser280 in XRKS with NADH and c) NADPH

679 680

Fig. 8

The RMSD trajectory of both two structures: The RMSD of apo enzyme is

681

represented in red lines and the RMSD trajectory of

682

represented in black lines. RMSF fluctuations for each active site residues: Trp

683

24, Asp 51, Tyr 52, His 114 and Asn 315. For RMSD, the x-axis represent

684

time (in ns) and y-axis represent RMSD value (in nm). For RMSF, x-axis

685

represent the frame number of simulation and y-axis represent RMSF value (in

686

nm)

687

Fig. 9

D-xylose

complex is

The fluctuation graph of distance between D-xylose substrate and the active

688

site residues. It is the distance variation between residue’s atom and nearest

689

substrate atom. The distance range of fluctuation has been provided (in Å)

690

with minimum-maximum values. a) Xyl325 O2 – His109 NE2 : The graph for

691

oxygen (O2) of xylose with the nitrogen (NE2) of His 109. b) Xyl325 O2 –

692

Asp46 OD2 : The graph for oxygen (O2) of xylose with the oxygen (OD2) of

693

Asp46 c) Xyl325 O3 – His109 NE2 : The graph for oxygen (O3) of xylose

694

with the nitrogen (NE2) of His 109 d) Xyl325 O3 – Tyr47 OH : The graph for

695

oxygen (O2) of xylose with the oxygen (OH) of Tyr47 e) Xyl325 O4 – Trp19

696

NE1: The graph for oxygen (O4) of xylose with the nitrogen (NE1) of Trp19 f)

697

Xyl 325 O5 – Asn310 OD1 : The graph for oxygen (O5) of xylose with the

698

oxygen (OD1) of Asn310. The x-axis represents the frame number of

699

simulation and y-axis represent the distance (in Å) between nearest pair of D-

700

xylose atom and active site residue atom. 29



Page 30 of 50


701

Fig. 10

Comparative fermentation characteristics between KSIIPE453 (a, c) and

702

KSIIPE453t (b, d) with cell recycling; G, growth phase; S1, cell settling; F1,

703

first fermentation cycle; S2, cell settling; F2, second fermentation cycle with

704

cell recycling; S3, cell settling; F3, third fermentation cycle with cell recycling;

705

BT, batch termination; (a, b) growth and fermentation in SCB derived

706

xylose; (c, d) growth in SCB derived

707

saccharified glucose.

708 709 710 711

D-xylose

D-

and fermentation in SCB

Table legends Table 1

Justification of strain engineering

712

Table 2

Yeasts and bacterium involved in overexpression studies

713

Table 3

Parent and constructed plasmids

714

Table 4

Oligonucleotides with introduced restriction sites as underlined and complete sequence of XRKS gene

715 716

Table 5

Real time expression analysis of XRKS for KSIIPE453 and KSIIPE453t

717

Table 6

Non-bond interactions predicted in the active site for NADH and NADPH

718

with modelled XRKS. The specific interactions of XRKS with NADPH are

719

highlighted in blue and predicted interactions for NADH are represented in

720

green.

721

Table 7

KSIIPE453t from biomass derived sugars

722 723

Comparative xylitol and ethanol production in fermenter by KSIIPE453 and

Table 8

Material balance for sugar management by KSIIPE453t and KSIIPE453 from

724

biomass derived sugars; Hydrolysate, SCB acid hydrolysate rich in D-xylose;

725

Saccharified broth, SCB saccharified broth rich in D-glucose; G, growth

726

phase; F1, first fermentation cycle; F2, second fermentation cycle with cell 30


Page 31 of 50


727

recycling; F3, third fermentation cycle with cell recycling; |Δ(KSIIPE453t –

728

KSIIPE453)|, difference in alcohol yields between the transformed and wild

729

type variety.

31



Page 32 of 50 Xylitol fermentation with engineered yeast

Criteria Choice of yeast

Yeast A thermophilic ethanologen with natural assimilator of pentose and hexose for growth and fermentation

Genetic Homologous engineering overexpression of D-xylose reductase (XR) gene to enhance xylitol yield

Remarks  Kluyveromyces marxianus IIPE453 (KSIIPE453) can grow on pentose and hexose sugar and ferment pentose to xylitol and hexose to ethanol  A multiproduct biorefinery based on lignocellulosic material can be envisaged with this strain. Cost of ethanol could be supplemented by the cost of other non-energy product  A single strain can be deployed for developing two products, ethanol, and xylitol.  Sugar management or optimum utilization of total fermentable sugars from lignocellulosic biomass to achieve maximum product titer was considered as key to the biorefinery concept.  Development of a selective marker is necessary to overexpress XR gene and thus a Ura3-mutant is developed  Modification of lower pathway like XDH deletion would hamper the strain’s inherent feature of pentose sugar assimilation. In such case, strain should be allowed to grow in hexose sugars by compromising ethanol titer.  Genetic modification should not affect the ethanol fermentation capacity in terms of yield and titer and upset the single strain multi-product biorefinery approach  Entire material balance for sugars and products have been compared for native (KSIIPE453) and modified (KSIIPE453t) in proposed biorefinery approach with enhanced xylitol titer (for KSIIPE453t)

References

20

21

22

this study this study

Table 1

32


Page 33 of 50


Strains KSIIPE453 KSIIPE453_Ura3KSIIPE453t Escherichia coli TOP 10

Genotype Wild-type Kluyveromyces marxianus IIPE453 (MTCC 5314) KSIIP453, ura3KS:: KanMX pYES2_ XRKS_S11 (Ura3Sac, XRKS overexpressed) F- mcrA Δ( mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 recA1 araD139 Δ(araleu)7697 galUgalKrpsL (StrR) endA1 nupG

References 23

This study This study InvitrogenTM

Table 2

33




Plasmids/cassette pDrive pUG6 Parent plasmid pYES2/CT/lacz pSPGK1 pDrive_ Ura3KS pDrive_ Ura3KS_KanMX Constructed vectors pYES2_ XRKS pYES2_ XRKS_S11

Description UA cloning vector Plasmid containing Kanamycin marker Yeast expression vector Plasmid containing ori_S11 pDrive carrying Ura3KS gene pDrive carrying partial Ura3KS gene and KanMX disruption cassette pYES2/CT/lacz carrying XRKS gene pYES2_ XRKS with ori_S11

Reference QiagenTM EuroscarfTM InvitrogenTM 24

This study This study This study This study

Table 3

34


Page 35 of 50


primer Ura3KSF Ura3KSR LoxPKanMX_F LoxPKanMX_R XRKSF XRKSR Gal1F CYC1R Ura3_pYES2_XR_F Ura3_pYES2_XR_R pSPGK1_S11_F pSPGK1_S11_R GAPDHKSF GAPDHKSR gene

XRKS

restriction sites

sequence

target gene

source

annealing temperature (°c)

5’-GCAAGAGGAAGTATCATCAGCTAGCC-3’ Ura3KS gDNA 56 5’-GGAGAACTCTTAAGCGGATCTG-3’ 5’-GCGATATCCAGCTGAAGCTTCGTACGC-3’ KanMX with EcoRV pUG6 58 loxP sites 5’-GCGATATCGCATAGGCCACTAGTGGATCTG-3’ 5’-CCAGGTACCCCATGACATACCTCGCACCAACAG-3’ KpnI XRKS gDNA/ RNA 62 5’-CCAGCGGCCGCCTTAGATAAAGGTTGGGAATTCGTTGC-3’ NotI 5’-ACGGATTAGAAGCCGCCGAG-3’ XRKS pYES2_XRKS 55 5’-ACTAGTGGATCATCCCCACG-3’ 5’-ATGTCGAAAGCTACATATAAGGAACG-3’ Ura3Sac pYES2_XRKS 55 5’-GTTTTGCTGGCCGCATCTTCTC-3’ 5’-GTTAACGATCACAGCGGACGGTGG-3’ KspAI pSPGK1 55 S11 5’-TACGTATACACGAAGAGGGAAAATTGACTCG-3’ Eco105I 5’-ATGGTTTCTATTGCTATTAACGGTT-3’ 52 GAPDHKS RNA 5’-CTAAGCAACGTGTTCGACCAAG-3’ sequence 5’ATGACATACCTCGCACCAACAGTTACCTTGAACAATGGTTCCGAGATGCCGCTAGTCGGCTTGGGATGCTGGAAAATCCCAAACGA AGTGTGTGCCGAACAGGTGTACGAAGCCATCAAGTTGGGCTACCGCTTGTTCGACGGCGCGCAGGACTACGCCAACGAAAAAGAGG TGGGTCAAGGTATTAACAGAGCCATCAAGGAAGGAATCGTCAAGAGAGAAGACTTGGTCGTCGTTTCTAAGTTGTGGAACAGTTTCC ACCACCCAGACAACGTGCGTACCGCAGTCGAAAGAACTTTGAACGACTTGCAATTGGACTACTTGGACTTGTTCTACATCCATTTCCC ATTGGCTTTCAAGTTCGTGCCACTAGACGAGAAGTACCCTCCAGGTTTCTACACAGGTAAGGACAATTTCGCCAAGGAAATCATCGA AGAGGAGCCTGTCCCAATCTTGGACACCTACAGAGCCCTTGAGAAGTTGGTCGACGAAGGTTTGATCAAATCTTTGGGTATCTCAAA CTTTTCGGGTGCATTGATCCAGGACTTGTTGCGTGGCGCCCGTATCAAGCCAGTCGCCTTGCAGATCGAACACCACCCATACTTGGTC CAGGACCGCTTGATCACGTACGCCCAAAAGGTGGGCTTGCAAGTCGTCGCCTACTCCAGTTTCGGCCCACTATCCTTTGTCGAGTTGA ACAACGAAAAGGCCTTGCACACAAAGACTTTGTTCGAAAACGACACCATCAAGGCCATCGCTCAAAAACACAACGTCACCCCATCCC ACGTCTTGTTGAAGTGGTCCACCCAACGTGGTATCGCCGTCATTCCAAAGTCCTCCAAGAAGGAACGTCTCCTCGAGAACTTGAAGA TCGAAGAGACCTTTACCTTGTCCGACGAAGAGATCAAGGAGATCAACGGCTTGGACCAGGGATTGAGATTTAACGACCCATGGGACT GGTTGGGCAACGAATTCCCAACCTTTATCTAA3’

reference this study this study this study this study this study this study this study reference

NCBI GenBank Acc. No. KJ563917

GAATTC : EcoRI

Table 4

35




time CT XRKS CT GAPDHKS ∆CT ∆∆CT 2-∆∆CT xylose conc. xylitol conc. h mean ± SD mean ± SD g/L g/L 24 19 ± 0.17 13 ± 0.13 5.97 10.2 1.8 KSIIPE453 48 15.9 ± 0.3 10.26 ± 0.1 5.64 0.80 3.76 24 16.97 ± 0.07 12.1 ± 0.02 4.90 -1.07 2.1 7.59 3.45 KSIIPE453t 48 14.55 ± 0.01 9.54 ± 0.025 4.885 -0.84 1.79 0.44 6.89 Initial D-xylose concentration S0 = 22 g/L, D-galactose induction: 1% strain

Table 5

36


Page 37 of 50


Sl. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

XRKS with NADH Cys23:N - NADH:O2D Trp24:N - NADH:O3D Asp47:O - NADH:O2D Asp47:OD2 - NADH:O2D Ser173:O - NADH: N7N Asn174:ND2 - NADH: O7N Gln195:OE1 - NADH:N7N Ser222:N - NADH:O5D Ser222:OG - NADH:O2N Phe224:N - NADH:O1A Ser228:OG - NADH:O2N Glu231:OE2 - NADH:O2B Ser261:OG - NADH:N1A Lys278:N - NADH:O2A Lys278:NZ - NADH:O3B Arg284:NH1 - NADH:N3A Arg284:NH1 – NADH:O2B Asn288:ND2 – NADH:N7A -

Distance 3.2 3.3 3.1 2.7 3.0 3.0 2.7 3.4 3.3 3.6 2.4 2.4 2.4 2.8 3.1

3.4 3.3 3.1

XRKS with NADPH Cys23:N - NADPH : O2D Trp24:N - NADPH :O3D Asp47:O - NADPH :O2D Asp47:OD2 - NADPH :O2D Ser173:O - NADPH : N7N Asn174:ND2 - NADPH : O7N Gln195:OE1 - NADPH :N7N Tyr221:OH - NADPH :O7N Ser228:OG - NADPH :O2N Glu231:OE1 - NADPH :O3B Glu231:OE2 - NADPH :O3B Ser261:OG - NADPH :N1A Lys278:N - NADPH :O2A Ser280:OG - NADPH :O2X Ser280:N - NADPH :O2X Glu287:OE1 - NADPH :N6A Asn288:OD1 - NADPH :N6A Asn288:ND2 - NADPH :N7A

Distance 3.3 3.0 3.1 2.6 2.9 3.0 3.0 3.4

2.9 2.4 2.4 2.4 2.4 2.4 3.1

3.5 3.0 3.0

Table 6 37




Kinetic parameters

units

Product Yield coefficient Volumetric productivity Rate of substrate utilization

g/L g/g -1 g/L.h -1 g/L.h

KSIIPE453 11.1 ± 0.07 0.315 ± 0.01 0.19 ± 0.002 0.59 ± 0.04

KSIIPE453t 17.4 ± 0.28 0.51 ± 0.02 0.335 ± 0.01 0.66 ± 0.05

Xylitol | Δ (KSIIPE453t – KSIIPE453)| 6.3 ± 0.15 0.19 ± 0.01 0.145 ± 0.008 0.07 ± 0.01

KSIIPE453 21.6 ± 0.1 0.445 ± 0.004 0.83± 0.08 1.9 ± 0.25

Ethanol KSIIPE453t |Δ(KSIIPE453t – KSIIPE453)| 21.1 ± 0.2 0.5 ± 0.015 0.444 ± 0.003 0.001 ± 0.0001 0.81± 0.05 0.02 ± 0.001 1.85 ± 0.05 0.09 ± 0.001

Table 7

38


Page 39 of 50


Growth (G) Strain

KSIIPE453

KSIIPE453t

Sugar

Cell

Xylitol

Hydrolysate

g 97.0 ± 0.5

g 22.0 ± 0.1

g 15.40 ± 0.05

Hydrolysate

96.4 ± 0.6

21.7 ± 0.3

15.2 ± 0.05

Hydrolysate

96.8 ± 0.2

21.8 ± 0.3

15.60 ± 0.05

Hydrolysate

97.6 ± 0.1

21.4 ± 0.5

15.95 ± 0.05

Fermentation Xylitol Sugar F1 + F2 g g Hydrolysate 298.3 ± 0.3 95.21 ± 0.05 Saccharified 642.6 ± 0.5 broth Hydrolysate Saccharified broth

298.6 ± 0.3 643.5 ± 0.1

152.88 ± 0.2 -

Overall titers Ethanol

Xylitol

Ethanol

g -

g 110.61 ± 0.1

g -

289.2 ± 0.75

15.2 ± 0.05

289.2 ± 0.75

Total -

125.81 ± 0.15 168.48 ± 0.25

289.2 ± 0.75 -

287.64 ± 0.25

15.95 ± 0.05

287.64 ± 0.25

184.43 ± 0.3

287.64 ± 0.25

58.62 ± 0.15

1.56 ± 0.5

F1 + F2 +F3

Total Δ|KSIIPE453t – KSIIPE453|

Table 8

39



Figure 1 Strain engineering scheme 387x223mm (96 x 96 DPI)


Page 40 of 50

Page 41 of 50


PCR amplification and restriction digestion during over expression process; (L) Ladder (GeneRuler 1kb); a, loxP-KanMX-loxP cassette (1.62 kb); b, KSIIPE453_Ura3- with integrated disruption cassette (2.3 kb); c, Ura3KS in KSIIPE453 (0.86 kb); d, XRKS in KSIIPE453 (0.99 kb); e, restriction digestion of pYES2_XRKS with NdeI and SalI (3.6 Kb and 3.2 kb); f, S11 amplification from pSPGK1 (0.9 kb); g1 - g7 & g9 - g16, validation of pYES2_XRKS_S11 in KSIIPE453t (0.8 kb); g8, false positive colony without amplification; h, restriction digestion of the isolated pYES2_XRKS_S11 with PagI (7.1 kb); i1-i5, XRKS (0.99 kb) amplified from PYES2_XRKS_S11 isolated from 5 successive generations of KSIIPE453t 206x57mm (96 x 96 DPI)



Constructed vectors; (a) pDrive¬_Ura3KS_KanMX; (b) pYES2_XRKS; (c) pYES2_XRKS_S11 254x190mm (300 x 300 DPI)


Page 42 of 50

Page 43 of 50


Zymogram of purified XRKS from KSIIPE453 and KSIIPE453t; (Lane L: Native PAGE ladder; W24, W48 and T24, T48; purified XRKS from KSIIPE453 and KSIIPE453t during 24 and 48 h of fermentation from SCB acid hydrolysate. 138x118mm (96 x 96 DPI)



(a) Cartoon representation for the modelled structure of XRKS; (b) Electrostatic potential surface of the enzyme 236x110mm (96 x 96 DPI)


Page 44 of 50

Page 45 of 50


Electrostatic potential of NADH and NADPH binding groove in XRKS (A, C); Molecular interaction of NADH and NADPH with the enzyme (B, D). 234x155mm (96 x 96 DPI)



a) Multiple sequence alignment of XR from different hemiascomycetes yeast. The numbering has been done using template sequence as Candida tenuis. The sequence in red highlights XRKS. protein sequence. The blue box represents Ser280 position. b) Interaction of Ser280 in XRKS with NADH and c) NADPH 212x174mm (96 x 96 DPI)


Page 46 of 50

Page 47 of 50


The RMSD trajectory of both two structures: The RMSD of apo enzyme is represented in red lines and the RMSD trajectory of D-xylose complex is represented in black lines. RMSF fluctuations for each active site residues: Trp 24, Asp 51, Tyr 52, His 114 and Asn 315. For RMSD, the x-axis represent time (in ns) and yaxis represent RMSD value (in nm). For RMSF, x-axis represent the frame number of simulation and y-axis represent RMSF value (in nm) 342x170mm (96 x 96 DPI)



The fluctuation graph of distance between D-xylose substrate and the active site residues. It is the distance variation between residue’s atom and nearest substrate atom. The distance range of fluctuation has been provided (in Å) with minimum-maximum values. a) Xyl325 O2 – His109 NE2 : The graph for oxygen (O2) of xylose with the nitrogen (NE2) of His 109. b) Xyl325 O2 – Asp46 OD2 : The graph for oxygen (O2) of xylose with the oxygen (OD2) of Asp46 c) Xyl325 O3 – His109 NE2 : The graph for oxygen (O3) of xylose with the nitrogen (NE2) of His 109 d) Xyl325 O3 – Tyr47 OH : The graph for oxygen (O2) of xylose with the oxygen (OH) of Tyr47 e) Xyl325 O4 – Trp19 NE1: The graph for oxygen (O4) of xylose with the nitrogen (NE1) of Trp19 f) Xyl 325 O5 – Asn310 OD1 : The graph for oxygen (O5) of xylose with the oxygen (OD1) of Asn310. The x-axis represents the frame number of simulation and y-axis represent the distance (in Å) between nearest pair of D-xylose atom and active site residue atom. 352x170mm (96 x 96 DPI)


Page 48 of 50

Page 49 of 50


Comparative fermentation characteristics between KSIIPE453 (a, c) and KSIIPE453t (b, d) with cell recycling; G, growth phase; S1, cell settling; F1, first fermentation cycle; S2, cell settling; F2, second fermentation cycle with cell recycling; S3, cell settling; F3, third fermentation cycle with cell recycling; BT, batch termination; (a, b) growth and fermentation in SCB derived D-xylose; (c, d) growth in SCB derived Dxylose and fermentation in SCB saccharified glucose. 254x190mm (300 x 300 DPI)



Page 50 of 50

Graphical abstract

Yeast expression vector

Yeast expression vector with XR XR Cloning

Transformation

Overexpression

XR Fold expression

Ura3Sac

Ura3Sac Wild transformed

Xylitol


Xylitol Production from Lignocellulosic Pentosans: A Rational Strain

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