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Restoring of Glucose Metabolism of an engineered Yarrowia lipolytica for Succinic Acid Production via a Simple and Efficient Adaptive Evolution Strategy Xiaofeng Yang, Huaimin Wang, Chong Li, and Carol Sze Ki Lin J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 7, 2017

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Journal of Agricultural and Food Chemistry

Restoring of Glucose Metabolism of an engineered Yarrowia lipolytica for Succinic Acid Production via a Simple and Efficient Adaptive Evolution Strategy

Xiaofeng YANG 1, 2, #, Huaimin WANG 2, #, Chong LI 2, Carol Sze Ki LIN 2, *

1

Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering,

School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, People’s Republic of China 2

School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue,

Kowloon, Hong Kong

#

Authors contributed equally

*Corresponding author: Carol Sze Ki LIN, School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, [email protected]

1 Environment ACS Paragon Plus


1

ABSTRACT

2

Succinate dehydrogenase inactivation in Yarrowia lipolytica has been demonstrated for

3

robust succinic acid production, whereas the inefficient glucose metabolism has hindered its

4

practical application. In this study, a simple and efficient adaptive evolution strategy via cell

5

immobilisation was conducted in shake flasks, with an aim to restore the glucose metabolism

6

of Y. lipolytica mutant PGC01003. After 21 days with 14 generations evolution, glucose

7

consumption rate increased to 0.30 g/L/h in YPD medium consisting of 150 g/L initial

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glucose concentration, while poor yeast growth was observed in the same medium using the

9

initial strain without adaptive evolution. Succinic acid productivity of the evolved strain also

10

increased by 2.3 folds, with stable cell growth in YPD medium with high initial glucose

11

concentration. Batch fermentations resulted in final succinic acid concentrations of 65.7 g/L

12

and 87.9 g/L succinic acid using YPD medium and food waste hydrolysate, respectively. The

13

experimental results in this study show that a simple and efficient strategy could facilitate the

14

glucose uptake rate in succinic acid fermentation using glucose-rich substrates.

15 16

KEYWORDS: Adaptive evolution, Cell immobilisation, Glucose metabolism, Succinic acid,

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Yarrowia lipolytica


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1. INTRODUCTION

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The increasing energy demand and cost of petroleum have spurred the need for a shift from

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petroleum refinery to bio-based economy.1 This trend requires the development of highly

21

efficient utilization processes to exploit fully the potential of agricultural residues or food

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supply chain waste into chemicals, materials and fuels.2 Succinic acid (SA) was identified as

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one of the top twelve potential chemical building blocks by the US Department of Energy.3, 4

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The SA market size was estimated at US$ 191 million in 2013, production volume is

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expected to grow rapidly.5 However, the practical application of the natural producers in SA

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fermentation have the following disadvantages: (i) sensitivity of microorganisms;

27

(ii) expensive nutrient requirements; (iii) complicated product recovery and purification; (iv)

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large amount of wastes generated.6, 7 In the last decade, intensive research effort has been

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made in metabolic engineering of Escherichia coli for SA production.6 However,

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fermentation of E. coli faces several challenges,6,

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bacteriophage infections, fermentation at near-natural pH, complicated downstream

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processing and rigorous carbon catabolite repression.

8

including the susceptibility to

33 34

Recently, metabolic engineering of yeast for SA production has received significant scientific

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interest as an alternative strategy. In contrast to prokaryotes, yeast is highly tolerant of low

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pH values and generally recognised as safe (GRAS), making it superior for industrial

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application.9,10

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reconstruction12 and microarray gene transcription analysis13 were integrated with metabolic

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engineering to improve SA production and cell growth rate of Saccharomyces cerevisiae.

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Although considerable research progress has been made, none of the engineered yeast

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S. cerevisiae have yet achieved commercial application.14 Yan et al.9 used another strategy by

42

using a potentially high yield pathway (i.e. via the reductive TCA pathway) with a theoretical

Metabolic

profiling

analysis,11

genome-scale


metabolic

network


43

maximum glucose to SA yield of 1.71 mol/mol, but only 0.3 mol/mol SA yield on glucose

44

was achieved. To date, the highest SA titer achieved by engineered S. cerevisiae fermentation

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was 43 g/L, which was reported in the US patent by Van De Graaf et al.15

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Yuzbashev et al.10 demonstrated a strictly aerobic yeast Yarrowia lipolytica that could be an

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alternative SA producer via oxidative TCA pathway, in which a maximum SA titer of 17.4

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g/L was obtained without pH control. Regulation of the SDH2 expression allowed the mutant

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to produce 25 g/L SA under oxygen limitation conditions.16 Moreover, Kamzolova et al17-19

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developed a two-stage process for succinic acid production, which involved the combined

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microbial synthesis of α-ketoglutaric acid (KGA) by Y. lipolytica and the subsequent

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decarboxylation of KGA by hydrogen peroxide to SA. Under strictly controlled conditions,

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up to 71.7 g/L succinate was produced from ethanol with the yield of 70%, and 69.0 g/L

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succinate was produced from rapeseed oil.17, 18 Our former studies have demonstrated the

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possibility of SA fermentation from glycerol-based medium by deletion of Ylsdh5 encoding

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succinate dehydrogenase subunit 5 in Y. lipolytica.20,21 By using in-situ fibrous bed bioreactor

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(isFBB), up to 198.2 g/L SA was accumulated from crude glycerol using this strain.21

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So far, all the metabolic engineering of Y. lipolytica for SA production has led to partial or

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total loss of its ability to grow in glucose-based medium, which limits its industrial

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application. This glucose metabolism problem of engineered strain for SA production has

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been reported also in E. coli and S. cerevisiae.6 Methods such as increasing the reducing-

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force,22 intracellular ATP controlling,23 NADH/NAD+ ratio regulation24 by metabolic

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engineering or adaptive evolution

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metabolism. Compared to other three methods, adaptive evolution is more suitable for the

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built-in metabolic pathway.22, 25 Adaptive evolution in immobilised cell mode by FBB has

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been demonstrated as a highly efficient system for improvement of glucose uptake, alcohol

22, 25

have been attempted in order to improve the glucose


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and organic acid productivity and tolerance, as compared to the conventional free cell

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fermentation.26-28 However, operation conditions include monitoring of FBB bioreactor by

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highly-skilled labour, large volume of feeding medium and long-duration of fermentation

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process are essential for operating immobilised cell mode by FBB, makes it far from

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industrial application.

73 74

On the other hand, glucose-rich feedstocks are the most important carbon source in microbial

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fermentation.29 As vast majority of the carbon fixed by plant photosynthesis is available in

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the form of sugar polymers consist of cellulose, hemicellulose and starch.

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utilisation of glucose-rich feedstocks such as agro-waste residues and food waste have been

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investigated extensively to improve the economic competitiveness of bio-based SA

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production.2, 30 An alternative strategy is the integration of waste-based biorefinery concept,

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as food waste contains valuable nutrients and our former study successfully demonstrated the

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production of glucose-rich hydrolysate using food waste as feedstock.2 Herein, a simple and

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efficient adaptive evolution strategy was evaluated to enhance the glucose uptake rate of

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Y. lipolytica PGC01003 in SA fermentation. The evolved strain was investigated on the

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subsequent SA fermentation using glucose-rich food waste hydrolysate in bench-top

85

fermenter. In this study, the technical feasibility of SA production using food waste

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hydrolysate and engineered Y. lipolytica was examined. The results would demonstrate the

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potential using a low-cost feedstock for succinic acid fermentation as an environmentally

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friendly and economically sound bioconversion process.

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2. METHODS AND MATERIALS

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Strains and media


Therefore,


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The engineered strain of Y. lipolytica PGC01003, in which the Ylsdh5 genes encoding

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succinate dehydrogenase was knocked out for SA production in the previous work.20 The

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seed culture was stored in 30% (v/v) glycerol at -80 oC, which was activated in 50 mL

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modified YPG medium containing (w/v): 1% yeast extract, 2% tryptone, and 2% glycerol at

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28 oC and 220 rpm. The yeast strains were cultivated in YPG or YPD with various initial

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concentrations of glycerol or glucose at 28 oC and 220 rpm. Carbon and nitrogen sources

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were prepared separately and sterilized at 121 oC for 15 minutes.

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Food waste handling and hydrolysis

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Mixed food waste was collected from restaurants in Hong Kong Science Park. The storage

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and handling method was described by Sun et al.31 and the composition of food waste was

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determined as described in our previous study.32 Food waste with a solid-to-liquid ratio of

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50% (w/v) was hydrolysed in a 2.5-L bioreactor (BioFlo/CelliGen 115, New Brunswick

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Scientific, Edison, NJ, USA) at 55 oC. In order to control the nutrient composition of food

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waste hydrolysate being used in experiments, a consortium of commercial enzymes kindly

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provided by the Shangdong Longda Bio-Products Co., Ltd. were added at the beginning of

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hydrolysis. The consortium of enzymes contained (per gram dry food waste): Glucoamylase

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(120 U), amylase (10 U) and protease (150 U) .31 Samples were taken every 2-3 hours for

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sugar and free amino nitrogen (FAN) analyses. Finally, the hydrolysate was sterilised by

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membrane filtration (0.22 µm) and stored at -20 oC before use.

112 113

Shaking flasks fermentation of Y. lipolytica

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To compare Y. lipolytica fermentation in YPD and YPG media, shaking flasks fermentation

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was conducted in 250-mL shake flasks consisted of 50-mL YPD or YPG medium at 28 oC

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and 220 rpm. The fermentation began with inoculation of 1 mL of seed culture, and 1 mL


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broth was sampled every few hours for cell dry weight (CDW) measurement and high

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performance liquid chromatography (HPLC) analysis.

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To assess the stability of the evolved strain PSA02004, the cells were first transferred to YPG

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medium with 20 g/L initial glycerol concentration, and then 1 mL of the culture was

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transferred into YPD medium with 150 g/L initial glucose. The fermentation was carried out

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in 250-mL shake flasks with 50-mL YPD or YPG medium at 28 oC and 220 rpm. The

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fermentation began with addition of 1 mL of seed culture, and 1 mL broth was sampled every

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few hours for CDW measurement and HPLC analysis.

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Adaptive evolution of Y. lipolytica to glucose-rich YPD medium

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A series of experiments using adaptive evolution in either free cell fermentation or

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immobilised cell fermentation were conducted in 250-mL shake flasks with 50-mL YPD

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media containing various initial glucose concentrations, and shaking incubation at 220 rpm

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and 28 oC. To obtain higher cell biomass, 10 g/L of glycerol was supplemented into 1% YPD

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in the zero generation. Then it was transferred into 2.5% YPD medium which was marked as

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the first generation. Once the OD600 reached 1.8-2.2 within 24 hours, the culture was

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transferred into YPD medium with higher glucose concentration, and the initial glucose

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concentration increased stepwise from 25, 50, 75, 100 to 150 g/L.

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In free cell fermentation, the inoculum ratio of Y. lipolytica PGC01003 was 5% (v/v). In

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immobilised cell fermentation, 1% (w/v) of absorbent cotton was added in the culture to

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adsorb and immobilise cells in the first generation. Then, the absorbent cotton with

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immobilised cells was transferred to the fresh medium in the subsequent generations. After

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14 generations of adaptive evolution, 0.2 mL of the cell culture was plated on solid YPD

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medium containing 150 g/L glucose, and single colonies were selected. Six single colonies of



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each fermentation mode were picked up and screened by monitoring the cell growth rate in

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YPD medium with 150 g/L initial glucose concentration. The initial strain PGC01003 was

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used as a control.

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Batch fermentation

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A 2-L Sartorius Biostat B benchtop fermenter (B. Braun Melsungen AG, Melsungen,

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Germany) with 1.0 L working volume was used in batch fermentation. The pH value was

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automatically controlled at 6.0 with the addition of 5 mol/L NaOH, and the foaming was

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controlled with the addition of antifoam A (Sigma, Germany). Bench-top scale fermentations

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were performed at 28 oC, stirring speed at 600 rpm and aeration rate at 2.0 L/min using either

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defined medium or food waste hydrolysate.

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Analytical methods

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The analytical methods have been described in our previous work.20 Microbial growth was

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determined by OD600 and CDW. The specific growth rate (µ) was calculated by Equation (1):

157

= ×

(1)

158 159

where X is DCW and t is fermentation time.

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Glucose and metabolite concentrations were quantified using the ultra-performance liquid

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chromatography (UPLC) system (Waters, MA, USA), which was equipped with an Aminex

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87H column (Bio-Rad, Hercules, CA, USA) and a refractive index detector. H2SO4 (5 mM)

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was served as mobile phase at a flow rate of 0.6 mL/min. Temperature of column and

165

detector were kept at 60 oC and 35 oC, respectively. All samples and mobile phase were


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filtered by 0.22-µm membrane before loading. The FAN concentration of the hydrolysate was

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analysed using the ninhydrin colorimetric method.33

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3. RESULTS AND DISCCUSSION

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Adaptive evolution by free cell and immobilised cell modes

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As shown in Table 1, PGC01003 grew well in YPG medium and consumed 18.6 g/L glycerol

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to produce 5.4 g/L SA with 4 g/L cell biomass. Whereas only 5.7 g/L glucose was consumed

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in YPD medium after 84 hours, and the SA titer and CDW were only 35% and 31% of that in

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YPG medium, respectively. Disruption of succinate dehydrogenase in Y. lipolytica not only

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accumulated SA, but also caused partial loss of glucose metabolism ability.16, 20 The FAD

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recycled into FADH2 in the oxidation of SA into fumaric acid by SDH in the native

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Y. lipolytica, which has been disturbed in the SDH inactivated mutant (Fig. 1).34,35 Glycerol

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metabolism could restore the FAD/FADH2 recycle via the phosphorylation and successive

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oxidation reaction into dihydroxyacetone phosphate (DHAP).36 Moreover, the fast growth in

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glycerol could be caused by more transporters for glycerol uptake than that for glucose

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uptake in Y. lipolytica.36 Comprehensive metabolic and transcriptomic analyses also indicated

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the glucose transporters are not enough in Y. lipolytica.37 Yuzbashev et al. (2016) suggested

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acidification could negatively affect the cell growth of yeast on glucose-based media.

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Therefore, this would limit the practical application of Y. lipolytica PGC01003 in SA

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fermentation using naturally derived sugar-rich substrates. As most of the renewable

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feedstocks, such as corn straw, wheat straw and food waste are glucose-rich in nature as they

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predominantly consist of cellulose and starch as sugar polymers. Therefore, in order to

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enhance the glucose uptake rate in microbial SA fermentation, adaptive evolution of Y.

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lipolytica PGC01003 was performed. A highly efficient cell immobilisation method using

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cotton as absorbent was developed in this study. After cell growth to exponential phase, they

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were then transferred to the fresh medium. Free cell fermentation was carried out as a control

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group. Moreover, a rational design in composition of cultivation medium was applied for the


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initial medium, which was supplemented with 10 g/L glycerol. This design could facilitate

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higher biomass production in the first generation, which is the key factor for a successful and

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efficiently evolved adaptive process. After 14 transfers, the microbial growth in YPD

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medium with 150 g/L glucose in both fermentation modes were observed (Fig. 2). The

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immobilised cell fermentation took 21 days, which was 3 days shorter than that of free cell

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fermentation. The final evolved strains were designated as PSA01 and PSA02 for the free cell

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fermentation and immobilised cell fermentation, respectively.

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In the free cell fermentation, the glucose uptake rate was only 0.07 g/L/h in the third

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generation with medium containing 50 g/L initial glucose concentration, which increased four

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times to 0.29 g/L/h by PSA01 with 150 g/L initial glucose concentration. With the similar

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procedure, the glucose uptake rate increased three times to 0.30 g/L/h by PSA02. This high

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sugar consumption rate would lead to significant improvement of cell growth and SA

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productivity. Compared to the third generation, the cell growth rate and SA productivity

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increased by 2.9 and 4.0 times using the PSA01 strain, which were 4.8 and 5.0 times higher

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than those fermentation using the PSA02 strain. Moreover, the cell growth rate also

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significantly increased 2.2 and 2.3 times in free cell fermentation and immobilised cell

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fermentation, respectively. This suggests adaptive evolution by both fermentation modes

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could enhance the glucose uptake rate, the cell growth rate and SA productivity. Additionally,

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this immobilised cell fermentation has proved to be successful and highly efficient for

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adaptive evolution of Y. lipolytica.

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Evaluation of the evolved Y. lipolytica strains

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The evolved strains of PSA01 and PSA02 were cultivated separately on solid YPD agar

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plates with 150 g/L glucose for screening single colonies with stable phenotype. Six single

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colonies from both PSA01 and PSA02 strains were selected randomly for investigation of



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adaptive evolution in shake flask fermentation. As shown in Table 2, rapid cell growth was

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observed in cultivation broth consisting of YPD medium with 150 g/L initial glucose

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concentration, and 2.78-2.95 g/L CDW was obtained after 24 hours (Table 2). These results

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also indicated that the adaptive evolution was feasible and efficient.

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As shown in Fig. 3, two of each evolved strains with the highest cell growth rate, namely

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PSA01009, PSA01020, PSA02004 and PSA02007 were selected for future evaluation in

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comparison to fermentation using the initial strain PGC01003. These four evolved strains

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were transferred for 10 generations in YPD medium containing 150 g/L initial glucose

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concentration, then fermentations were carried out in medium using various initial glucose

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concentrations range of 25-300 g/L. Results demonstrated that all of these four evolved

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strains showed relatively high glucose tolerance i.e. cell growth was observed up to 200 g/L

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initial glucose concentration. In addition, similar cell growth rate was observed as compared

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to the growth in medium containing 25 g/L initial glucose concentration, suggesting the

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evolved strains could resist high initial glucose concentration in fermentation medium. The

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µmax profile showed a plateau of 0.23-0.30 h-1 from 25-200 g/L glucose, and dropped rapidly

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to 0.06 ± 0.005 h-1 when the glucose concentration increased to 300 g/L. However, the

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PGC01003 strain grew slowly with a maximum specific growth rate (µmax) reached only 0.09

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h-1 in medium consisting of 25 g/L initial glucose concentration. Decrease in µmax was

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observed in cultivation medium with increasing initial glucose concentration from 25 to

239

150 g/L, and eventually dropped to zero in medium consisting of 175 g/L initial glucose

240

concentration. Results from this study clearly demonstrate that the adaptive evolution by both

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fermentation modes were efficient for enhancing cell growth rate and glucose uptake rate of

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Y. lipolytica. Additionally, 5.5 ± 0.2 g/L SA was produced after 72 hours cultivation by these

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evolved strains in medium containing 150 g/L initial glucose concentration. Therefore, the


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PSA02004 strain was selected in the subsequent investigation using food waste hydrolysate

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based on the highest experimentally observed cell dry weight. In order to assess the stability

246

of the evolved strain in glucose-rich YPD medium, the evolved strain was cultivated in YPG

247

medium containing glycerol as the major carbon source, and 1 mL of broth was then

248

transferred into YPD medium containing 150 g/L initial glucose concentration. After 24

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hours cultivation, the resultant CDW was 4.67 ± 0.04 g/L, which indicates good stability of

250

the PSA02004 strain.

251 252

SA production from food waste hydrolysate by the evolved strain PSA02004

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As shown in Figure 4, SA production from glucose-rich media by the evolved strain

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PSA02004 was conducted in a 2-L fermenter using semi-defined YPD medium. Under

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oxygen-limited condition i.e. aeration rate at 2 L/min aeration and stirring speed at 600 rpm,20

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130.7 g/L glucose was consumed for production of 65.8 g/L SA with 22.5 g/L CDW after 96

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hours (Fig. 4A). SA production significantly increased in batch fermentation in bench-top

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fermenter. Apart from the high SA titer obtained in batch fermentation, high SA productivity

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of 0.69 g/L/h was resulted using the PSA02004 strain. Moreover, the SA yield reached 0.50

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g/g glucose which is equivalent to 0.76 mol/mol glucose, representing 76% of the theoretical

261

yield.10

262 263

Furthermore, its potential in SA production from glucose-rich food waste hydrolysate was

264

investigated, and hydrolysate from mixed food waste (i.e. leftover in restaurant) was used as

265

substrate. Food waste are potential sources of starch and protein-rich compounds that would

266

be used as nutrients in biotechnological processes, in which could be further hydrolysed by

267

the consortium of commercial enzymes consisting of glucoamylase, amylase and protease.2, 38

268

Food waste used in this study was collected from the same restaurant as our previous study,



269

which is rich in carbon and nitrogen sources of around 50% carbohydrate, 35–36% starch,

270

12–14% protein.32 After 24 hours hydrolysis by the commercial enzymes, 157.6 g/L glucose

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and 1.3 g/L FAN were obtained in the food waste hydrolysate. Then the hydrolysate was used

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as substrate with supplement of 5 g/L yeast extract in SA fermentation. After 126 hours

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cultivation, 157 g/L glucose was consumed completely to produce 87.8 g/L SA with 16.1 g/L

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of CDW (Fig. 4B). The SA yield and productivity reached 0.56 g/g glucose (equivalent to 85%

275

of theoretical yield) and 0.70 g/L/h, respectively. Compared to the batch fermentation using

276

defined medium, the SA titer, yield and productivity increased by 32%, 12% and 4%,

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respectively. The highest SA yield in Y. lipolytica achieved was up to 85% of theoretical

278

yield. These results suggest that the evolved Y. lipolytica PSA02004 strain could achieve the

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highest theoretical yield in SA production using food waste hydrolysate as feedstock.14, 21

280

Moreover, AA was completely depleted at the end of fermentation, which should be

281

consumed as a carbon source.20 These results also demonstrated that the use of evolved

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Yarrowia yeast strain would open the opportunity for efficient and environmentally friendly

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processes in commercial production of SA.

284 285

Since the first reported study for utilization of metabolic engineered Y. lipolytica for SA

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production in 2010, the mechanism of partial or total loss of glucose metabolism ability has

287

not yet been revealed.10 Adaptive evolution has been demonstrated for enhancing glucose

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uptake successfully by FBB. However, adaptive evolution strategy by FBB has several

289

disadvantages including highly-skilled labour, large volume of feeding medium and long

290

duration of fermentation process. In this study, the possibility of developing a simple and

291

efficient immobilised cell fermentation by shake flasks was investigated using adaptive

292

evolution strategy. After 21 days evolution, the glucose uptake rate, cell growth rate and

293

SA productivity of the SDH inactive Y. lipolytica were significantly increased, suggesting


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that the glucose metabolism was restored successfully (Fig. 2). Furthermore, the experimental

295

results reported in this study show that the evolved strains have stable phenotype in

296

YPD medium containing high initial glucose concentration (i.e. up to 150 g/L) (Table 2, Fig.

297

3). To our knowledge, this is the first report that using adaptive evolution for SA production

298

from glucose-rich medium by engineered Y. lipolytica. Furthermore, the evolved strain

299

produced 87.9 g/L SA from food waste hydrolysate with 85% of theoretical yield, which is

300

the highest SA yield in yeast, to date (Fig. 4). This suggests that this evolved strain has a high

301

potential for SA fermentation using glucose-rich agricultural residues and food waste derived

302

feedstocks. Since there are around 3.7 billion tons of agricultural residues worldwide, with a

303

composition of about 40% cellulose39, 40. The annual global production of food wastes is 1.3

304

billion tons, and these food wastes contain around 30–60% of starch41, 42. These agricultural-

305

and food residues could potentially be hydrolysed as glucose-rich nutrient source that

306

representing a promising feedstock in biotechnological processes.2, 40, 43 Very recently, study

307

of this evolved yeast strain has been carried out in a novel mixed fruit and vegetable waste

308

biorefinery concept in SA fermentation by our group (Li et al. 2017).44 Moreover, in order to

309

decrease the cost of downstream processing, the evolutionary approach in this study would be

310

used for enhanced SA production in low pH environment using Y. lipolytica. As showcased

311

in our previous study, the PGC01003 strain has been reported for its ability in fermentative

312

SA production under low pH environment (i.e. pH 4).20 Concluding, the experimental results

313

reported in this study clearly demonstrate a proof of concept which paves the way for future

314

development of more process-friendly Y. lipolytica yeast strains in fermentative SA

315

production. This interesting approach would open the opportunity for generating useful

316

knowledge for the whole scientific yeast metabolic engineering community.

317



318

ABBREVIATIONS AND NOMENCLATURE

319

Acetic acid (AA), adenosine triphosphate (ATP), cell dry weight (CDW), dihydroxyacetone

320

phosphate (DHAP), flavin adenine dinucleotide (FAD), free amino nitrogen (FAN), fibrous-

321

bed bioreactor (FBB), generally recognized as safe (GRAS), nicotinamide adenine

322

dinucleotide (NAD), succinic acid (SA), succinate dehydrogenase (SDH), tricarboxylic acid

323

(TCA).

324 325

ACKNOWLEDGEMENT

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The work described in this paper was fully supported by a grant from the City University of

327

Hong Kong [Project No. CityU 7004694].


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Thankappan, S.; Mohamed, Z.; Brocklesby, R.; Luque, R., Food waste as a valuable

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458

Figures captions

459

Fig. 1 Metabolic pathways showing the flow of NAD+/NADH and FAD/FADH2 with

460

utilisation of glucose and glycerol in Y. lipolytica PGC01003. The red cross represents the

461

oxidation of SA into fumaric acid, which was blocked by deletion of Ylsdh5 in Y. lipolytica

462

PGC01003. The metabolic pathway from glucose to GAP in glycolysis was indicated by

463

three arrows, in which neither NAD+/NADH nor FAD/FADH2 pathways were accompanied.

464

DHAP: dihydroxyacetone phosphate, GAP: glyceraldehyde-3-phosphate, Glycerol-3-P:

465

Glycerol 3-phosphate, Q: ubiquinone.

466 467

Fig. 2 Comparison of adaptive evolution progress of Y. lipolytica in free cell fermentation and

468

immobilised cell fermentation. Relative growth rate (A), in which the cell growth rate of the

469

first generation was set as 100%. [Relative growth rate = (cell growth rate in any

470

generation)/(cell growth rate in the first generation) ×100%]. Glucose consumption rate (B)

471

and SA productivity (C) were presented as the average value in each generation.

472 473

Fig. 3 Comparison of maximum specific growth rate of the adapted and wild-type strains in

474

defined media with various initial glucose concentrations of 25-300 g/L in shake flask

475

fermentation.

476 477

Fig. 4 Bench-top scale fermentation profile using the adapted strain PSA02004 in YPD

478

medium

(A)

and

food

waste


hydrolysate

(B).


Page 24 of 30

Table 1. Fermentative SA production by Y. lipolytica PGC01003 in YPG and YPD media. Fermentation CDW Medium time (h) (g/L)

Glycerol or glucose consumption (g/L)

SA titer (g/L)

SA yield (g/g)

SA productivity (g/L/h)

AA titer (g/L)

YPG

48

4.0

18.6

5.4

0.29

0.06

3.8

YPD

84

1.8

5.7

1.9

0.33

0.02

4.4


Page 25 of 30


Table 2. Summary of CDW and µmax of the evolved strains in YPD medium with 150 g/L initial glucose concentration. Strain

CDW(g/L)

µmax (h-1)

PSA01002

2.91

0.55

PSA01005

2.88

0.47

PSA01009

2.93

0.67

PSA01010

2.89

0.46

PSA01011

2.92

0.47

PSA01020

2.89

0.74

PSA02004

2.95

0.72

PSA02005

2.78

0.61

PSA02006

2.85

0.55

PSA02007

2.81

0.62

PSA02008

2.81

0.61

PSA02011

2.81

0.61



Page 26 of 30

Fig.1 Glucose

Glycerol NADH

GAP

NAD+

Glycerol-3-P

DHAP

NAD+ NADH

Pyruvate

FADH2

NAD+

FAD

NADH

Acetyl-CoA QH2 Oxaloacetate

NADH

Q

Citrate

NAD+

Isocitrate

Malate

NAD+

TCA cycle NADH

Fumarate FADH2 FAD

ketoglutarate NAD+

Succinate

Succinyl-CoA

NADH


Mitochondrial

Page 27 of 30


Fig. 2 ˚

175

300

150

250

125

200

100 150 75 100

50

50

25 0

Relative growth rate (%)

Glucose concentration (g/L)

A

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Generation number

0.4


175 150

0.3

125 100

0.2 75 50

0.1

25 0

0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Glucose consumption rate (g/L/h)

B

Generation number

C 0.20 Glucose concentration 150 Free cell mode 125

0.15

Immobilised cell mode

100 0.10 75 50

0.05

25 0

0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Generation number


SA productivity (g/L/h)


175


Fig. 3


Page 28 of 30

Page 29 of 30


Fig. 4

20

90

15

60

10

30

5 0

24

48 72 Time (h)

96

0

30 Glucose SA AA CDW

150 Glucose, SA, AA (g/L)

25

120

0

180

120

25 20

90

15

60

10

30

5

0

0

24


48

72

Time (h)

96

120

0

CDW (g/L)

30

Glucose SA AA CDW

150 Glucose, SA, AA (g/L)

B

˚

180

CDW (g/L)

A


TOC Graphic

Page 30 of 30

Evolved strains Adaptive evolution Y. lipolytica PGC01003

SA production Food waste

Hydrolysis

ACS Paragon Plus Environment

Food waste hydrolysate

Yarrowia lipolytica - American Chemical Society

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