Enhanced Î²-Amyrin Synthesis in Saccharomyces cerevisiae by

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Biotechnology and Biological Transformations

Enhanced #-Amyrin Synthesis in Saccharomyces cerevisiae by Coupling An Optimal Acetyl-CoA Supply Pathway Hu Liu, Jingjing Fan, Chen Wang, Chun Li, and Xiaohong Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00653 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Journal of Agricultural and Food Chemistry

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Enhanced β-Amyrin Synthesis in Saccharomyces cerevisiae by

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Coupling An Optimal Acetyl-CoA Supply Pathway

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Hu Liu1a, Jingjing Fan1a, Chen Wanga, Chun Lia b, Xiaohong Zhoua*

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aInstitute

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Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081,

9

China

for Synthetic Biosystem, Department of Biochemical Engineering, School of

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bKey

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Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China

Laboratory of Systems Bioengineering (Ministry of Education), School of

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1These

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*Corresponding author.

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E-mail address: [email protected] (Xiaohong Zhou).

authors contributed equally to this article.

1

ACS Paragon Plus Environment


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ABSTRACT: β-Amyrin is a plant-derived triterpenoid skeleton with wide applications

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in food and medical industry. β-Amyrin biosynthesis in Saccharomyces cerevisiae is

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derived from mevalonate pathway with cytosolic acetyl-CoA as a precursor. In this

21

work, endogenous and several heterologous acetyl-CoA synthesis pathways were

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coupled to β-amyrin production and a combinational acetyl-CoA supply route was

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demonstrated to be optimal due to more balanced redox cofactors, much lower energy

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consumption and glucose utilization as well as significantly enhanced β-amyrin

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production (200% increase than the original β-amyrin-producing strain). Further

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disruption of an acetyl-CoA competing pathway led to a 330% increase in β-amyrin

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production as compared with the original strain. Finally, the engineered strain harboring

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the optimal pathway configuration achieved a final β-amyrin production of 279.0±13.0

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mg/L in glucose fed-batch fermentation, which is the highest as ever reported. This

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work provides an efficient platform for triterpenoid biosynthesis in Saccharomyces

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cerevisiae.

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KEYWORDS:

Acetyl-CoA;

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Saccharomyces cerevisiae

β-Amyrin;

Stoichiometric

2


analysis;

Triterpenoid;

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 INTRODUCTION

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Triterpenoids are highly diverse natural products widely distributed in plants with

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various biological activities.1, 2 For instance, glycyrrhizin, a triterpenoid extracted from

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licorice, is applied as medicine and natural sweetener due to its various pharmacological

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effects and strong sweetness.3 β-Amyrin is also a kind of triterpenoid with similar

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pharmacological activities as glycyrrhizin and serves as a key precursor to a wide range

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of triterpenoid products.4 As a low-abundant secondary metabolite, β-amyrin extraction

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from natural plants is usually low-yielding, and thus requires considerable consumption

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of natural sources.5 To circumvent the limitations of traditional extraction processes,

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metabolic engineering efforts have been made to achieve the production of many plant-

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derived valuable compounds using microorganisms.6-10 Although β-amyrin production

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in Escherichia coli (E. coli) has been reported, the productivity is extremely low11.

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Recently, several β-amyrin synthases from various plants have been identified and

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introduced in Saccharomyces cerevisiae (S. cerevisiae) for the construction of β-amyrin

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biosynthesis pathways.12-15

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β-Amyrin biosynthesis in engineered S. cerevisiae is derived from mevalonate (MVA)

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pathway with cytosolic acetyl-CoA as a precursor. Previous efforts to enhance β-amyrin

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production have generally focused on genetic modifications of the MVA pathway or its

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downstream section to increase the metabolic flux towards β-amyrin.6, 16 However, there

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remains a bottleneck in the supply of acetyl-CoA to MVA pathway. When it is relieved,

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β-amyrin production should be further enhanced.17 The commonly employed strategies

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for enhancing the supply of acetyl-CoA to MVA pathway have involved engineering a 3



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PDH bypass,18 introduction of the ATP-citrate lyase (ACL) pathway19 and functional

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expression of the pyruvate dehydrogenase complex in the cytosol.20-24 The

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overexpression of either acetyl-CoA synthetase or the ACL gene resulted in more than

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50% increase in cytosolic acetyl-CoA level and a significant improvement in squalene

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production.25 To reduce the energy cost of endogenous PDH bypass, Meadows et al.

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rewired the pathway to substitute the PDH-bypass and increased the cytosolic acetyl-

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CoA pool by combining several acetyl-CoA supply pathways, resulting in yeast

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fermentations that generate 15% v/v farnesene.26 Although these strategies have been

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demonstrated to enhance the acetyl-CoA level, they promoted the productions of acetyl-

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CoA-derived chemicals with varying degrees of success. Thus, choosing the best

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approach for acetyl-CoA supply from various alternatives to maximize β-amyrin

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production would be quite challenging as it requires a global analysis of the matching

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between precursor supply and product formation rather than just concentrating at one

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single point, like strengthened precursor supply.27

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Therefore, in this work, endogenous and various heterologous acetyl-CoA supply

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pathways were coupled to β-amyrin biosynthesis respectively in an engineered β-

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amyrin-producing S. cerevisiae strain (Figure 1) and a global analysis of their matching

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in terms such as energy, redox cofactor and product yield was performed. The obtained

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optimal acetyl-CoA synthesis pathway, combined with deletion of an acetyl-CoA

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competing pathway and glucose fed-batch fermentation, led to the highest β-amyrin

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production ever reported. Furthermore, the strategy of optimizing the precursor-supply

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pathway for specific product formation based on their matching relations in terms of

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energy, redox cofactor and yield is conducive for the design of efficient cell factories.

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 MATERIALS AND METHODS

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Strain, media and yeast cultivation. S. cerevisiae strain SGib is a β-amyrin-

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producing strain constructed by our group and was used as the parent strain.6 The yeast

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strains were cultured and fermented at 30 ℃and 200 rpm in YPD medium consisting of

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10 g/L yeast extract (Merck Millipore), 20 g/L peptone (Difco) and 20 g/L glucose

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(Merck Millipore). For strain culture and activation, all the S. cerevisiae strains were

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first pre-cultured in 5 mL YPD medium for 20 hours and then inoculated into 50 mL

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fresh YPD medium at the ratio of 1:100 for 120 hours incubation. Fed-batch

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fermentations were carried out in a well-controlled 5-L bioreactor (Minifors,

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Switzerland) with a working volume of 3 L. The temperature was kept constant at 30 ℃

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and the air flow was 3 L/min (1 vvm) with agitation speed of 250 rpm. The pH was

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maintained at pH 6.0 by automatic addition of 5 M NH4OH. After 24 hours

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fermentation, 5 g/L glucose or ethanol was supplemented every 12 hours.

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Plasmids and strains construction. Genes of alcohol dehydrogenase (ADH),

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acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthase (ACS) were cloned from

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S. cerevisiae strain SGib. E. coli pyruvate dehydrogenase complex (PDH) genes (aceE,

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aceF, lpd) and acetylating acetaldehyde dehydrogenase (A-ALD) gene (eutE) were

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obtained by polymerase chain reaction (PCR). The modified NADP-dependent PDH

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(PDHm) genes were acquired by replacing lpd gene with a variant lpdm which

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corresponds to five amino acid substitution identified for NADP binding.28 The lpdm 5



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gene was obtained by overlap extension PCR. Genes of ACL from Aspergillus nidulans,

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NADH-dependent HMG-CoA reductase variant (NADH-HMGr) from Silicibacter

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pomeroyi,

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phosphotransacetylase (PTA) from Clostridium kluyveri and the yeast promoter Pmini

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were chemically synthesized. Other yeast promoters and terminators used in this study

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were PCR-amplified from the genome of strain SGib. PCRs were conducted with a 30-

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cycle program: 98℃ for 1 min, 98 for 10s, 55℃ for 10 s, 72 ℃ for 1 min/kb, and a final

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extension at 72℃ for 5 min. PrimerSTAR DNA polymerase (Takara) was used

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according to the manufacturer's protocol. The gene expression cassettes as promoter–

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gene–terminator were assembled by Gibson assembler strategy and integrated into a

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plasmid or genome by homologous recombination.

phosphoketolase

(PK)

from

Leuconostoc

mesenteroides,

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The plasmid-based strain BA11 was constructed by cotransforming gene expression

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cassettes PMini-aceE-TSLM5, PGPD1-aceF-TTEF2 and PTPI1-lpdm-TADH1 together with

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linearized multicopy pRS42K plasmid into S. cerevisiae. For genomic integration of

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acetyl-CoA synthesis pathways, the HO locus was used. While for engineered strains

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with zwf1 deletion, acetyl-CoA synthesis pathways were integrated directly into the

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zwf1 locus. The selection marker cassette PTEF1-kanMX-TTEF1 was cotransformed and

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integrated into the corresponding genome locus. The plasmids and strains used in this

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study are summarized in Table S1. All the primers used for the construction of

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expression cassettes can be found in Table S2.

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Metabolite extraction and analysis. After cultivation for 120 h, 10 mL of yeast cell

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culture was centrifuged and the pellet obtained was resuspended in 10 mL of a fresh 6


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solution of 20% (w/v) KOH in 50% ethanol and boiled for 10 min to cell lysis. After

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cooling, the sample was extracted two times with equal volume of hexane. The hexane

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phase was evaporated and trimethylsilylated for gas chromatography-mass spectrometry

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(GC-MS) analysis of β-amyrin and ergosterol. GC-MS was performed on a GCMS-

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QP2010 Ultra (Shimadzu Scientific Instruments) equipped with a SH-Rxi-5Sil MS (30

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m×0.25 mm×0.25 μm, SHIMADZU, Japan) GC column. Compound separation was

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achieved with an injection temperature of 250 ℃ and a 30-min temperature gradient

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program starting at 80 ℃ for 1 min followed by heating the column to 280 ℃ at 20 ℃

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min-1 increase, holding at 280 ℃ for 15 min, reaching 300 ℃ at 20 ℃ min-1 increase

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and finally keeping at 300 ℃ for 5 min. The MS scan range acquired 45-450 m/z,

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helium flow rate was 1.0 mL/min, 1 μL sample was injected with 10:1 split stream

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mode. Peaks were identified by comparing the retention time and mass spectra with

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those of the authentic standards. β-Amyrin and ergosterol standards (Sigma-Aldrich)

136

were also used for quantitative analysis. The GC-MS profiles of β-amyrin and

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ergosterol from engineered strain BA01 were exhibited in Figure S1. Furthermore, high-

138

performance liquid chromatography (HPLC) was performed for the detection of glucose,

139

ethanol, glycerol and acetic acid using a SHIMADZU HPLC system with an Aminex

140

HPX-87H column (300 mm × 7.8 mm, Bio-Rad, Hercules, CA, USA) and a Waters

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2414 refractive index detector. The mobile phase was 5 mM H2SO4 and the flow rate

142

was 0.5 mL/min. The column temperature was set at 55 ℃. In order to quantify the dry

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cell weight (DCW, g/L), the optical density (OD) of cultivation samples was determined

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at 600 nm in duplicates using a UV-1800 SHIMADZU UV spectrometer. DCW was

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calculated using an OD-DCW correlation: DCW/OD600=0.444.

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Determination of acetyl-CoA concentrations and NAD(P)H/NAD(P)+ ratios.

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Acetyl-CoA and redox cofactor measurements were conducted with the Acetyl-

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Coenzyme A Assay Kit (Solarbio, Beijing, China) and NAD(P)H/NAD(P)+

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Quantification Kit (Solarbio, Beijing, China) respectively. Briefly, yeast cells were

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collected at mid-log growth phase by centrifugation (12000 g, 2 min) and sonicated in

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extraction buffers for the release of intracellular metabolites. After centrifugation, the

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supernatants were analyzed according to the protocols provided by the manufacturer.

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The concentration of acetyl-CoA was the average of biological duplicates and

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normalized by the DCW.

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Quantitative PCR. The relative expression ratios of genes in engineered strains were

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determined via quantitative PCR (qPCR) according to the method described earlier with

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the housekeeping gene ACT1 as a reference.5 The qPCR analysis was performed at the

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LightCycler 96 system (Roche) using LightCycler SYBR Green I Master Kit (Roche).

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Primers used for qPCR are shown in Table S2.

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 RESULTS AND DISCUSSION

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Overexpression of endogenous acetyl-CoA supply pathway to enhance β-amyrin

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production. β-Amyrin biosynthesis in yeast is derived from mevalonate pathway with

163

cytosolic acetyl-CoA as a precursor. S. cerevisiae SGib is a β-amyrin-producing strain

164

constructed by our group through introduction of a plant-derived β-amyrin synthase

165

gene (GgbAS) and overexpression of several genes downstream of mevalonate (genes 8


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encoding isopentenyl diphosphate isomerase (IDI), farnesyl pyrophosphate synthase

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(ERG20), squalene synthase (ERG9) and squalene monooxygenase (ERG1)) for the

168

enhancement of β-amyrin production.6 In this study, we focus on an appropriate acetyl-

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CoA supply pathway coupling with β-amyrin synthesis for further improvement of β-

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amyrin production. Cytosolic acetyl-CoA in native S. cerevisiae is synthesized from

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PDH bypass or during ethanol re-oxidization via ADH, ALD and ACS. Overexpression

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of these enzymes in the pathway has been demonstrated to be capable of improving the

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acetyl-CoA level.29,

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overexpression in SGib to generate engineered strains BA01(ALD6+ACS1),

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BA02(ALD6+ACS2) BA03(ADH2) and BA04 (ADH2+ALD6+ACS1). In S. cerevisiae,

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ACS is encoded by two genes, ACS1 and ACS2. At high concentrations of glucose,

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ACS1 is transcriptionally repressed and ACS2 is constitutively expressed irrespectively

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of the kind of carbon source. However, ACS1 was shown to have a 30-fold higher

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affinity to acetate than ACS2.18 Therefore, ACS1 was chosen for overexpression in

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strain BA04. It is shown in Figure 2A that the production of β-amyrin in SGib was

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determined to be 3.1 ± 0.2 mg/g DCW. And the engineered strains BA01 to BA04

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showed improved β-amyrin productions but with limited extents, with BA04 showing

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the highest β-amyrin production of 4.4±0.2 mg/g DCW, 42% higher than that of SGib.

184

Furthermore, ADH2 overexpression in strain BA03 led to a decreased biomass, which is

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probably caused by the accumulation of acetic acid (Figure 2B). A temporary acetic

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acid accumulation was also observed at 24 h in other engineered strains, which is

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probably due to the imbalance in the enzyme activities of ALD6 and ACS1/ ACS2.

30

Therefore, ALD6, ACS1/ACS2 and ADH2 were chosen for

9



188

β-Amyrin synthesis from acetyl-CoA involves more than 10 distinct metabolic

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steps as shown in Table S3. The overall stoichiometry for β-amyrin production from

190

acetyl-CoA is summarized in equation 1 of Table 1, which indicates that, besides large

191

amounts of acetyl-CoA, high levels of ATP and NADPH are required in this energy-

192

intensive pathway to produce β-amyrin. The endogenous PDH bypass for acetyl-CoA

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synthesis is low efficient owing to the ATP-requiring reactions catalyzed by ACS (2

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ATP for one acetyl-CoA formation). Therefore, PDH bypass coupling with β-amyrin

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synthesis leads to equation 2 in Table 1, indicating a high energy input requirement (36

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ATP) for endogenous PDH bypass-coupled β-amyrin synthesis, which might explain the

197

limited enhancing effect on β-amyrin production (almost 42% increase) despite the

198

efforts on genetic perturbations for increasing metabolic flux from glucose to acetyl-

199

CoA. Owing to the low efficiency of endogenous PDH bypass, several heterologous

200

acetyl-CoA biosynthesis pathways with lower energy input requirement were

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introduced in SGib subsequently to couple with β-amyrin production for a better match.

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Introduction of heterologous acetyl-CoA supply routes to couple with β-amyrin

203

synthesis. The first heterologous acetyl-CoA synthesis pathway introduced in SGib was

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ACL route, which converts the TCA cycle intermediate citrate to acetyl-CoA at the cost

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of one ATP molecule,31 implying a more energy-efficient pathway than native PDH

206

bypass. However, the resulting strain BA05 (ACL) did not show an improved β-amyrin

207

production. While the supplementation of citrate to the medium increased β-amyrin

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level with the highest production of 4.2 ± 0.4 mg/g DCW at 1 g/L citrate addition,

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almost 36% higher than that of SGib (Figure 3). By contrast, citrate supplementation did 10


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not show any enhancing effect on β-amyrin production in SGib. These results indicate

211

that the limited improvement in flux to β-amyrin synthesis is probably due to the

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insufficient citrate supply to ACL in cytosol of yeast as citrate generated in TCA cycle

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needs to be exported out of mitochondria. Although mitochondrial engineering has been

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attempted for enhancing the export of mitochondrial citrate to the cytosol,20 it is still a

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complicated task. And considering the high cost of citrate supplementation, it is not an

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ideal option to employ ACL pathway for β-amyrin production from an industrial point

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of view.

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In contrast to ATP-dependent PDH bypass and ACL pathways, the E. coli PDH

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possesses the function of converting pyruvate to acetyl-CoA without ATP involvement.

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The PDH pathway coupling to β-amyrin synthesis generates equation 3 in Table 1.

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Although ATP is not needed, equation 3 demonstrates the requirement of large amounts

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of NADPH for the synthesis of β-amyrin with concomitant release of NADH. Due to

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the lack of transhydrogenase activity for interconverting NADH to NADPH directly in

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yeast,32 this imbalance between NADPH consumption and NADH generation might

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lead to inefficient β-amyrin production. To address this redox imbalance, a strategy of

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changing the cofactor specificity of enzyme was employed.33 The native E. coli PDH is

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NAD-dependent and a modified PDH (PDHm) that accepts NADP for acetyl-CoA

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production was reported recently to alter the intracellular NADPH/NADP+ ratio.34

229

Accordingly, PDH was replaced by PDHm to rebalance the redox cofactors (equation 4,

230

Table 1). Furthermore, the PDH-coupled β-amyrin synthesis (equation 3) is an NADPH-

231

consuming reaction while the PDHm-coupled β-amyrin synthesis (equation 4) implies an 11



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NADPH-producing process and needs NADP+ to get into the reaction. Therefore,

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further deletion of zwf1 gene encoding an NADP+-dependent enzyme would lead to

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NADP+ accumulation and ensure sufficient NADP+ supply to PDHm. Based on the

235

stoichiometric analysis above, engineered strains BA06 (PDH), BA07 (PDHm) and

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BA08 (PDHm+△zwf1) were constructed. It is shown in Figure 4A that the introduction

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of PDH or PDHm alone did not show any enhancing effects on β-amyrin production,

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while the combination of PDHm and zwf1 deletion in BA08 increased the level of β-

239

amyrin, 61% higher than that of SGib strain. To explore what was driving those

240

differences, the transcriptional expression of PDH or PDHm in the engineered strains

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were investigated and displayed in Figure 4B. It is interesting to note that BA08 with

242

the combination of PDHm introduction and zwf1 deletion showed a much higher PDHm

243

expression level than BA06 and BA07. Since PDH and PDHm were expressed in BA06

244

and BA07 respectively under the control of the same promoter, zwf1 deletion was

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further conducted in strain BA06 (PDH) and the PDH expression level was tested to

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examine whether zwf1 deletion had an impact on the promoter of PDH(m). The obtained

247

strain BA09, however, did not exert a higher PDH expression level (Figure 4B),

248

indicating that zwf1 deletion made no difference to the promoter. The zwf1gene encodes

249

glucose-6-phosphate dehydrogenase involved in pentose phosphate pathway and is a

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main source for NADPH generation in S. cerevisiae. In a recent study by Gold et al., the

251

deletion of zwf1 gene was performed to create an NADPH deficiency and force the cell

252

to couple its growth to tyrosine production via overexpression of an NADP-dependent

253

prephenate dehydrogenase.35 Moreover, it was reported that NADPH deficiency in S. 12


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cerevisiae with zwf1 gene disruption can result in methionine auxotrophy and increased

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sensitivity to oxidizing agents. While overexpression of NADP-dependent ALD6 gene

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can restore the Met+ phenotype of the △ zwf1 strain.36 Enlightened by these, we

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speculated that the enhanced PDHm expression in BA08 was due to the NADPH

258

deficiency associated with zwf1 deletion. To confirm this, the intracellular

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NADPH/NADP+ ratios in engineered strains were tested. It is observed in Figure 4C

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that, compared with SGib, the overexpression of NADH-generating PDH in BA06

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exerted minimal impact on NADPH/NADP+ ratio, while the overexpression of

262

NADPH-generating PDHm in BA07 increased the NADPH/NADP+ ratio from 0.63 ±

263

0.03 to 0.72 ± 0.05. zwf1 deletion in BA06 (PDH) resulted in BA09 and produced a

264

much reduced NADPH level. Similarly, zwf1 deletion in SGib resulted in BA10, which

265

also displayed a significant decrease in NADPH level, demonstrating that zwf1 deletion

266

led to NADPH deficiency. However, the combination of zwf1 deletion with NADPH-

267

generating PDHm introduction in BA08 restored much of the NADPH level (Figure 4C),

268

indicating that the high PDHm expression level in BA08 was probably due to the

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metabolic regulation in cells for redox balance in response to NADPH deficiency

270

associated with zwf1 deletion. Although zwf1 deletion led to a higher PDHm expression

271

level and a promoted β-amyrin production, the overexpression of PDHm in multicopy

272

plasmid pRS42K (BA11) did not show a significant enhancing effect on β-amyrin

273

production probably due to NADP+ deficiency in S. cerevisiae. Additionally, BA10 with

274

zwf1 deletion alone did not produce an enhanced β-amyrin production. Based on these

275

results, it is suggested that the PDHm overexpression should be coupled to sufficient 13



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NADP+ supply caused by zwf1 deletion or other engineering strategies. This

277

combination could enable the PDH pathway operating at its maximum efficiency and

278

boost the PDH-dependent flux for β-amyrin production .

279

Besides the discussed pathways above, an alternative route through expression of PK

280

and PTA was shown to shunt carbon flux through the pentose phosphate pathway and

281

contribute to acetyl-CoA production in S. cerevisiae.37,

282

pathways, the PK and PTA (PK/PTA) pathway is attractive as it represents a higher

283

attainable yield due to the absence of NADH generation.39 However, the PK/PTA

284

pathway requires ATP, which inspires researchers to propose the integration of PK/PTA

285

pathway with other ATP-independent acetyl-CoA supply routes.39 Recently, the

286

PK/PTA pathway was combined with an ATP-independent A-ALD pathway for

287

enhancing β-farnesene production.26 Therefore, in this study we investigated the effect

288

on β-amyrin production of this combinational strategy, which includes expression of a

289

xylulose-5-phosphate (X5P)-specific PK (xPK) and a PTA to provide a route from X5P

290

to acetyl-CoA along with A-ALD expression to catalyze the conversion of acetaldehyde

291

to acetyl-CoA. Moreover, the native NADPH-dependent HMGr was replaced with an

292

NADH-specific version to rebalance the redox cofactors between A-ALD pathway and

293

β-amyrin synthesis. Five engineered strains BA12 (PK/PTA), BA13 (A-ALD), BA14

294

(A-ALD+NADH-HMGr),

295

ALD+NADH-HMGr) were constructed to examine the effects of single pathway

296

introduction and the combination of them on β-amyrin production (Figure 5A). It is

297

shown that introduction of either PK/PTA or A-ALD pathway in strain BA12 and BA13

BA15

(NADH-HMGr)

14


38

and

Compared with other

BA16

(PK/PTA+A-

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298

led to remarkably reduced biomass when compared with SGib. The replacement of

299

native NADPH-dependent HMGr with an NADH-specific variant in BA14 led to

300

rebalanced redox cofactors (equation 5, Table 1) and a dramatically improved biomass

301

(Figure 5A), implying redox imbalance to be the major cause of cell growth repression.

302

To confirm this, the intracellular NADH/NAD+ ratios in engineered strains were tested.

303

Compared with SGib, the introduction of A-ALD pathway in BA13 caused a

304

remarkable increase in NADH level. Nevertheless, the combination of A-ALD with

305

NADH-dependent HMGr in BA14 restored the NADH level and rebalanced redox

306

cofactors. Another engineered strain BA15 with only NADH-HMGr overexpression in

307

genome of SGib at HO site showed a sharp decrease in NADH level and a disrupted cell

308

growth, further verifying that the growth repression effect of BA13 was due to affected

309

energy metabolism associated with the redox imbalance. For BA12 harboring the

310

PK/PTA pathway, the split of carbon from the glycolytic pathway to the pentose

311

phosphate pathway might contribute to its growth attenuation. A biomass reduction of S.

312

cerevisiae expressing the PK pathway was also observed in previous studies.37, 40

313

Although the introduction of PK/PTA or A-ALD pathway alone did not effectively

314

improve β-amyrin synthesis, the combination of them along with NADH-HMGr

315

introduction in BA16 exhibited a remarkable enhancing effect on β-amyrin production

316

(9.3 ± 0.5 mg/g DCW), almost 200% higher than that of SGib (Figure 5A). As the

317

PK/PTA pathway doesn't produce NADH, it is a high-carbon-yield pathway but with

318

ATP consumption (equation 6, Table 1). While the A-ALD pathway is ATP-

319

independent and could produce NADH (equation 3, Table 1), which can be reoxidized 15



320

by respiration to provide ATP for PK/PTA pathway.39 Therefore, the combination of

321

these two pathways makes them complement each other to achieve an integral acetyl-

322

CoA synthesis route where a portion of NADH generated in A-ALD pathway is

323

supplied to NADH-HMGr and the surplus NADH is reoxidized to provide ATP for

324

PK/PTA pathway and downstream β-amyrin synthesis. When the ATP supply from

325

excess NADH reoxidation just matches the ATP consumption, a highest yield is

326

attained (equation 7, Table 1). In this optimal scenario, the distributions of acetyl-CoA

327

synthesis between PK/PTA and A-ALD pathways are required to be 40% and 60%

328

respectively (P/O ratio assumed to be 141). Although it is difficult to achieve the precise

329

distributions between these two pathways, the engineered strain BA16 which harbors

330

the combinational pathway still displayed the best performance in β-amyrin

331

biosynthesis among all the engineered strains, indicating a perfect match between this

332

integrative acetyl-CoA supply pathway and β-amyrin production.

333

After optimization of acetyl-CoA supply pathway for β-amyrin production, the

334

glyoxylate shunt was disrupted in BA16 to further remove an acetyl-CoA competing

335

pathway. The corresponding genes, MLS1 and CIT2, encode malate synthase and

336

peroxisomal citrate synthase, which are key enzymes in the glyoxylate shunt. However,

337

MLS1 deletion in BA16 resulted in a repressed cell growth (data not shown). While

338

CIT2 deletion in BA16 generated BA17 strain, which showed a further 42% increase in

339

β-amyrin production than BA16 and a nearly 330% increase than SGib (Figure 6A).

340

However, CIT2 deletion in SGib did not increase β-amyrin production (data not shown),

341

which is inconsistent with the result of BA17. Since the glyoxylate shunt shows 16


Page 16 of 40

Page 17 of 40


342

minimal activity during growth on glucose and is predominantly active during growth

343

on C2 compounds such as ethanol and acetate, the physiological performances of BA16

344

and SGib were tested and it is observed that BA16 exhibited a higher accumulation of

345

glycerol, ethanol and acetic acid than SGib (Figure S2), indicating a higher glyoxylate

346

shunt activity in strain BA16. Therefore, CIT2 deletion in BA16 produced a more

347

obvious effect than that on SGib strain.

348

Comparison of various acetyl-CoA supply pathways for coupling with β-amyrin

349

synthesis. For further analysis of the coupling between various acetyl-CoA supply

350

pathways and β-amyrin synthesis, the engineered strains harboring different acetyl-CoA

351

supply routes for β-amyrin synthesis were compared for β-amyrin and ergosterol

352

production. Herein, the level of ergosterol was followed for monitoring the flux

353

branched from 2, 3-oxidosqualene towards sterol pathway (Figure 1), which is a

354

competing pathway against triterpenoid synthesis. As can be seen in Figure 6A,

355

different acetyl-CoA supply routes displayed different enhancing effects on β-amyrin

356

production and yield with the combinational PK/PTA and A-ALD pathway showing the

357

highest production of 9.3±0.2 mg/g DCW and yield of 2.2±0.2 mg/g glucose. On the

358

basis of this optimal acetyl-CoA supply route, further CIT2 deletion achieved a β-

359

amyrin production of 13.2 ± 0.1 mg/g DCW and yield of 3.5 ± 0.2 mg/g glucose.

360

Moreover, The optimal strain BA17 dramatically boosted β-amyrin production (330%

361

increase) but did not cause a significant increase in ergosterol level (27% increase). To

362

investigate whether the optimal β-amyrin production may result from the maximum

363

acetyl-CoA supply, the intracellular acetyl-CoA levels were measured in engineered 17



364

strains (Figure 6B). It is observed that all the acetyl-CoA levels were improved in

365

engineered strains but with different degrees. Notably, the maximum acetyl-CoA level

366

does not correspond to the optimal β-amyrin production. Therefore, it is speculated that,

367

besides precursor supply, an optimal matching between precursor supply and product

368

formation should also play a crucial role for product accumulation in microbial cell

369

factories.

370

The stoichiometry for converting glucose to β-amyrin at chemical limit is 7C6H12O6

371

→ C30H50O+12CO2+17H2O, which indicates that the theoretical maximum yield of β-

372

amyrin on glucose is 0.338 g/g glucose, consistent with the value reported earlier.42

373

With the native PDH bypass for acetyl-CoA supply, the high ATP cost (36 ATP,

374

equation 2, Table 1) is a major factor limiting the attainable yield of β-amyrin. Even

375

when all 18 NADH generated are used for reoxidation to provide ATP (P/O ratio

376

assumed to be 141), additional glucose is needed for respiratory dissimilation to generate

377

the remaining 18 ATP, which limits the attainable yield of β-amyrin to only 0.234 g/g

378

glucose (Table 2). When ATP-independent PDH or A-ALD route for acetyl-CoA

379

supply is used, the NADH/NADPH imbalance becomes a risk. Due to the absence of

380

ATP consumption, all 36 NADH generated (equation 3, Table 1) are reoxidized to

381

provide ATP for biomass formation, whereas the required 14 NADPH (equation 3,

382

Table 1) are generated from a large additional flux through pentose phosphate pathway,

383

both of which lead to an even lower attainable yield (0.232 g/g glucose) than PDH

384

bypass. The replacement of PDH with PDHm or combination of A-ALD and NADH-

385

HMGr alleviates the redox imbalance and significantly reduces or even eliminates the 18


Page 18 of 40

Page 19 of 40


386

NADPH demand from pentose phosphate pathway (equation 4 and 5), leading to much

387

higher attainable yields of β-amyrin (0.263 and 0.257 g/g glucose) than those of PDH

388

and A-ALD pathways. The absence of NADH generation makes PK/PTA pathway

389

achieve the highest attainable yield (0.275 g/g glucose) among all the single pathway-

390

coupled β-amyrin biosynthesis. Whereas the high ATP cost (24 ATP, equation 6, Table

391

1) restrains it from fitting perfectly with the same energy-intensive downstream

392

pathway for β-amyrin synthesis (equation 1, Table 1). Through combination of PK/PTA

393

and A-ALD pathways together with NADH-HMGr, the ATP consumption in PK/PTA

394

pathway is satisfied by the reoxidation of NADH generated in A-ALD pathway, leading

395

to an overall pathway of glucose conversion to β-amyrin with an even higher attainable

396

yield (0.296 g/g glucose), more balanced redox cofactors and a much lower ATP

397

consumption than any single pathway-coupled β-amyrin synthesis (Table 2, highlighted

398

in red). Therefore, this combinational acetyl-CoA supply pathway was demonstrated to

399

be optimal for β-amyrin synthesis and finally boosted β-amyrin production to its

400

maximum level. All the acetyl-CoA supply routes attempted in this study (except for

401

ACL pathway) were listed in Table 2 for comparisons of ATP, redox cofactors and

402

glucose consumption as well as attainable β-amyrin yield, which clearly indicate the

403

limitations and genetic manipulation targets for each acetyl-CoA supply pathway to

404

couple with β-amyrin production.

405

Fed-batch fermentation. To explore the highest performance of the optimal β-

406

amyrin-producing strain (BA17), fed-batch fermentations were performed in a 5 L

407

fermentator containing 3 L medium based on glucose or ethanol feeding. After 24 hours 19



408

fermentation, 6 g/L ethanol or 5 g/L glucose was supplemented at the fixed speed every

409

12 hours. Figure 7 displays the time courses of β-amyrin production in BA17 and SGib.

410

For SGib strain, the ethanol feed mode was more efficient than glucose fermentation,

411

achieving a β-amyrin production of 102.9±8.9 mg/L with a productivity of 0.86±0.07

412

mg/(L·h). While for BA17 strain, glucose fermentation was far more efficient than

413

ethanol fermentation for producing β-amyrin. As is shown in Figure 1, for PK/PTA

414

pathway, the substrate is xylulose-5-phosphate which is derived only from glucose.

415

While for A-ALD pathway, the substrate is acetaldehyde which can be derived from

416

either glucose by glycolytic reaction or ethanol by ADH2-catalyzed reaction. Therefore,

417

when using ethanol as the sole carbon source, only the A-ALD pathway can be utilized

418

for acetyl-CoA formation. While glucose utilization can activate the PK/PTA and A-

419

ALD pathways simultaneously and make them complement each other, leading to an

420

appropriate flux distribution under the optimal pathway configuration. Furthermore, it is

421

noted in Figure 7 that glucose utilization did not cause the increase of ergosterol

422

accumulation, demonstrating a product-dependent effect of the optimal acetyl-CoA

423

supply pathway, which directed flux to β-amyrin production instead of sterol pathway.

424

As a result, the engineered strain BA17 produced β-amyrin of 279.0±13.0 mg/L with a

425

productivity of 3.32±0.15 mg/(L·h), almost 2.9- and 3.3-fold higher than those of SGib

426

strain, and nearly two folds of the highest β-amyrin production ever reported earlier.6

427

Additionally, in glucose fed-batch fermentation mode, strain BA17 achieved a β-amyrin

428

yield of 4.65±0.22 mg/g glucose, 34% higher than that obtained in batch fermentation

429

without glucose feeding. 20


Page 20 of 40

Page 21 of 40


430

The aim of this research was to screen an appropriate acetyl-CoA supply pathway for

431

β-amyrin production. It has been demonstrated that a combinational acetyl-CoA supply

432

route was optimal when coupled with β-amyrin synthesis due to more balanced redox

433

cofactors, much lower energy consumption and glucose utilization as well as

434

significantly enhanced β-amyrin production (200% increase than the original β-amyrin-

435

producing strain). After disruption of an acetyl-CoA consuming pathway, the

436

engineered strain containing the combinational pathway achieved a final β-amyrin

437

production of 279.0 ± 13.0 mg/L in glucose fed-batch fermentation, which is the

438

maximum production ever reported. This research settled the problem that the mismatch

439

between precursor supply and β-amyrin biosynthesis limits β-amyrin production in

440

engineered S. cerevisiae, leading to a deeper understanding for design of efficient cell

441

factories.

442 443



ACKNOWLEDGMENT

444

This work was supported by the National Science Foundation of Distinguished

445

Young Scholars of China (21425624) and the National Natural Science Foundation of

446

China (Nos. 21476026 and 21706012).

447 448



449

Supporting Information

450 451

ASSOCIATED CONTENT

Plasmids and strains used in this study; primers applied for DNA assembly and qPCR; reactions involved in β-amyrin biosynthesis; and part of experimental results 21



452

(fermentation data of engineered strains, GC-MS analysis of β-amyrin and ergosterol,

453

batch cultivation results of SGib and BA16). This material is available free of charge on

454

the ACS Publications website.

455 456



457

[1] Lan, X.; Deng, K.; Zhao, J.; Chen, Y.; Xin, X.; Liu, Y.; Khan, I. A.; Yang, S.; Wang, T.; Xu,

458

Q., New Triterpenoid Saponins from Green Vegetable Soya Beans and Their Anti-Inflammatory

459

Activities. J. Agric. Food Chem. 2017, 65, 11065-11072.

460

[2] Pérez, A. J.; Pecio, Ł.; Kowalczyk, M.; Kontek, R.; Gajek, G.; Stopinsek, L.; Mirt, I.;

461

Oleszek, W.; Stochmal, A., Triterpenoid Components from Oak Heartwood (Quercus robur)

462

and Their Potential Health Benefits. J. Agric. Food Chem. 2017, 65, 4611.

463

[3] Zhao, Y.; Lv, B.; Feng, X.; Li, C., Perspective on Biotransformation and De Novo

464

Biosynthesis of Licorice Constituents. J. Agric. Food Chem. 2017, 65, 11147-11156.

465

[4] Seki, H.; Ohyama, K.; Sawai, S.; Mizutani, M.; Ohnishi, T.; Sudo, H.; Akashi, T.; Aoki, T.;

466

Saito, K.; Muranaka, T., Licorice beta-amyrin 11-oxidase, a cytochrome P450 with a key role in

467

the biosynthesis of the triterpene sweetener glycyrrhizin. Proc. Natl. Acad. Sci. U.S.A. 2008,

468

105, 14204.

469

[5] Zhou, J.; Du, G.; Chen, J., Novel fermentation processes for manufacturing plant natural

470

products. Curr. Opin. Biotechnol. 2014, 25, 17-23.

471

[6] Genlin, Z.; Qian, C.; Jingzhu, L.; Baiyang, L.; Jun, L.; Chun, L., Refactoring β‐amyrin

472

synthesis in Saccharomyces cerevisiae. AICHE J. 2015, 61, 3172-3179.

REFERENCES

22


Page 22 of 40

Page 23 of 40


473

[7] Wang, R.; Gu, X.; Yao, M.; Pan, C.; Liu, H.; Xiao, W.; Wang, Y.; Yuan, Y., Engineering of

474

β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces

475

cerevisiae. Frontiers of Chemical Science and Engineering 2017, 11, 89-99.

476

[8] Liu, D.; Li, B.; Liu, H.; Guo, X.; Yuan, Y., Profiling influences of gene overexpression on

477

heterologous resveratrol production in Saccharomyces cerevisiae. Frontiers of Chemical

478

Science and Engineering 2017, 11, 117-125.

479

[9] Ye, L.; Lv, X.; Yu, H., Assembly of biosynthetic pathways in Saccharomyces cerevisiae

480

using a marker recyclable integrative plasmid toolbox. Frontiers of Chemical Science and

481

Engineering 2017, 11, 126-132.

482

[10] Zhao, Y.; Fan, J.; Wang, C.; Feng, X.; Li, C., Enhancing oleanolic acid production in

483

engineered Saccharomyces cerevisiae. Bioresour. Technol. 2018, 257, 339-343.

484

[11] Takemura, M.; Tanaka, R.; Misawa, N., Pathway engineering for the production of β-

485

amyrin and cycloartenol in Escherichia coli—a method to biosynthesize plant-derived triterpene

486

skeletons in E. coli. Appl. Microbiol. Biotechnol. 2017, 101, 6615-6625.

487

[12] Dai, Z.; Wang, B.; Liu, Y.; Shi, M.; Wang, D.; Zhang, X.; Liu, T.; Huang, L.; Zhang, X.,

488

Producing aglycons of ginsenosides in bakers' yeast. Sci. Rep. 2014, 4, 3698-3698.

489

[13] Kirby, J.; Romanini, D. W.; Paradise, E. M.; Keasling, J. D., Engineering triterpene

490

production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua. FEBS J.

491

2008, 275, 1852-1859.

492

[14] Madsen, K. M.; Udatha, G. D.; Semba, S.; Otero, J. M.; Koetter, P.; Nielsen, J.; Ebizuka,

493

Y.; Kushiro, T.; Panagiotou, G., Linking genotype and phenotype of Saccharomyces cerevisiae

23



494

strains reveals metabolic engineering targets and leads to triterpene hyper-producers. PLoS One

495

2011, 6, e14763.

496

[15] Liu, Y.; Chen, H.; Wen, H.; Gao, Y.; Wang, L.; Liu, C., Enhancing the accumulation of

497

beta-amyrin in Saccharomyces cerevisiae by co-expression of Glycyrrhiza uralensis squalene

498

synthase 1 and beta-amyrin synthase genes. Yao Xue Xue Bao 2014, 49, 734-41.

499

[16] Shao, Z.; Zhao, H.; Zhao, H., DNA assembler, an in vivo genetic method for rapid

500

construction of biochemical pathways. Nucleic Acids Res. 2009, 37, e16.

501

[17] Vickers, C. E.; Williams, T. C.; Peng, B.; Cherry, J., Recent advances in synthetic biology

502

for engineering isoprenoid production in yeast. Curr. Opin. Chem. Biol. 2017, 40, 47-56.

503

[18] Shiba, Y.; Paradise, E. M.; Kirby, J.; Ro, D. K.; Keasling, J. D., Engineering of the

504

pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of

505

isoprenoids. Metab. Eng. 2007, 9, 160-168.

506

[19] Rodriguez, S.; Denby, C. M.; Van Vu, T.; Baidoo, E. E. K.; Wang, G.; Keasling, J. D.,

507

ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production

508

in Saccharomyces cerevisiae. Microbial Cell Factories 2016, 15, 48.

509

[20] Rodriguez, S.; Denby, C. M.; Vu, T. V.; Baidoo, E. E. K.; Wang, G.; Keasling, J. D., ATP

510

citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production

511

inSaccharomyces cerevisiae. Microbial Cell Factories 2016, 15, 48.

512

[21] Kozak, B. U.; Van, H. R.; Benjamin, K. R.; Wu, L.; Daran, J. M.; Pronk, J. T.; Van, A. M.,

513

Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways

514

for cytosolic acetyl-CoA synthesis. Metab. Eng. 2014, 21, 46-59.

24


Page 24 of 40

Page 25 of 40


515

[22] Song, J. Y.; Park, J. S.; Kang, C. D.; Cho, H. Y.; Yang, D.; Lee, S.; Cho, K. M.,

516

Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in

517

Saccharomyces cerevisiae. Metab. Eng. 2016, 35, 38-45.

518

[23] Lv, X.; Fan, W.; Zhou, P.; Ye, L.; Xie, W.; Xu, H.; Yu, H., Dual regulation of cytoplasmic

519

and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces

520

cerevisiae. Nature Communications 2016, 7, 12851.

521

[24] Tang, X.; Chen, W. N., Investigation of fatty acid accumulation in the engineered

522

Saccharomyces cerevisiae under nitrogen limited culture condition. Bioresour. Technol. 2014,

523

162, 200-206.

524

[25] Huang, Y.-Y.; Jian, X.-X.; Lv, Y.-B.; Nian, K.-Q.; Gao, Q.; Chen, J.; Wei, L.-J.; Hua, Q.,

525

Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered

526

acetyl-CoA metabolism. J. Biotechnol. 2018, 281, 106-114.

527

[26] Meadows, A. L.; Hawkins, K. M.; Tsegaye, Y.; Antipov, E.; Kim, Y.; Raetz, L.; Dahl, R.

528

H.; Tai, A.; Mahatdejkulmeadows, T.; Xu, L., Rewriting yeast central carbon metabolism for

529

industrial isoprenoid production. Nature 2016, 537, 694.

530

[27] Wallenius, J.; Maaheimo, H.; Eerikäinen, T., Carbon 13-Metabolic Flux Analysis derived

531

constraint-based metabolic modelling of Clostridium acetobutylicum in stressed chemostat

532

conditions. Bioresour. Technol. 2016, 219, 378-386.

533

[28] Bocanegra, J. A.; Scrutton, N. S.; Perham, R. N., Creation of an NADP-dependent pyruvate

534

dehydrogenase multienzyme complex by protein engineering. Biochemistry 1993, 32, 2737-

535

2740.

25



536

[29] Liu, W.; Bo, Z.; Jiang, R., Improving acetyl-CoA biosynthesis in Saccharomyces cerevisiae

537

via the overexpression of pantothenate kinase and PDH bypass. Biotechnology for Biofuels 2017,

538

10, 41.

539

[30] Lv, X.; Xie, W.; Lu, W.; Guo, F.; Gu, J.; Yu, H.; Ye, L., Enhanced isoprene biosynthesis in

540

Saccharomyces cerevisiae by engineering of the native acetyl-CoA and mevalonic acid

541

pathways with a push-pull-restrain strategy. J. Biotechnol. 2014, 186, 128-136.

542

[31] Lian, J.; Si, T.; Nair, N. U.; Zhao, H., Design and construction of acetyl-CoA

543

overproducing Saccharomyces cerevisiae strains. Metab. Eng. 2014, 24, 139.

544

[32] Celton, M.; Goelzer, A.; Camarasa, C.; Fromion, V.; Dequin, S., A constraint-based model

545

analysis of the metabolic consequences of increased NADPH oxidation in Saccharomyces

546

cerevisiae. 2012; Vol. 14, p 366-79.

547

[33] Park, J.; Choi, Y., Cofactor engineering in cyanobacteria to overcome imbalance between

548

NADPH and NADH: A mini review. Frontiers of Chemical Science and Engineering 2017, 11,

549

66-71.

550

[34] Cardenas, J.; Silva, N. A. D., Engineering cofactor and transport mechanisms in

551

Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis. Metab. Eng.

552

2016, 36, 80-89.

553

[35] Gold, N.; Gowen, C.; Lussier, F.-X.; C Cautha, S.; Mahadevan, R.; J J Martin, V.,

554

Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.

555

2015; Vol. 14, p 73.

26


Page 26 of 40

Page 27 of 40


556

[36] Grabowska, D.; Chelstowska, A., The ALD6 Gene Product Is Indispensable for Providing

557

NADPH in Yeast Cells Lacking Glucose-6-phosphate Dehydrogenase Activity. J. Biol. Chem.

558

2003, 278, 13984-13988.

559

[37] Kocharin, K.; Siewers, V.; Nielsen, J., Improved polyhydroxybutyrate production by

560

Saccharomyces cerevisiae through the use of the phosphoketolase pathway. Biotechnol. Bioeng.

561

2013, 110, 2216-2224.

562

[38] de Jong, B. W.; Shi, S.; Siewers, V.; Nielsen, J., Improved production of fatty acid ethyl

563

esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway

564

and expression of the heterologous phosphoketolase pathway. Microbial Cell Factories 2014,

565

13, 39.

566

[39] Rossum, H. M. V.; Kozak, B. U.; Pronk, J. T.; Maris, A. J. A. V., Engineering cytosolic

567

acetyl-coenzyme A supply in Saccharomyces cerevisiae : Pathway stoichiometry, free-energy

568

conservation and redox-cofactor balancing. Metab. Eng. 2016, 36, 99.

569

[40] Papini, M.; Nookaew, I.; Siewers, V.; Nielsen, J., Physiological characterization of

570

recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase

571

pathway: validation of activity through 13C-based metabolic flux analysis. Appl. Microbiol.

572

Biotechnol. 2012, 95, 1001-1010.

573

[41] Verduyn, C.; Stouthamer, A. H.; Scheffers, W. A.; Dijken, J. P. V., A theoretical

574

evaluation of growth yields of yeasts. Antonie Van Leeuwenhoek 1991, 59, 49-63.

575

[42] Paramasivan, K.; Mutturi, S., Progress in terpene synthesis strategies through engineering

576

of Saccharomyces cerevisiae. Crit. Rev. Biotechnol. 2017, 37, 1-16.

27



Page 28 of 40

Caption to Figures: Figure 1. Overview of various acetyl-CoA supply pathways coupling to β-amyrin production in engineered S. cerevisiae. Overexpressed genes in endogenous acetyl-CoA synthesis pathway are shown in red. The heterologous acetyl-CoA synthesis pathways introduced in this study are shown in pink, blue and green. Among them, PK, PTA and A-ALD constitute a combinational pathway for acetylCoA synthesis. The genes CIT2 and MLS1 involved in an acetyl-CoA consuming pathway are shown in yellow for deletion. The solid and dotted arrows indicate single and multiple steps, respectively. ADH2, alcohol dehydrogenase gene; ALD6, acetaldehyde dehydrogenase gene; ACS, acetyl-CoA synthase gene; ACL, ATPcitrate lyase gene from Aspergillus nidulans; PDH, genes of pyruvate dehydrogenase complex from E. coli; PDHm, modified NADP-dependent PDH gene; PK, phosphoketolase gene from Leuconostoc mesenteroides; PTA, phosphotransacetylase gene from Clostridium kluyveri; A-ALD, acetylating acetaldehyde dehydrogenase gene from E. coli; NADH-HMGr, NADH-dependent HMG-CoA reductase variant gene from Silicibacter pomeroyi;

CIT2, citrate

synthase gene; MLS1, malate synthase gene. Figure 2. β-Amyrin production (A) and acetic acid accumulation (B) in engineered strains overexpressing endogenous acetyl-CoA synthesis pathway. All the strains were cultured in YPD medium for 120 hours. t-tests were conducted to evaluate the statistical significance at a p ＜ 0.05. The asterisk shows a statistical

28


Page 29 of 40


significance between SGib and engineered strains. Error bars represent standard deviations of three replicates. Figure 3. β-Amyrin production in engineered strain BA05 harboring the heterologous ACL pathway for acetyl-CoA synthesis with and without citrate supplementation. The strains were cultured in YPD medium for 120 hours. t-tests were conducted to evaluate the statistical significance at a p＜0.05. The asterisk shows a statistical significance between SGib and the engineered strain. Error bars represent standard deviations of three replicates. Figure 4. β-Amyrin production (A), relative expression level of PDH(m) (B) and intracellular NADPH/NADP+ ratio (C) in engineered strains harboring the heterologous PDH(m) pathway for acetyl-CoA synthesis. All the strains were cultured in YPD medium for 120 hours. t-tests were conducted to evaluate the statistical significance at a p＜0.05. The asterisk shows a statistical significance between SGib and engineered strains. Error bars represent standard deviations of three replicates. Figure 5. β-Amyrin production (A) and intracellular NADH/NAD+ ratio (B) in engineered strains harboring the heterologous PK/PTA and A-ALD pathways for acetyl-CoA synthesis. All the strains were cultured in YPD medium for 120 hours. t-tests were conducted to evaluate the statistical significance at a p ＜ 0.05. The asterisk shows a statistical significance between SGib and engineered strains. Error bars represent standard deviations of three replicates.

29



Figure 6. β-Amyrin production and yield on glucose, ergosterol accumulation (A), and intracellular acetyl-CoA level (B) in engineered strains harboring different acetylCoA supply pathways. All the strains were cultured in YPD medium for 120 hours. Error bars represent standard deviations of three replicates. Figure 7. β-Amyrin production of engineered strain BA17 in 5 L bioreactor by ethanol (A) and glucose (B) fed-batch fermentations. β-Amyrin production of SGib was taken as a control. Samples from three biological replicates were taken every 12 h for product quantification. The error bars indicate standard deviations.

30


Page 30 of 40

Page 31 of 40


Table 1 Overall reaction stoichiometry for the formation of 1 mole of β-amyrin from acetyl-CoA or glucose by various acetyl-CoA supply pathways. No.

Pathway

Reaction stoichiometry

1

β-amyrin synthesis

18Acetyl-CoA+14(NADPH+H+)+18ATP+6H2O=C30H50O

from acetyl-CoA

(β-amyrin) +18CoA+14NADP++18(ADP+Pi)+6CO2

PDH bypass

9C6H12O6+18NAD++4NADP++36ATP+24H2O=C30H50O

2

(β-amyrin)+18(NADH+H+)+4(NADPH+H+)+ 36(ADP+Pi)+24CO2 3

PDH or A-ALD

9C6H12O6+14(NADPH+H+)+36NAD+=C30H50O (βamyrin)+ 14NADP++36(NADH+H+)+24CO2+12H2O

4

PDHm

9C6H12O6+18NAD++4NADP+=C30H50O (β-amyrin)+ 18(NADH+H+)+4(NADPH+H+)+24CO2 +12H2O

5

6

A-ALD+NADH-

9C6H12O6+2(NADPH+H+)+24NAD+=C30H50O (β-amyrin)

HMGr

+2NADP++ 24(NADH+H+)+24CO2+12H2O

PK/PTA

6C6H12O6+14(NADPH+H+)+24ATP=C30H50O (β-amyrin)+ 14NADP++24(ADP+Pi)+6CO2+6H2O

7

PK/PTA+ A-

7.8C6H12O6+2(NADPH+H+)+9.6NAD++9.6ATP=C30H50O

ALD+NADH-

(β-amyrin)+2NADP++9.6(NADH+H+) + 9.6(ADP+Pi)+

HMGr

16.8CO2+ 9.6H2O

(combinational pathway)

31



Page 32 of 40

Table 2 Comparison of various acetyl-CoA supply pathways for coupling with β-amyrin production in terms of ATP, redox cofactors (NADH/NADPH) and glucose consumption as well as attainable β-amyrin yield.a Minimum mols required per mol βamyrin Pathway ATP

NADH NADPH b

b

Maximum theoretical performance

Glucose c

Attainable yield (g/g glucose) d

Chemical limit

0

0

0

7

0.338

PDH bypass

18

0

-4

10.1

0.234

PDH or A-ALD

0

-36

14

10.2

0.232

PDHm

0

-18

-4

9.0

0.263

A-ALD+NADH-

0

-24

2

9.2

0.257

24

0

14

8.6

0.275

0

0

2

8.0

0.296

HMGr PK/PTA PK/PTA+AALD+NADHHMGr (combinational pathway) a

The demands for ATP, NADH/NADPH and glucose are theoretical values obtained

from reaction stoichiometries. The ATP requirement is preferentially met by oxidation of NADH generated. And additional ATP demand or surplus NADH not needed for ATP generation is provided. b The

negative values represent amount of NADH or NADPH generated.

32


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c

For calculation of glucose demand, additional glucose needed for respiratory

dissimilation to provide ATP and metabolism through pentose phosphate pathway to generate NADPH was also considered. The stoichiometries for glucose dissimilation to provide one NADPH and one ATP were assumed to be 0.061 and 0.086 respectively. d

Theoretical values calculated according to glucose demand.

Figure 1

33



Figure 2

34


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Figure 3

35



Figure 4

36


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Figure 5

37



Figure 6

38


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Figure 7

39



TOC Graphic

40


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Enhanced Î²-Amyrin Synthesis in Saccharomyces cerevisiae by

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