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Metabolic engineering for optimizing isobutanol

production in Synechocystis PCC 6803

Hao Xie

Degree project in Biology, Master of Science (2 years), 2018

Examensarbete i biologi 45 hp till magisterexamen, Uppsala universitet, 2018

Biology Education Center and Microbial Chemistry, Molecular Biomimetics, Dept of Chemistry-Ångström Laboratory

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Content

Abstract ... 5

Abbreviations... 7

Aim ... 9

Introduction ... 11

Cyanobacterial cell factory ... 11

Biofuels ... 12

Isobutanol... 13

2-keto acid pathway for renewable fuels production ... 13

2-keto acid pathway for sustainable isobutanol production in Synechocystis ... 13

Genetic tools for Synechocystis ... 16

Materials and methods ... 19

Chemicals and reagents ... 19

Cyanobacterium strain and growth condition ... 19

PCR amplification ... 19

Gel electrophoresis ... 20

DNA purification ... 20

Plasmid construction ... 20

E. coli transformation ... 21

Construction of transgenic Synechocystis strain ... 22

Optical density measurement ... 22

Isobutanol quantification assay ... 22

Crude protein extraction and SDS-PAGE/Western-immunoblot ... 23

Results ... 25

Enhancement of isobutanol production by optimizing cultivation conditions ... 25

Potential bottleneck identification of upstream genes in 2-keto acid pathway for isobutanol production ... 27

Confirmation of bottleneck existing in 2-keto acid pathway ... 30

Cumulative isobutanol production in strain pEEK2-ST ... 31

Discussion ... 33

Acknowledgement ... 39

Reference ... 41

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Abstract

The diminishing of fossil fuels and growing concerns towards climate change have intensified biofuel production from renewable resources. Recently, progresses are made in microbial production of biofuels. Among various biofuels, isobutanol is gaining an increasing attention due to its high energy content and suitable chemical and physical properties, enabling it to be a suitable substitution of fossil fuel. In this study, instead of using heterotrophic microorganisms, we performed metabolic engineering of Synechocystis PCC 6803 (Synechocystis) for isobutanol production under autotrophic condition. After introduced 2-keto acid pathway, Synechocystis is able to produce isobutanol when provided with water, carbon dioxide and solar energy. When cultivated in an optimal condition (50 µmol photons m-1s-2 and

adjusted pH to 7-8 with HCl), the engineered strain pEEK2-ST was able to produce 425 mg L -1 in-flask isobutanol titer and 911 mg L-1 cumulative isobutanol titer, respectively, in 46 days.

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Abbreviations

ADH Alcohol dehydrogenase

ALDH Aldehyde dehydrogenase

ALR Aldehyde reductase

AlsS Acetolactate synthase

BCD Bicistronic design

DCM Methylene dichloride

dH2O Sterilized demineralized water

DIBP Diisobutyl phthalate

dNTP Deoxyribonucleotide

E. coli Escherichia coli

GC Gas Chromatography

GOI Gene of interest

HCl Hydrogen chloride

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IlvC Acetohydroxy acid isomeroreductase

IlvD Dihydroxy-acid dehydratase KDC 2-keto acid decarboxylase α-KGD α-ketoglutarate decarboxylase Kivd α-ketoisovalerate decarboxylase LB Lysoeny broth

L. lactis Lacococcus lactis

3M1B 3-methyl-1-butanol

MIPS L-myo-inositol-1-phosphate synthase OD750 Optical density at 750 nm wavelength

PCR Polymerase chain reaction RBS Ribosome binding site

S. cerevisiae Saccharomyces cerevisiae

SDS-PAGE Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis

Synechocystis Synechocystis PCC 6803 Synechococcus Synechocystis PCC 7942

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Aim

The overall aim of this work presented in this thesis is optimizing isobutanol production in

Synechocystis, which includes the following perspectives:

1. Optimize cultivation conditions for isobutanol production in Synechocystis

2. Identify potential bottlenecks in 2-keto acid pathway for isobutanol production in

Synechocystis

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Introduction

Cyanobacterial cell factory

Cyanobacterium, also known as blue-green alga, is the oldest photosynthetic organism which originated around 2.6-3.5 billion years ago (Hedges et al. 2001). And it is widely accepted that the photosynthetic organelle, chloroplast, evolved from cyanobacterium, according to “Endosymbiotic theory” (Löffelhardt et al. 1994). Cyanobacteria represent complex lineages and have a diverse distribution on the earth, ranging from fresh water to ocean, from hot springs to alkaline lakes and from soils to rocks. Morphologically, they can be characterized as unicells or filaments, and they can occur singly or grouped as colonies (Burja et al. 2001) (Figure 1). Apart from its photosynthetic ability, cyanobacterium is special referring to its various modes of metabolism. When living environment changes form light to dark or anoxic condition, cyanobacterium is capable to change its energy generating metabolism, from photosynthesis to fermentation (Stal et al. 1997). Take Nostoc punctiforme for an example, with sufficient nitrogen source, it can grow phototrophically and the filament of it is consist of vegetative cells (Meeks et al. 2001). When exposed to environmental changes, the vegetative cell may differentiate into other cell types in response to stress conditions (Meeks et al. 2002). One of the differentiation process is the formation of dormant spore like cells called akinetes (Perez et al. 2016).

Due to its photosynthetic ability and simple structures, cyanobacterium is becoming a model microorganism for photosynthesis research and also being used as a cell factory for chemical production. Conventional cell factories, such as Escherichia coli (E. coli) and Saccharomyces

cerevisiae (S. cerevisiae), are known as heterotrophic microorganisms and rely on organic

carbon source for growth and chemical production. On the contrary, cyanobacterium can serve as a promising platform for sustainable production of chemicals as long as being provided with sunlight, carbon dioxide, water and minimum nutrients, eliminating the cost of foreign organic carbon source providing. Furthermore, cyanobacterium owns higher photosynthetic rate that can convert up to 3-9 % solar energy to biomass, compared to 0.25-3 % achieved by plants (Dismukes et al. 2008). Considering its many advantages, cyanobacterium is an attractive candidate as a cell factory for various chemical productions.

Figure 1: Different forms of cyanobacteria. (A) Synechocystis PCC 6803. (B) Synechococcus PCC 7002 (C)

Nostoc punctiforme ATCC 29133.

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manipulate genetic codes and control gene expression level in cyanobacteria so that the carbon flow can be redirected to desirable pathways to produce valuable chemicals, such as biofuels (Quintana et al. 2011), commodity chemicals (Zhou et al. 2012) and polyesters (Luengo et al. 2003).

Biofuels

Fuel produced through contemporary biological processes instead of geological process is called biofuel, presently mainly from plants, microorganisms, animals and wastes. Efforts aiming to develop sustainable biofuels have intensified due to the diminishing supply of fossil fuels and the environmental problems caused by overused traditional fossil fuels (Stephanopoulos 2007). Based on the origin material and production technology of biofuels, they are generally classified as the first, second, third and fourth generation biofuel (EASAC 2012). The first-generation biofuel is made from food crops grown on arable land, such as sugar, starch and vegetable oil which can be further converted into biodiesel and ethanol using yeast fermentation. The second-generation biofuel refers to biofuel made from various types of biomass. Biomass here indicates any source of organic carbon that renews rapidly as part of the carbon cycle. The third-generation biofuel is based on algae biomass production, which is under extensive research with the aim to improve the production titer with lower cost and develop efficient approaches to separate biofuels from non-fuel components. The fourth-generation biofuel is based on raw material that is inexhaustible, cheap and widely available. The production of the fourth-generation biofuel is achieved by either rewriting the metabolic pathways in photosynthetic organisms to produce photobiological solar fuels or building synthetic cell factory for production of solar fuels.

As a promising platform for producing the fourth-generation biofuel, cyanobacterium attracts an increasing attention, due to its fast growth rate, ability to fix carbon dioxide, genetic tractability (Nozzi et al. 2013). Cyanobacteria have been engineered successfully for the production of a number of different biofuels and biofuel-related compounds (Machado et al. 2012). The first attempt of biofuel production in cyanobacteria was achieved by introducing pyruvate decarboxylase and alcohol dehydrogenase into Synechococcus PCC 7942 (Synechococcus) for ethanol production (Deng et al. 1999). However, considering the disadvantages of ethanol, e.g. hygroscopicity and low energy density, efforts for biofuel production are shifted from ethanol production to other long-chain fuels production (Table 1).

Table 1: Cyanobacterial strains engineered for long-chain fuels production.

Compound Organism Titer Reference

Ethanol Synechocystis 5.5 g L-1 (Gao et al. 2012)

Acetone Synechocystis 36 mg L-1 (Zhou et al. 2012)

1-butanol Synechococcus 317 mg L-1 (Lan et al. 2013)

Isobutanol Synechococcus 450 mg L-1 (Atsumi et al. 2009)

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Isobutanol

Butanol (also called butyl alcohol) is a four-carbon alcohol with a formula of C4H9OH, which

is generally used as an industrial solvent in products, such as lacquer and enamels, and a promising candidate for transportation fuels as well. Isobutanol is one of the isomers of butanol, which is considered as an attractive alternative of gasoline due to its many advantages. Firstly, the energy density of isobutanol reaches 29.2 MJ L-1, about 90 % of gasoline (32.5 MJ L-1),

which is higher than that of both diesel and ethanol (Dürre 2007). Secondly, isobutanol can be used in pure form or blended with gasoline in any ratio. Thirdly, isobutanol has a relatively low vapor pressure and is not hygroscopic, making it easier and safer for transportation and storage. Lastly, isobutanol is more compatible with current infrastructure.

Apart from the application in fuel industry, isobutanol plays roles in other areas for various application as well. Specifically, isobutanol is commonly used as a precursor of derivative esters—isobutyl esters. Take diisobutyl phthalate (DIBP) for instance, it is used as plasticizers in plastics and rubbers. Isobutanol is also a good candidate of solvent for chemical extraction in production of organic compounds. Furthermore, isobutanol is widely served as an additive in many different aspects including paint additive, automotive polish additive and automotive paint cleaner additive. As an important chemical in industry, isobutanol can be dehydrated to isobutene, which is further for the production of C12 used in jet fuel or C16 as biodiesel. In

conclusion, it is isobutanol’s versatile applications distinguish it from other isoforms of butanol and make it deserve an increasing attention for renewable bio-production (Wang et al. 2016).

2-keto acid pathway for renewable fuels production

Amino acid biosynthesis pathway is one of the most widely distributed metabolic pathways existing in nature. The branched-chain amino acids, including Valine, Isoleucine, L-Leucine, Phenylalanine and L-Norvaline, are formed primarily from pyruvate and is further converted to larger 2-keto acid precursors (Figure 2). 2-keto acid is an organic compound that has a ketone group adjacent to a carboxylic acid group. On the other hand, Ehrlich pathway (Ehrlich 1907) is responsible for amino acid degradation in some bacteria and fungi. Amino acids are firstly deaminated to corresponding 2-keto acids, which are followed by decarboxylation into aldehyde catalyzed by 2-keto acid decarboxylase (KDC). Finally, aldehyde can be either reduced to alcohol by alcohol dehydrogenase/aldehyde reductase (ADH/ALR) or oxidized to acid by aldehyde dehydrogenase (ALDH) (Figure 3).

For both amino acid synthesis pathway and Ehrlich pathway, 2-keto acids are important intermediates, which can be further converted to a wide range of chemicals by decarboxylation, oxidation, reduction, chain elongation or condensation. What attracts most attention is the production of various alcohols generated from 2-keto acids by KDC and alcohol dehydrogenase (ADH) (Figure 4). 2-keto acid pathway has been successfully implemented into various model microorganisms including E. coli (Atsumi et al. 2008), S. cerevisiae (Avalos et

al. 2013) and Synechococcus (Atsumi et al. 2009) for advanced biofuels production.

2-keto acid pathway for sustainable isobutanol production in Synechocystis

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emission and the shortage of fossil fuels revitalize the novel sustainable production of biofuels. Sustainable isobutanol production was first observed in yeast through deamination of valine in small quantity (Dickinson et al. 1998). Only in the last decades that a big progress is made for isobutanol production from microorganisms and gradually isobutanol is regarded as a promising substitution of gasoline (Atsumi et al. 2008).

Figure 2: Branched-chain amino acid Biosynthesis. IlvIH, acetolactate synthase large and small subunits; IlvC, 2-hydroxy-3-keto-acid reductoisomerase; IlvD, dihydroxy-acid hydratase; AlsS, acetolactate synthase; IlvGM, acetolactate synthase II large and small subunits; LeuA, 2-isopropylmalate synthase; LeuB, 3-propylmalate dehydrogenase; LeuCD, isopropylmalate isomerase; CimA, citramalate synthase; IlvE, branched-chain amino acid aminotransferase; TyrB, tyrosine aminotransferase (Tashiro et al. 2014).

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Figure 3: Ehrlich amino acid degradation pathway. TA, transaminase; KDC, 2-keto acid decarboxylase; ADH, alcohol dehydrogenase; ALR, aldehyde reductase; ALDH, aldehyde dehydrogenase; ACL, acyl-CoA ligase; ATF, alcohol O-acyltransferase (Tashiro et al. 2014).

Figure 4: Conversion of 2-keto acids to long-chain alcohols (Atsumi et al. 2008).

Acetolactate synthase (AlsS), is an enzyme widely distributing in plants and microorganisms. It has a specific catalytic function in the first step of synthesis of branched-chain amino acids, converting two pyruvate molecular into an acetolactate molecule and carbon dioxide (Chipman et al. 1998).

2 pyruvate 2-acetolactate + CO2

Acetohydroxy acid isomeroreductase (IlvC), also known as ketol-acid reductoisomerase, belongs to the family of oxidoreductases.

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Dihydroxy-acid dehydratase (IlvD), belonging to the family of lyases, which cleaves carbon-oxygen bonds.

2,3-dihydroxyisovalerate 2-ketoisovalerate + H2O

α-ketoisovalerate decarboxylase (Kivd) belongs to broad-substrate-range KDC, which is common in plants, yeasts and fungi but is rare in bacteria (König 1998). Thus, Kivd is the key enzyme required to be introduced into non-native hosts for alcohol production. Kivd, from

Lacococcus lactis (L. lactis), is a thiamine diphosphate (ThDP)-dependent 2-keto acid

decarboxylase which can catalyze reactions with a wide range of substrates including α-ketoisovalerate, α-ketoisocaproate, α-ketomethylvalerate, and α-phenylpyruvate (Miao et al. 2018). The name ‘Kivd’ originates from its high affinity towards substrate α-ketoisovalerate (De La Plaza et al. 2004).

2-ketoisovalerate isobutyraldehyde + CO2

Alcohol dehydrogenase (Adh) belongs to the group of dehydrogenase enzymes, which occurs in many organisms and facilitates the inversion between alcohols and aldehydes. When catalyzing the formation of aldehydes form alcohol, there is a constant supply of NADP+ at the

same time.

Isobutyraldehyde + NADPH isobutanol + NADP+

Figure 5: Overview of the isobutanol and 3M1B producing pathway named 2-keto acid pathway examined in this study. kivd uses α-ketoisovalerate, which is an important intermediate for L-Valine and L-Leucine synthesis in

Synechocystis, to produce isobutanol and uses Ketoisocaproate to produce 3M1B.

Genetic tools for Synechocystis

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sequence of mRNA into amino acid sequence and polypeptide folds to form functional protein spontaneously (Anfinsen CB 1973).

As the first cyanobacterium with sequenced genome (Kaneko et al. 1996), Synechocystis has been used as a typical phototrophic host for both metabolic engineering and synthetic biology studies (Liu et al. 2018). However, genetic toolbox available for Synechocystis is still limited when comparing with other model microorganisms, such as E. coli and S. cerevisiae. Thus, with the aim to establish an optimal platform for biofuel production in cyanobacteria, it is necessary to develop “BioBricks” for cyanobacteria. “BioBricks” stands for the standardized DNA parts with common interface, which can be assembled for various aims in living organisms, including promoter, transcription terminator, RBS and other regulatory factors (Wang et al. 2012).

Promoter is the most well characterized genetic tool in Synechocystis, which can be generally divided into endogenous one and heterogenous one. Many endogenous promoters have been successfully applied in Synechocystis, including strong constitutive promoters, such as PpsbA2, PrbcL, PcpcB (Huang et al. 2010; Zhou et al. 2014) and inducible promoters, such as PnrsB, PcoaT and PpetE (Englund et al. 2016). Apart from native promoters, there are limited foreign promoters are characterized to be used in Synechocystis, due to the large difference of holopolymerases in Synechocystis compared to other microorganisms. Ptac/Ptrc promoter, a strong promoter widely used in E. coli, is successfully introduced and applied in Synechocystis to show strong expression of certain genes (Huang et al. 2010). PL03 (TetR regulated) promoter is another foreign promoter functioning in Synechocystis, but it does not perform as well as in

E.coli (Huang et al. 2013). As for the selection of RBS, apart from RBS originates from psbA2

and rbcL genes, a synthetic RBS named RBS* is a better candidate due to its perfectly complementary sequence to the ribosomal anti-Shine-Dalgarno sequence, ensuring its strong initiation of translation in Synechocystis (Heidorn et al. 2011).

Gene expression in Synechocystis can be achieved by either integrating genes into the genome of Synechocystis using integrative vector or conjugating self-replicative vector containing the gene of interest (GOI) into Synechocystis. In this study, a self-replicative plasmid named pEEK2 (Huang et al. 2010, Rui et al. 2017) is used for gene expression in Synechocystis.

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Materials and methods

Chemicals and reagents

All chemicals are analytical pure (Sigma or Merck). All chemicals and reagents used in this project are listed in Appendix 1. The recipes for medium or buffer are listed in Appendix 2 and Appendix 3.

Cyanobacterium strain and growth condition

Synechocystis cell (seed culture) was maintained in BG11 medium at 30 °C under continuous

light (at a light intensity of 50 µmol photons m-1s-2) in shake flasks or on agar plates.

PCR amplification

PCR for fragments

The PCR amplification of different promoters, gene fragments, etc. were achieved by Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase. The PCR reactions were performed in optimized condition as stated in Table 2 (Pipetting instructions) and Table 3 (Cycling instructions), according to the protocol provided by Thermo Scientific Company.

Table 2: Pipetting instructions

Components Amount dH2O Add to 50 µL 5x Phusion HF Buffer 10 µL 10 mM dNTPs 1 µL Forward Primer (10 µM) 2.5 µL Reverse Primer (10 µM) 2.5 µL Template DNA 1 pg-10 ng

Phusion Hot Start II DNA Polymerase 0.5 µL

Table 3: Cycling instructions

Step Temperature, °C Time Number of cycles Initial denaturation 98 30 s 1

Denaturation 98 10 s

Annealing the lower Tm of primers 30 s 30 Extension 72 15-30 s kb-1

Final extension 72 5-10 min 1

Colony PCR

Colony PCR was performed with Dream Taq DNA Polymerase, for both E. coli stain and

Synechocystis strain. The PCR reaction was performed in optimized condition as stated in

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Table 4: Pipetting instructions

Components Amount dH2O 14.5 µL

10x Dream Taq Buffer 2 µL 10 mM dNTPs 0.4 µL Forward Primer (10 µM) 1 µL Reverse Primer (10 µM) 1 µL Template DNA 1 µL

Phusion Hot Start II DNA Polymerase 0.1 µL

Table 5: Cycling instructions

Step Temperature, °C Time Number of cycles

Initial denaturation 95 3 min 1 Denaturation 95 30 s

Annealing 56 30 s 25 Extension 72 1 min kb-1

Final extension 72 5-10 min 1

Gel electrophoresis

PCR amplification products were analyzed on the 1 % agarose gel. The 1 % agarose gel was made by dissolving 0.2 g agarose into 20 mL 1x TAE buffer containing 0.16 % Thiazole Orange for DNA detection. The running Buffer used is 1x TAE buffer. The stock solution of TAE buffer was 50 times concentrated (Appendix 3). Electrophoresis was performed under 100 V in 1x TAE buffer for 20 min and then visualized under UV-light.

DNA purification

The PCR product purification was performed with Thermo Scientific GeneJET PCR Purification Kit. For purification of certain DNA fragment in digestion products, gel purification was conducted with ZymocleanTM Gel DNA Recovery Kit.

Plasmid construction

Plasmid constructions were based on shuttle vector called “pEEK2” (Figure 7). “pEEK2”, a self-replicative plasmid, contains a gene functioning as kanamycin resistance, a series of restriction sites and a series of genes functioning for conjugation.

Fast digestion

Digestion reactions of PCR products and plasmids were performed by Thermo Scientific FastDigest restriction enzymes, with optimized reaction components and amounts based on protocol provided by Thermo Scientific Company (Table 6). After mixing gently and spinning down, the mixtures were incubated at 37 °C in water thermostat for 30 min. After digestion reactions, the digestion products were purified with DNA Clean & ConcentratorTM-5. The final

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Table 6: Protocol for Fast Digestion of different DNA

Components Plasmid DNA PCR product Water, nuclease-free add to 40 µL add to 90 µL 10x FastDigest Buffer 4 µL 6 µL DNA 2 µg 600 ng FastDigest Enzyme 2 µl 3 µl Total volume 40 µl 90 µl

Quick ligation

Ligation was performed by Quick LigationTM Kit from BioLabs. 50 ng of vector and 3-fold

molar excess of insert were mixed and the total volume was adjusted to 10 µL with dH2O,

blended with 10 µL of 2x Quick Ligation Buffer and 1 µL Quick T4 DNA Ligase. The ligation mixture was incubated at room temperature for 1 hour before the next transformation step.

E. coli transformation

Competent cell preparation

The E. coli strain used for cloning were DH5a and DH5aZ1. The competent cells were prepared by CCMB method. Inoculated seed culture in 5 mL LB culture and grew overnight in 37 °C. The second day, inoculated a new large volume culture with the overnight cultivated seed culture in 250 mL fresh LB medium, grew the culture till OD600=0.3. Gently re-suspend

in 80 mL ice cold CCMB buffer after centrifugation (3000x rcf at 4 °C for 10 min) and then incubated on ice for 20 min. Repeat the centrifuge again and re-suspend in 10 mL of ice cold CCMB buffer. After incubating for another 20 min, the cells were divided into Eppendorf tubes (100 µL for each) and stored in -80 °C freezer.

Transformation

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which was incubated in 37 °C incubator overnight. Picked up colonies the next day for screen check via colony PCR.

Plasmid preparation and sequencing

After screening, positive colonies were cultivated in LB medium overnight in 37 °C incubator. Plasmid extraction is performed with GeneJET GelTM Plasmid Miniprep Kit. The plasmid then

was sent to Eurofins Genomics for sequencing to confirm the success of cloning.

Construction of transgenic Synechocystis strain

Conjugation

E. coli cargo cells and E. coli HB101 helper cells (containing plasmid pRL443-AmpR) were

cultivated overnight at 37 °C. Both E. coli cells were centrifuged at 3000x rpm for 10 min at room temperature and re-suspend in LB without antibiotic. Mix cargo cells (100 µL), helper cells (100 µL) with condensed wide type Synechocystis cells (100 µL) in Eppendorf tube and incubate for 1.5 h at 30 °C under low light. The mixture was then spread on a filter on a BG11 agar plate without antibiotic for about 2 days at 30 °C. For colony selection, the filter was transferred to BG11 agar plate containing antibiotic.

Screen of positive colonies

After single colony appearing on BG11 agar plates, colony PCR was performed to select positive colony with accurate plasmid construct.

Optical density measurement

Synechocystis optical density was measured at 750 nm (OD750) using a micro-plate reader (HIDEX, Plate Chameleon), in 96-well plates with 200 µL culture for each well.

Isobutanol quantification assay

Isobutnol extraction

2 mL culture was centrifuged at 10000x rpm for 10 min. Then 1306 µL supernatant was transferred into 15 mL screw tube, mixed with 45 µL 3000 mg L-1 1-pentanol internal standard

and 450 µL dichloromethane (DCM). The mixture was shaken on Multi-Tube Vortexer VX-2500 (VWR) at maximum speed for 5 min and next centrifuged at 5000x rpm for 10 min. Transfer up to 400 µL DCM phase into 1.5 mL clear glass gas chromatography (GC) vials (VWR).

Isobutanol quantification

The extracted sample was analyzed on a PerkinElmer GC 580 system equipped with a flame ionization detector and an Elite-WAX Polyethylene Glycol Series Capillary column, 30 m X 0.25 mm X 0.25 µm (PerkinElmer). Nitrogen was used as carrier gas, with 10 mL min-1 flow

rate. The starting oven temperature was 50 °C and then raised to 100 °C with a rate of 10 °C min-1, followed by a rise to 180 °C with a rate of 20 °C min-1. The gas chromatography (GC)

results were analyzed using TotalChrom Navigator version 6.3.2.

In-flask and cumulative isobutanol production

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8 % of isobutanol was removed every two days due to nutrition replenishment and isobutanol measurement. Thus, cumulative isobutanol production take that part isobutanol into account for productivity calculation.

Crude protein extraction and SDS-PAGE/Western-immunoblot

Crude protein extraction

Proteins were extracted from day 4 cultures. 2 mL culture was harvested by centrifuging at 5000x rpm for 10 min and the pellet was washed by 2 mL PBS buffer. Repeat the same centrifugation and re-suspend the pellet in 200 µL PBS, which was then frozen in -80 °C for 10 min, followed by heating at 37 °C for 10 min. After that, 2 µL Protease Arrest (GBioscience) and glass beads (Sigma-Aldrich) were mixed with the cells. Break the cells by using Precellys-24 Beadbeater (Bertin Instruments), program 3 x 30 s. Centrifugation (5000x rpm, 4 °C, 10 min) then was performed twice to collect green supernatant as crude soluble protein. The protein concentration was determined by the DC protein assay (Bio-Rad).

SDS-PAGE/Western-immunoblot

With protein concentration determined, 5 µg protein of each sample in each well was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using Mini-PROTEAN TGXTM gels (Bio-Rad), and transferred to PVDF membrane (Bio-Rad). The

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Results

Enhancement of isobutanol production by optimizing cultivation conditions

Cultivation conditions are important for growth pattern of cyanobacteria, especially affect the cultivation period of cyanobacteria maintaining in stationary stage. Growth pattern is further related with production rate and final titer of isobutanol produced in Synechocystis.

Synechocystis seed culture was generally cultivated in BG11 medium, at 30 °C under

continuous light (light intensity of 50 µmol photons m-1s-2). Considering light intensity and

medium pH condition are two important factors concerning growth pattern of Synechocystis, we hypothesized that they are playing a big role for isobutanol production in Synechocystis. Totally four different engineering strains, named pEEK2-kivd, pEEK2-VI, pEEK2-ST and pEEK2-SV, were used for exploring the optimized cultivation condition for isobutation production in Synechocystic (Figure 10A). Due to the similar effects of light and pH to four different strains, the results of the engineered Synechocystis strain pEEK2-ST will be showed below.

Light intensity

pEEK2-ST is one of the engineered strains which shows the highest isobutanol production. Three different light intensities were applied on pEEK2-ST. Light intensity makes a big difference for growth patterns (Figure 8A). When comparing with the growth of pEEK2-ST under 50 µmol photons m-1s-2, the lower (15 µmol photons m-1s-2) intensity light gave longer

living period and the cell maintained in growth phase for 9 days with a relative slow growth rate. On the other hand, strain pEEK2-ST grown under relative high (100 µmol photons m-1s -2) light intensity grew faster and accordingly the culture bleached faster.

Figure 8: Growth and isobutanol production of engineered Synechocystis strain pEEK2-ST under different light intensities: 15 µmol photons m-1s-2, 50 µmol photons m-1s-2, 100 µmol photons m-1s-2. (A) Growth curves of

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In consist with growth pattern, the isobutanol production pattern showed similar tendency. Strain pEEK2-ST cultivated under 50 µmol photons m-1s-2 reached the highest isobutanol titer

52 mg L-1 at day 8 (Figure 8B). Besides, at day 10, strain pEEK2-ST under 50 µmol photons

m-1s-2 produced 21 mg L-1OD-1 isobutanol (Figure 8C), which is almost two folds higher than

strain under other light intensities. In conclusion, light intensity of 50 µmol photons m-1s-2 is

the most suitable light condition for isobutanol production in our engineered Synechocystis strain.

pH condition

Synechocystis culture can reach around pH = 10 after growing two days, which is probably the

major reason limiting growth of synechocystis, because the optimal pH for Synechocystis’s growth is around 7-8 (Bano et al. 2004). Two different chemicals, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and hydrogen chloride (HCl), were used respectively for adjusting Synechocystis culture to pH between 7 and 8. The growth curve (Figure 9A) indicates strain pEEK2-ST with adjusted pH could maintain in growth phase for a longer period before stepping into stationary phase, and OD750 reached 5-6, which was much higher than the

culture without pH adjustment (OD750=3).

Figure 9: Growth and isobutanol production of engineered Synechocystis strain pEEK2- ST under different pH conditions: culture without buffering by HEPES or adjustment by HCl, culture buffered by HEPES, culture adjusted by HCl. (A) Growth curves of engineered strain pEEK2-ST under different pH conditions. Optical density was measured every day by micro-plate reader. (B) Isobutanol titer of engineered strain pEEK2-ST under different pH conditions at Day 2, Day 4, Day 6, Day 8 and Day 10. (C) Isobutanol production of engineered strain pEEK2-ST under different pH conditions at Day 2, Day 4, Day 6, Day 8 and Day 10. All the results represent the mean of three biological replicates and three technical replicates. Error bars represent the standard deviation.

On the other hand, the total isobutanol accumulation of strain pEEK2-ST reached 115 mg L-1

and 194 mg L-1 till day 10, when pH is adjusted to 7-8 with HEPES and HCl, respectively. In

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till day 8, and on day 10 the total isobutanol titer drops to 44 mg L-1 (Figure 9B). It was expected

that after day 10, the culture adjusted with HCl could reach a higher isobutanol titer, whereas the strain pEEK2-ST without adjusted pH was going to die quickly. Furthermore, when comparing the culture with adjusted pH by different chemicals, HCl shows a better effect on controlling pH, resulting in better growth pattern and higher isobutanol production in

Synechocystis.

With the aim to analysis the efficiency of isobutanol production, there is another unit for measuring isobutanol production: mg L-1OD-1. It is reasonable that strain pEEK2-ST with

adjusted pH by HCl behaves the best that each single Synechocystis cell produced the most isobutanol, which reaches 42 mg L-1OD-1 at day 10 (Figure 9C). Meanwhile it is unexpected

to observe the similar production from the culture without adjusted pH and the culture with adjusted pH by HEPES, 21 mg L-1OD-1 and 19 mg L-1OD-1 respectively at day 10. In summary, Synechocystis strain with adjusted pH by HCl can achieve the highest isobutanol production.

Potential bottleneck identification of upstream genes in 2-keto acid

pathway for isobutanol production

Kivd has been previously identified as one bottleneck for isobutanol production in

Synechocystis (Miao et al. 2018). Considering 2-keto acid pathway involves a series of

different enzymes converting pyruvate into isobutanol (Figure 5), including AlsS, IlvC, IlvD, Kivd, Adh, there are probably some other bottlenecks existing in the pathway. Overexpressing endogenous Adh enzyme (encoded by slr1192) was helpless for improving isobutanol production in the recent stage, indicating the endogenous Adh in Synechocystis was not saturated in catalyzing the formation of isobutanol from isobutyraldehyde (Miao et al. 2017). Therefore, identifying other potential bottlenecks from three upstream enzymes was the task of this part work. We selected four different acetolactate synthase encoded genes (alsS from

L.lactis, sll0065, slr2088, sll1981 from Synechocystis), two different acetohydroxy acid

isomeroreductase encoded genes (ilvC from E. coli, sll1363 from Synechocystis), two different dihydroxy-acid dehydratase encoded genes (ilvD from E. coli, slr0452 from Synechocystis). Noted that sll0065 was identified to encode the small subunit of the native AlsS and slr2088,

sll1981 were identified to encode two large subunits of the native AlsS, respectively. One of

the upstream genes together with the best performing engineered kivd gene ST were assembled into the self-replicative vector pEEK2 (Figure 10B), which is a broad host range self-replicative vector based on the previous reported pPWQAK1 (Huang et al. 2010). Meanwhile, empty pEEK2 vector and pEEK2 vector containing ST were conjugated into Synechocystis as control strains. For each construct, a strong constitutive promoter Ptrccore with a BCD construct

(Mutalik et al. 2013) and RBS* (Heidorn et al. 2011) were used for driving gene transcription and translation, respectively.

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strain with empty pEEK2 grew a bit better than other strains and strain pEEK2-ST grew a bit worse than other strains in general (Figure 11A).

Figure 10: Schematic presentation of all engineered Synechocystis strains. Original kivd, encodes α-ketoisovalerate decarboxylase (L. lactis); kivd (ST), single replacement of Ser 286 to threonine; kivd (VI), single replacement of Val 461 to isoleucine; kivd (SV), double replacements of Ser 286 to threonine and Val 461 to isoleucine; alsS, encodes acetolactate synthase (B. subtilis); sll0065, encodes acetolactate synthase small subunit (Synechocystis); slr2088, encodes acetolactate synthase large subunit (Synechocystis); sll1981, encodes acetolactate synthase large subunit (Synechocystis); ilvC, encodes acetohydroxy acid isomeroreductase (E. coli);

sll1363, encodes acetohydroxy acid isomeroreductase (Synechocystis); ilvD, encodes dihydroxy acid dehydratase

(E. coli); slr0452, encodes dihydroxy acid dehydratase (Synechocystis). (A) Constructs with different versions of

kivd driven by Ptrccore-BCD on self-replicative vector. (B) Constructs with kivd (ST) and one of the upstream

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Isobutanol production was measured every two days and data below showed isobutanol production every four days (Figure 11B). For each strain, most isobutanol was accumulating during growth phase. Surprisingly, Synechocystis strain with only ST overexpressed (pEEK2-ST) showed the highest isobutanol titer on each measurement day and reached 312 mg L-1 on

day 20, whereas pEEK2-ST-2088, pEEK2-ST-ilvC, pEEK2-ST-1363, pEEK2-ST-ilvD, pEEK2-ST-0452 showed slightly lower isobutanol titer compared with pEEK2-ST. Combining isobutanol production (Figure 11B, 11C) of each strain with relative ST expression level (Figure 11G), it is concluded that the more ST expressed, the more isobutanol was produced. As for pEEK2-ST-0065, its relatively lower isobutanol titer is probably due to the low expression level of both sll0065 and ST. It is interesting to observe the totally different isobutanol production performance of strain pEEK2-ST-1981, which showed much lower isobutanol production of 64 mg L-1 on day 20 (Figure 11B), when comparing with all other

engineered strains.

When introducing 2-keto acid pathway into Synechocystis, apart from the target product isobutanol produced, there is one byproduct named 3M1B produced as well. From intermediate 2-ketoisovalerate, it can be catalyzed by Kivd and Adh for isobutanol production or by LeuABCD, Kivd and Adh for 3M1B production. Because there is no overexpression of

LeuABCD in all engineered strains, in theory there should be no difference of the ratio of

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Figure 11: Comparison of growth, isobutanol production and the molar ratio of isobutanol and 3M1B in engineered Synechocystis strains pEEK2, pEEK2-ST, pEEK2-ST-0065, pEEK2-ST-2088, pEEK2-ST-1981, pEEK2-ST-ilvC, pEEK2-ST-1363, pEEK2-ST-ilvD, pEEK2-ST-0452. (A) Growth curves during 22 days of cultivation. Optical density was measured every day using micro-plate reader. (B) Isobutanol titer at Day 4, Day 8, Day 12, Day 16 and Day 20 from different engineered strains. (C) Isobutanol production at Day 20 from different engineered strains. (D) Molar ratio of isobutanol and 3M1B produced in different engineered strains, calculated based on the isobutanol titer on Day 20. (E) SDS-PAGE results for different engineered strains. It consists of 3 SDS-PAGEs from 3 different colonies. Each lane represents the result from a single engineered strain. Strain pEEK2 is negative control and strain pEEK2-ST is positive control. (F) Western-immunoblot results for different engineered strains. The first row of Strep-tag Western-immunoblot showed the expression of ST and the second row showed the expression of sll1981, sll0065, slr2088, sll1363, slr0452, ilvC, ilvD. (G) Relative expression level of ST in engineered strains. Results are the mean of three biological replicates and three technical replicates. Error bars represent standard deviation.

Confirmation of bottleneck existing in 2-keto acid pathway

Apart from overexpressing specific genes in Synechocystis cell for identifying potential bottlenecks in 2-keto acid pathway for isobutanol production, exogenous addition of substrates is another approach for characterizing potential bottlenecks and exploring the maximum enzyme activity. Since strain pEEK2-ST still showed the highest isobutanol production among all the engineered strains, it was used in this further investigation of bottlenecks. External 2,3-dihydroxyisovalerate (100 mg L-1 and 200 mg L-1) and pyruvate (1 g L-1 and 2 g L-1), substrates

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measured after 48 hour. Unfortunately, there was no difference of isobutanol production between culture with and without the addition of external substrates (Figure 12), which indicates that some enzymes have achieved the highest catalytic activity, which further confirms the existing of the potential bottlenecks in 2-keto acid pathway for isobutanol production in Synechocystis.

Figure 12: Isobutanol production of strain pEEK2-ST after addition of 2,3-dihydroxyisovalerate (100 mg L-1 and

200 mg L-1) and pyruvate (1 g L-1 and 2 g L-1). The cultures were inoculated as OD750=0.1 and cultivated for 2

days until OD750=1.5. Then 10 mL culture was taken from each flask and resuspended with 5 mL fresh BG11

medium. Then 2,3-dihydroxyisovalerate and pyruvate were added into couture to reach different final concentrations. After 48 h cultivation, isobutanol production was measured. A culture without addition of 2,3-dihydroxyisovalerate and pyruvate was used as a control. Results are the mean of three biological replicates and three technical replicates. Error bars represent standard deviation.

Cumulative isobutanol production in strain pEEK2-ST

From previous results, strain pEEK2-ST showed the highest isobutanol production capacity, therefore it was cultivated continuously for 46 days until no further isobutanl was produced, and the culture was buffered with HCl every day to control pH within the range of 7-8. With adjusted pH, the culture maintained at stationary stage for a long period (Figure 13A). The in-flask isobutanol titer reached 435 mg L-1 and the cumulative isobutanol titer accumulated to

911 mg L-1 in 46 days (Figure 13B). Even though the in-flask isobutanol titer decrease from

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Figure 13: In-flask and cumulative isobutanol titer for pEEK2-ST. (A) Growth curve during 46 days of cultivation. Optical density was measured every day using micro-plate reader. (B) In-flask and cumulative isobutanol titer of strain pEEK2-ST during 46 days of cultivation. In-flask isobutanol production is the data we directly required form GC measurement, while cumulative isobutanol production takes into account the dilutions made to the

Synechocystis culture by feeding. For detailed description, see the materials and methods section. Results are the

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Discussion

Synechocystis has been successfully used to produce several alcohols, including ethanol (Gao

et al. 2012), 1-butanol (Anfelt et al. 2015), isobutanol (Varman et al. 2013). In this study, we focused on isobutanol production in Synechocystis. Specifically, a combination of genetic tools and cultivation condition modification are applied for improvement of isobutanol productivity. Finally 435 mg L-1 in-flask titer and 911 mg L-1 cumulative titer of isobutanol were achieved

in Synechocystis, which break the record of the highest isobutanol titer in Synechocistis (Varman et al. 2013). From their results, the engineered Synechocystis strain can only accumulate 90 mg L-1 isobutanol titer from 50 mM bicarbonate in a gas-tight shaking flask and

when supplied with glucose, the isobutanol titer is moderately promoted to 114 mg L-1 (Varman

et al. 2013).

Synechocystis is adaptive to various environment regarding different light intensities and pH

conditions. However, for the aim of higher isobutanol production in Synechocystis, it is vital to provide an optimal cultivation condition which is most suitable for production efficiency. Lower light intensity is a safe choice for Synechocystis of biofuel production because in lower light intensity, Synechocystis can survive longer and the isobitanol production is increasing stably, though with a relative slower synthesis rate (Figure 8B). Due to the adaptation of

Synechocystis strains used in this study to the light condition in the lab, 100 µmol photons m -1s-2 is considered to be a high light intensity and it takes time for them to get used to it. When

exposed to 100 µmol photons m-1s-2, it is impossible for cell to absorb additional light after Synechocystis cell reaches its maximum photosynthetic capacity. Extra light probably causes

light stress towards Synechocystis cell, affecting both growth pattern and metabolism pattern of cells. According to the results above, light intensity of 50 µmol photons m-1s-2 is the best

suitable light in our lab for efficient isobutanol production in Synechocystis.

HEPES and HCl are two different chemicals used in this study for adjusting the pH of

Synechocystis culture. HEPES buffering agent is widely used for cell culture, due to its better

performance in maintaining physiological pH despite changes in carbon dioxide concentrations. Unfortunately, since we used quite high concentration of NaHCO3 in the culture, HEPES was

not working very well at controlling the pH in this case. Besides, using HEPES to buffer the pH effects the culture volume maintaining. Specifically, each time 2 mL of HEPES, even more than 2 mL, was added into the closed tissue flask, which makes it difficult and complicated for both maintaining the culture volume at 25 mL constantly and the calculation of final isobutanol titer. Furthermore, when we added HEPES, the salinity in the culture was increased as well, which could be a stress for the cells. On the other hand, HCl is a better choice in our system, the adjusted pH condition with HCl not only could make cell live for a longer period and also the final isobutanol titer was almost four times more than strain without adjusted pH condition (Figure 9B). There are a few possible reasons contributing to much higher isobutanol production for the strain with adjusted pH by HCl. Firstly, HCl is efficient in controlling pH of

Synechocystis culture, providing a comfortable environment for cell growth. A pleasant living

condition enables cell maintain its metabolic activity actively, providing enough intermediates, especially pyruvate, for isobutanol production. Secondly, the availability of HCO3- is affected

by pH significantly. Without adjustment, the pH of Synechocystis culture can reach 10-11 after one day cultivation and in this alkalic condition, the majority of HCO3- is changed to CO32-,

which cannot be used by Synechocystis for carbon metabolism. On the contrary, when

Synechocystis culture with adjusted pH by HCl, the majority of carbon is existing as HCO3

-and CO2 in the medium. And so far, there are five different carbon transporters have been

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strain with adjusted pH condition is believed to have the highest carbon source assimilation efficiency, promising more carbon source can flow into 2-keto acid pathway for isobutanol production. Apart from isobutanol titer, these factors contribute to a higher isobutanol productivity of each single cell as well (Figure 9C).

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Figure 14:Schematic presentation of engineered Synechocystis strains. Constructs with kivd (ST) and one of the upstream genes overexpressed on self-replicative vector named pSNK01. kivd (ST), single replacement of Ser 286 to threonine; sll0065, encodes acetolactate synthase small subunit (Synechocystis); slr2088, encodes acetolactate synthase large subunit (Synechocystis); sll1981, encodes acetolactate synthase large subunit (Synechocystis); ilvC, encodes acetohydroxy acid isomeroreductase (E. coli); sll1363, encodes acetohydroxy acid isomeroreductase (Synechocystis); ilvD, encodes dihydroxy acid dehydratase (E. coli); slr0452, encodes dihydroxy acid dehydratase (Synechocystis). All α-ketoisovalerate decarboxylase was Strep-tagged at the N-terminal. All upstream enzymes were Flag-tagged at the N-terminal.

Something interesting and unexpected is the isobutanol production of strain pEEK2-ST-1981 (Figure 11B), which was much lower than all the other engineered strains. sll1981 encoded protein was purified (Wang et al. 2017) and characterized with versatile functions in

Synechocystis. The protein encoded by sll1981 has been reported to show acetolactate synthase

(AlsS) activity, L-myo-inositol-1-phosphate synthase (MIPS) activity (Chatterjee et al. 2006) and α-ketoglutarate decarboxylase (α-KGD) activity (Wang et al. 2017). And it was proposed to have a much higher activity as α-KGD than as ALS. However, the ALS activity of sll1981 encoded enzyme is essential for isobutanol production in 2-keto acid pathway to catalyze the formation of 2-acetolactate from pyruvate. On the other hand, due to the important role of α-KGD in TCA cycle of cyanobacteria (Zhang et al. 2018), it is probably that sll1981 encoded enzyme serves as a key enzyme in completing the TCA cycle in Synechocystis (Wang et al. 2017). Combining the facts above, we hypothesized that in the strain overexpressing sll1981, due to the higher α-KGD activity of sll1981 encoded enzyme for TCA cycle in Synechocystis, pyruvate, a starting substrate for isobutanol production, flows into TCA cycle instead of 2-keto acid pathway, resulting in less isobutanol production.

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Salmonella enterica and Corynebacterium glutamicum (Umbarger 1996). AlsS in those

microorganisms consists of a small subunit and a large subunit, which serve totally different functions. The large subunit is responsible for the catalytic function, while the small subunit mediates the feedback inhibition of the activity of the large subunit (Umbarger 1996). In

Synechocystis, there are totally 3 subunits of AlsS are characterized, among which sll0065

encodes one small subunit, sll1981 and slr2088 encode two large subunits respectively. However, it is not clear about the exact function of each subunit. It is possible that two of the subunits serve as catalytic role and one of the subunits serves as a reporter of amino acids (isoleucine, valine, leucine) amount for inhibition of the activity of catalytic subunits. After blasting each subunit from Synechocystis with IlvN, a small subunit from Corynebacterium

glutamicum, sll1981 encoded protein shares the most amino acid identity with IlvN, indicating sll1981 encoded protein probably serves as a reporter subunit of the amount of amino acid

(isoleucine, valine, leucine) to further affect the catalytic activity of other two catalytic subunits. The above hypothesis provides another explanation towards the much lower isobutanol production in strain pEEK-ST-1981. In detail, when sll1981 is overexpressed, more reporter subunit can be activated by the existing amino acid in Synechocystis cell, resulting in the increased inhibition of the activity of catalytic subunits and followed by decreased isobutanol production.

Based on isobutanol production of strains overexpressing ST and one of the upstream genes, we failed to identify other potential bottlenecks in the isobutanol synthesis pathway. External addition of 2,3-dihydroxyisovalerate and pyruvate contributes nothing towards isobutanol production of strain pEEK2-ST, which is possible a confirmation of the existing of the potential bottlenecks of upstream genes. Pyruvate is a central intermediate for carbon metabolism in

Synechocystis, and there is no increased isobutanol is produced after external addition of

pyruvate. One explanation is that some bottlenecks is existing among upstream genes. Another possible explanation is the low affinity between pyruvate and AlsS enzyme, resulting in the majority of pyruvate addition flows to other metabolic pathways instead of 2-keto acid pathway. As for the external addition of 2,3-dihydroxyisovalerate, it is risky to conclude anything because it is not known whether it can enter into Synechocystis cell or not.

In our study, all Synechocystis strains were cultivated in plug-sealed tissue culture flasks (close system) instead of cotton-cap E-flasks (open system), considering isobutanol is a volatile compound (Miao et al. 2017). At the same time, there was no air exchange between the close system and atmosphere. Instead of CO2, HCO3- is used as carbon source by converting it to

CO2 with the enzyme carbonic anhydrase. When the cells were cultivated in BG11 with 50

mM NaHCO3 and 2 mL 500 mM NaHCO3 was supplemented to the culture every second day,

there came along another problem that much Na+ accumulating in the culture can cause salt

stress towards cells. After 46 days of cultivation, the salinity of the culture reached 2.5%, whereas the salinity of fresh BG11 medium is only 0.17%. Thus, the 15 folds higher salinity of the culture probably an important factor causing the cell death in the end.

In our system, the produced isobutanol was left in the Synechocystis culture and surprisingly it was found that the isobutanol titer is not increasing for the whole cultivation period (Figure 8B). From day 8 to 10, the isobutanol titer deceased from 52 mg L-1 to 44 mg L-1 and it is

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that the fresh isobutanol produced in the culture cannot cover the amount we took out with the 2 mL culture. Furthermore, when combining growth curve and isobutanol titer (Figure 11A, 11B), it is obvious that the isobutanol production rate is higher in growth phase than in stationary phase. And the in-flask isobutanol titer is almost no change at all in latter cultivation period (Figure 13B). The possible reason is that the concentration of isobutanol in culture has reached the maximum capacity and is toxic to Synechocystis cell (Varman et al. 2013).

In the future, for further improvement of isobutanol production in Synechocystis, there are two approaches can be adopted. One approach is to establish an in situ alcohol-concentrating system using a solvent trap, which can not only avoid the toxic effect of isobutanol towards

Synechocystis cell, but also enable continuous isobutanol production without product feedback

inhibition. Oleyl alcohol (Ezeji et al. 2010) has been demonstrated as an efficient solvent for removal isobutanol from cultivation culture for improved isobutanol titer. Based on Varmans’s (Varman et al. 2013) results, the autotrophic culture with oleyl alcohol trap can achieve 2.2 times more isobutanol titer than culture without oleyl alcohol trap. Thus, in theory, strain pEEK2-ST can produce 957 mg L-1 in-flask titer and 2 g L-1 cumulative tier, respectively, if

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Acknowledgement

This master thesis project was carried out in Microbial Chemistry, Department Chemistry-Ångström. First and foremost, I would like to give my sincere thanks to Peter Lindblad, my supervisor, who gives me the opportunity to explore what I am interested and all the helps I get for both my present project and also my future scientific development. I would also express my appreciation form my heart to my colleague, PhD student Rui Miao, who guides me step by step on lab skills and experiment designing. I cherish the time we spend together for the same scientific goal. I would also give a warm hug to all other colleagues in Cyano Group, for both the scientific suggestions of my degree project and the happy hours we spent together. Furthermore, to those of my friends in Uppsala, it’s your company supporting me for the last two years and make me feel I am not lonely at all. Finally, my parents, who deserve the most

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Appendix

1. Chemicals and reagents used for this project

Chemicals/Reagents Company

DreamTaq DNA Polymerase Thermo Scientific

10x DreamTaq Buffer Thermo Scientific

dNTP Mix, 2mM each Thermo Scientific

Phusion Hot Start II DNA Polymerase Thermo Scientific

5x Phusion HF Buffer Thermo Scientific

FastDigest enzyme Thermo Scientific

10x FastDigest Buffer Thermo Scientific

Quick T4 DNA Ligase Biolabs

2x Quick Ligation Buffer Biolabs

GeneJET PCR Purification Kit Thermo Scientific

GeneJET Plasmid Miniprep Kit Thermo Scientific

Gel DNA Recovery Kit ZYMO RESEARCH

DNA Clean & ConcentratorTM-5 ZYMO RESEARCH

2. BG11o stock solution recipe

Stock No. Stock solution components (g L-1) for 1000x

Stock 1 K2HPO4 40

Stock 2 MgSO4.7H2O 75

Stock 3 CaCl2.2H2O Citric acid 6.0 36

Stock 4 Ferric ammonium citrate EDTA disodium salt 6.0 1.0

References

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