༤ ኈ ㄽ ᩥ㸦Doctoral Thesis㸧Basic and applied research on
psychrophile-based simple
biocatalysts for productionof valuable chemicals
ప ⳦ࢩࣥࣉࣝ㓝⣲ゐ፹ࢆ⏝࠸ࡓ᭷⏝໬Ꮫရ⏕⏘࡟㛵ࡍࡿᇶ♏ཬࡧᛂ⏝◊✲Luo Gonglinfeng
ᗈᓥ኱Ꮫ኱Ꮫ㝔ඛ➃≀㉁⛉Ꮫ◊✲⛉
Graduate School of Advanced Sciences of Matter
Hiroshima University
2021 ᖺ 9 ᭶
㸦September 2021㸧

┠ḟ
㸯㸬୺ㄽᩥ
Basic and applied research on psychrophile-based simple biocatalysts for
production of valuable chemicals㸦ప ⳦ࢩࣥࣉࣝ㓝⣲ゐ፹ࢆ⏝࠸ࡿ᭷⏝໬Ꮫရ⏕⏘࡟㛵ࡍࡿᇶ♏
ཬࡧᛂ⏝◊✲㸧    Luo Gonglinfeng㸰㸬බ⾲ㄽᩥ
(1) Accelerating itaconic acid production by increasing membrane
permeability of whole-cell biocatalyst based on a psychrophilic
bacterium Shewanella livingstonensis Ac10
Luo, G., Fujino, M., Nakano, S., Hida, A., Tajima, T., & Kato, J.
Journal of Biotechnology, 312, 56-62 (2020)(2) Unexpectedly high thermostability of an NADP-dependent malic
enzyme from a psychrophilic bacterium, Shewanella livingstonensis
Ac10Luo, G., Fujii, S., Koda, T., Tajima, T., Sambongi, Y., Hida, A., Kato, J.
Jounral of Bioscience and Bioengineering,
DOI: 10.1016/j.jbiosc.2021.07.005.

୺ㄽᩥContents
Chapter 1 General introduction ......................................................................................................... 4
Chapter 2 Effects of heat treatment on basic metabolic enzyme activities of Shewanella
livingstonensis Ac10 and discovery of an NADP-dependent malic enzyme with unexpected
thermostability .................................................................................................................................. 9
2.1 Introduction ......................................................................................................................... 9
2.2 Experimental procedures................................................................................................... 11
2.2.1 Medium and reagents ............................................................................................. 11
2.2.2 Strains and culture conditions ................................................................................ 11
2.2.3 Gene cloning and plasmid construction ................................................................. 11
2.2.4 Cell extraction preparation and activity assays ...................................................... 13
2.2.5 Protein purification................................................................................................. 14
2.2.6 Enzyme assay for NADP-MEs............................................................................... 15
2.2.7 Stability measurement through circular dichroism ................................................ 15
2.3 Results ............................................................................................................................... 17
2.3.1 Thermostability of S. livingstonensis Ac10 metabolic enzymes ............................ 17
2.3.2 Cloning, expression, and purification of S. livingstonensis Ac10 NADP-ME ....... 18
2.3.3 Kinetic characteristics of SL-ME ........................................................................... 21
2.3.4 Effects of temperatures on the activity of SL-ME.................................................. 23
2.3.5 Thermostability of SL-ME activity ........................................................................ 25
2.3.6 Thermal denaturation of SL-ME ............................................................................ 27
2.4 Discussion ......................................................................................................................... 31
Chapter 3 Efficient itaconic acid production by a psychrophile-based simple biocatalyst ............. 33
3.1 Introduction ....................................................................................................................... 33
3.2 Experimental procedures................................................................................................... 3513.2.1 Medium and reagents ............................................................................................. 35
3.2.2 Gene cloning and plasmid construction ................................................................. 35
3.2.3 Strains and culture conditions ................................................................................ 36
3.2.4 Preparation of the cell suspension .......................................................................... 37
3.2.5 Preparation of the cell extract................................................................................. 37
3.2.6 Itaconic acid production and organic acid analysis ................................................ 37
3.2.7 Protein purification................................................................................................. 38
3.2.8 Docking simulation ................................................................................................ 39
3.2.9 Library construction ............................................................................................... 39
3.2.10 Library screening ................................................................................................. 40
3.2.11 Extraction of total lipids and separation of phospholipids ................................... 41
3.2.12 Preparation of Fatty Acid Methyl Esters (FAME) and analysis of membrane
phospholipids composition.............................................................................................. 42
3.2.13 Gas Chromatography / Mass Spectrometry (GC/MS) analysis ............................ 43
3.3 Results ............................................................................................................................... 44
3.3.1 Cell membrane behaves as a barrier for substrate uptake ...................................... 44
3.3.2 Effects of heat treatment on improvement of membrane permeability .................. 45
3.3.3 Optimal conditions for heat treatment .................................................................... 46
3.3.4 Optimal reaction temperature for itaconic acid production .................................... 52
3.3.5 Recycling of cells after heat treatment ................................................................... 55
3.3.6 Itaconic acid production was limited by product inhibition ................................... 56
3.3.7 Identification of residues related to itaconic acid binding...................................... 58
3.3.8 Library screening ................................................................................................... 592

3.3.9 Shewanella frigidimarina based whole-cell biocatalyst is not permeabilized by heat
treatment.......................................................................................................................... 60
3.3.10 FAME composition of S. frigidimarina and S. livingstonensis ............................ 61
3.4 Discussion ......................................................................................................................... 62
Chapter 4 General discussion and conclusion ................................................................................. 65
Acknowledgements ......................................................................................................................... 69
References ....................................................................................................................................... 70
Appendix ......................................................................................................................................... 81

3

Chapter 1 General introduction
Replacing conventional petroleum-based chemical production with more sustainablebio-based one has been an attractive challenge for effective use of natural resources inchemical industry. Bio-based chemical processes are usually more environmentally
sustainable and effective than petrochemical processes since the catalysts used in biobased chemical processes, enzymes, are capable of catalyzing reactions at moderatetemperatures and produce few undesirable by-products. Thus, there is no need to deploy
expensive, high pressure tolerant, corrosion resistant equipment and complex
downstream processes. Conversions by purified enzymes and whole-cell biocatalyst are
the two major procedures to apply enzymes in chemical processes. Advances inrecombinant protein synthesis have dramatically reduced the cost of enzyme
purification from cells. It was reported that recombinant enzymes take up over 50% of
the enzyme market in 2011 (1).
Purified enzymes are applied to a wide range of industry including food (2, 3), textiles(4), detergent (5, 6), and pharmaceutical (7, 8) usually used directly or immobilized byother supportive materials to improve its cyclability and robustness. Since the cost of
enzymes purification from cells accounts for a significant portion of overall production
costs (9), and not all industries require high-grade purified enzymes, bio-conversion byrecombinant enzymes without purification from their host cells appears to be a more
economic approach.
Whole-cell biocatalyst, is a less expensive way to incorporate enzymes into chemical
processes since the costs of enzymes purification from cells are avoided. It was4estimated that whole-cell biocatalyst costs approximately a tenth of the cost of purified
enzyme (10). Whole-cell biocatalyst also provides a more protective reaction
environment to the enzymes comparing with purified enzymes which are directly
exposed to the reactor. It also allows multi-step reactions in one pot to produce complex
products from simple substrate or regenerate some expensive cofactors such as
NAD(P)+ and NAD(P)H for saving the production cost.
Other applications of whole-cell biocatalyst include catalyzing the reactions at
conditions with limited or even without any solvent to improve the final titer of the
products by lyophilized whole cells (11, 12) and incorporating immobilized cells into
bioreactors (13).
Whole-cell biocatalyst could be constructed based on all kinds of cells, traditionally,
Escherichia coli (14-16) and Saccharomyces cerevisiae (17, 18) are typically used as
the host for enzymes from prokaryotic and eukaryotic creatures respectively. Althoughwhole-cell biocatalyst seems more promising for chemical industry than purified
enzyme because it is less expensive and easy to be prepared on a large scale. There are
two major challenges remained to be overcome. First, as compared to purified enzyme,
whole-cell biocatalyst has advantages in the regeneration of expensive cofactors like
NAD(P)+ and NAD(P)H by multi-step reactions. Since these cofactors are involved ina variety of other biochemical reactions, suppressing or eliminating those undesirable
side reactions becomes a major challenge to increase the yield of target products. There
are mainly two approaches to overcome this obstacle: knocking out host enzymes which
are competitive with heterologous enzymes, and over-expressing heterologous enzymes.
5Tao et al. constructed a whole-cell biocatalyst based on E. coli co-expressing GlcNAc
2-epimerase and Neu5Ac aldolase to produce N-acetyl-D-neuraminic acid. The yield
was 1.6-fold improved by knocking out nagE gene of the host cell to eliminate the side
reaction (19). However, overexpressing heterologous proteins can sometimes beburdensome to host cells (20).
Psychrophiles are able to live and replicate in extreme cold, including temperatures
below the freezing point of water (21). Some interesting characteristics of psychrophilic
microorganisms have been confirmed as the host of whole-cell biocatalyst. Tajima et al.
constructed a psychrophile-based simple biocatalyst (PSCat) to produce aspartic acidfrom fumaric acid with nearly 100% yield by eliminating the side reaction within thehost cell through heat treatment at a moderate temperature (22). It was found thatfumarase of the psychrophilic bacterium, Shewanella livingstonensis Ac10, wasinactivated by heat treatment at 50 °C for 15 min. Therefore, total amount of substrate(fumaric acid in this case) was able to be consumed through desirable reaction by
aspartate ammonia-lyase (aspartase) leading to a nearly 100% yield. Traditionally, thiskind of side reactions are avoided by overexpressing heterologous enzymes, but as
mentioned before, overexpression is not always available. The possibility of side
reactions elimination easily provides us a new tool to construct whole-cell biocatalysts.
The foundation of elimination of side reactions is that metabolic enzymes ofpsychrophile are assumed to be less thermostable than mesophilic enzymes and could
be inactivated at temperatures which have less effects upon those mesophilic ones.
However, basic knowledge of psychrophilic enzymes, especially their thermostabilities6

is insufficient. Current research presented an investigation on the thermostability of anumber of organic acid relevant enzymes in S. livingstonensis Ac10, most of them hada reasonable limited thermostability and were inactivated at 50 °C, but an NADPdependent malic enzyme (SL-ME) was found with unexpected high thermostability
which will be discussed in chapter 2.
Second, the cell membrane that acts as a cage to trap useful enzymes also can be abarrier for substrate to enter the cells. This causes significant issues, full capacity of the
enzymes cannot be utilized because of inefficient substrate access, and certain
conversion processes can be disabled due to the inability of substrate to obtain accessto the enzymes in the cells. In order to solve the problem, surfactants and organic
solvents are widely applied to destroy the part of membrane structure deliberately. P.
Fontanille and C. Larroche tested several reagents including toluene, chloroform andTriton X-100 treatment to permeabilize Pseudomonas rhodesiae cells for production of
isonovalal. It was found that Triton X-100 treatment showed the highest improvement
on isonovalal production rate (23). Changing membrane structure through lipoprotein
mutation to facilitate substrate uptake is also a feasible way (24). Besides, surface
display of the enzymes is also proven an effective approach to enhance the accessibility
between the enzyme and the substrate (25).
Psychrophile-based simple biocatalyst can also help to solve the permeability issue. As
mentioned before that the cell membrane sometimes acted as a barrier for substrate
uptake. In chapter 3, a novel method to permeabilize the cell membrane of
psychrophilic bacterium through heat treatment was described. The heat-permeabilized7

PSCat was able to convert citrate to itaconate at a faster rate than mesophilic one basedon E. coli. Through current research, the author is aiming to not only explore basic
enzymatic characteristics of psychrophilic enzymes but also construct a practicalproduction process for valuable chemicals using psychrophilic cells as hosts ofbiocatalyst.

substrate

heterologous

host enzymes

enzymes

by-productproduct

Fig. 1.1 Basic concept of psychrophile-based simple biocatalyst. In psychrophile, the hostmetabolic enzymes could be inactivated at temperatures which have less effects on mesophileorigined heterologous enzymes. In the meantime, the cell membrane of psychrophile is easy tobe permeabilized through heat treatment, this will allow substrate to enter into the cells more
efficiently thus accelerating the conversion rate.

8Chapter 2 Effects of heat treatment on basic metabolic
enzyme activities of Shewanella livingstonensis Ac10 anddiscovery of an NADP-dependent malic enzyme with
unexpected thermostability

2.1 Introduction

Cold adaption enzymes in psychrophiles have attracted the interest of biochemists andbioengineers due to their unusually high specific activities at low temperatures and themechanism that underpins them (26, 27). In general, cold adaptation enzymes, have more
flexibility in their active sites to catalyze reactions at low temperatures while their
thermostabilities have to be compromised to some degree (28).
Taking advantage of the low thermostability of psychrophilic enzymes, psychrophile-based
simple biocatalysts has been developed. A psychrophilic bacterium, Shewanella livingstonensis
Ac10 was selected as the host organism. Psychrophile-based simple biocatalyst relies on easy
inactivation of psychrophilic host enzymes for the efficient conversion of valuable chemicals
at high yields. Mesophilic or thermophilic enzymes were heterologously expressed in PSCats,in which psychrophilic enzymes, usually leading to undesired byproducts, were heatinactivated through heat treatment at a moderate condition to efficiently obtain target products
catalyzed by the exogenous enzymes. Thus, it is important to have a general concept about how
thermostable the psychrophilic enzymes are and in what conditions can those homologous
enzymes be inactivated without decreasing the yield of target products.
9However, some unusual cases have been identified, such as thioredoxin in the psychrophilic
bacterium Pseudoalteromonas haloplanktis (29) and isocitrate dehydrogenase in Desulfotalea
psychrophila (30), both of which are stable at temperatures well above their optimum growthtemperatures. Such irregular enzymes need to be identified and considered in advance before
constructing a PSCat.
NADP-dependent malic enzyme (NADP-ME, EC: 1.1.1.40) is an NADP-dependent enzymethat catalyzes the reversible oxidative decarboxylation of L-malate to pyruvate in the meantime
converting NADP+ to NADPH. NADP-ME has been identified and separated from many
creatures, including mammals, plants, and bacteria (31-33). It is important for organic acidmetabolism and photosynthesis in plants (34). It also provides NADPH for long-chain fatty
acid synthesis in mammals, fungus, and lipogenesis creatures like algae (35, 36).
NADP-ME in thermophiles such as Sulfolobus solfataricus and Thermococcus kodakarensis
has also been found. Sulfolobus solfataricus NADP-ME catalyzes the reaction at extreme
conditions and showed a less preference to the reaction direction. On the other hand, NADPME in mesophiles such as Mortierella alpine, an oleaginous fungus, has a reasonably limited
thermostability and was completely inactivated above 45 °C (37).
In this chapter, a survey for thermostabilities of several metabolic enzymes in Shewanella
livingstonensis Ac10 was performed. During the survey, a thermostable NADP-ME (SL-ME)
was unexpectedly discovered and its unusual thermostability was investigated as well as its
kinetic characteristics. Research on thermostability of enzymes in psychrophiles is limited.
Exploring such irregular examples will increase our understanding of enzyme temperature
adaptations and could lead to the development of more attractive enzymes in the future.
102.2 Experimental procedures

2.2.1 Medium and reagents

Lysogeny Broth (LB; Tryptone 10 g/L, NaCl 10 g/L, Yeast Extract 5 g/L) was used for cultureof E. coli strains. Agar powder 20 g/L was added for solid medium. Tryptic Soy Broth (TSB;
Difco, US) was used for culture of psychrophile. DNA polymerase KOD-Plus and DNA ligase
Ligation High v2.0 were purchased from Toyobo Co. LTD (Osaka, Japan). Restriction
endonucleases, FastDigest, were purchased from Thermo Fisher Scientific (Massachusetts, US).
All other reagents are special grade chemicals if it is not mentioned.

2.2.2 Strains and culture conditions

Bacterial strains used in the current section are described in Table 2.1. Lysogeny Broth andTriptic Soy Broth (Difco Laboratories, Detroit, Michigan, US) were used to cultivate E. coli
strains at 37 °C and Shewanella livingstonensis Ac10 at 18 °C with 50 mg/L of rifampicin,
respectively. 50 mg/L kanamycin was added for recombinant E. coli strains.

2.2.3 Gene cloning and plasmid construction

E. coli DH5α and S. livingstonensis Ac10 genome DNA were used as template for gene
amplification. The bacteria cultures were collected by the centrifugation at 20,400 × g for 2
min. Collected cells were resuspended in 540 μL GTE (glucose 50 mM, Tris 25 mM, EDTA 10
mM, pH = 8.0) containing 10 g/L lysozyme. After incubation at 37°C for 2 hours, 5 μL of 10
11

mg/mL Proteinase K and 60 μL of 10% w/v SDS were added to the mixture, and it was
incubated at 50 °C for 1 hour. Cooling down suspension on ice was mixed with 600 μL
phenol/chloroform solution with shaking for 10 min. After centrifugation at 20,400 ×g for 5min, the supernatant was moved to new micro tube. Phenol/chloroform treatment was repeated
to increase the purity of DNA. To precipitate the DNA, 40 μL of 3M CH3COOK and 1 mL of
99.5% ethanol were added into 400 μL of supernatant and it was kept at -20 °C for 30 min. The
precipitate obtained by the centrifugation at 20,400 ×g for 5 min was washed with 70% w/v
ethanol and dissolved with 50 μL of TE (Tris-HCl 10 mM, EDTA 1 mM; pH = 8.0) containing
50 μg/mL RNase.
Since the genome sequence of S. livingstonensis Ac10 has not been released, primers for
amplification of the malic enzyme gene in the Ac10 strain (SL-ME) were designed using the
genome sequence of Shewanella frigidimarina NCIMB 400, a closely related species.
Sfri_3757 is the name of a putative NADP-ME gene in S. frigidimarina NCIMB 400. Primers,
plasmids, and strains used for cloning are described in Table 2.1. SL-ME gene was amplified
by Toyobo KOD Plus polymerase following the manufacturer’s instructions. The PCR productwas then purified using FastGene PCR/Gel extraction kit (Nippon Genetics, Tokyo, Japan) anddigested by EcoRI and XhoI FastDigest restriction enzymes (Thermo Fisher Scientific,
Massachusetts, US). DNA fragments were separated by electrophoresis on a 1% w/v agarose
gel and the gel bands containing the DNA fragments were cut out and extracted again. The PCR
product was ligated with pET28b (+) fragments digested by the same restriction enzymes.
At last the ligation product was added into 100 μL E. coli competent cell suspension. The
mixture was then kept on ice for 30 min. After that, the E. coli was heat-shocked at 42 °C for12

30 seconds then cooled down on ice immediately. After 1 min, 1 mL of LB was added, then the
mixture was incubated at 37 °C for 1 hour. After the incubation, the mixture was spread on a
LB plate containing kanamycin to select E. coli harboring the plasmid. Twelve hours later,colonies on the plate were picked up and cultivated. The plasmid was extracted and confirmedby restriction enzyme digestion to confirm the insertion of the DNA fragments. NADP-ME
from E. coli (MaeB) was cloned following the same procedure.

2.2.4 Cell extraction preparation and activity assays

S. livingstonensis Ac10 was first pre-cultivated in TSB with 100 mg/L ampicillin and 50 mg/L
rifampicin for 48 hours. The cultures were then used to inoculate in 200 mL TSB in 500 mL
Erlenmeyer flask (1%, v/v). The S. livingstonensis Ac10 cells were then collected bycentrifugation at 4°C, 5,000 × g for 10 min and the cell extraction was prepared throughsonication.
The cell extract was heat-treated at 50 or 60 °C before analyzing. Absorbances were monitored
by a UV-Vis spectrometer (Thermo Fisher Scientific, Massachusetts, US) at 340 nm foranalyzing of NADP-ME, lactate dehydrogenase (LDH), pyruvate dehydrogenase (PDH),
aconitase, isocitrate dehydrogenase (IDH) and 2-oxoglutarate dehydrogenase (OGDH), 412 nm
for citrate synthase, and 235 nm for SCS activities. All analyses were performed at 18 °C in 1
mL reaction mixture, enzyme activities of the cell extract without any heat treatment wereregarded as 100%.
The reaction mixture of analyses was described as below:

13

For the NADP-ME, the reaction mixture contained 1 mM NADP+, 10 mM L-malate, and 100
mM Tris-HCl (pH = 9.0). For the LDH, the reaction mixture contained 0.015 mM NADH, 10mM pyruvate, 25 mM potassium phosphate buffer (pH = 7.0). For the PDH, the reaction
mixture contained 0.2 mM TPP, 0.1 mM CoA, 1.0 mM MgCl2, 0.3 mM DTT, 2.5 mM NAD+,
100 μg BSA, 3.0 mM Lipoamide, 0.025 mM FAD+, 5.0 mM pyruvate, and 50 mM phosphatebuffer (pH = 8.0). For the aconitase, the reaction mixture contained 20 mM citrate, 1 mM
MnSO4, 100 mM Tris-HCl (pH = 9.0), 1.6 U IDH (Oriental Yeast, Tokyo, Japan), and 1 mM
NAD+.
For the IDH, the reaction mixture contained 1 mM NAD+, 20 mM isocitrate, 50 mM MgCl2
and 100 mM Tris-HCl (pH = 8.5). For the OGDH, the reaction mixture contained 0.5 mM
NAD+ , 2.5 mM 2-oxoglutarate, 0.25 mM MgCl2, 0.04 mM CoA, 2.5 mM cysteine, and 75 mM
potassium phosphate buffer (pH = 7.0). For the citrate synthase, the reaction mixture contained
0.5 mM oxaloacetic acid, 0.3 mM actyl-CoA, 0.1 mM 2-nitrobenzoic acid and 100 mM TrisHCl (pH = 9.0). For the succinyl-CoA synthase, the reaction mixture contained 10 mM
succinate, 10 mM MgCl2, 0.1 mM DTT, 0.1 mM CoA, 50 mM KCl, 0.4 mM ATP, and 50 mM
Tris-HCl (pH = 9.0).

2.2.5 Protein purification

Recombinant E. coli BL21 (DE3) cells were harvested by centrifugation at 4 °C, 5,000 × g for
5 min and stored at ‒80 °C. Cells were then resuspended in binding buffer (20 mM phosphate,
20 mM imidazole, 0.5 M NaCl, pH = 7.4) and lysed by French pressure cell press at 20000 psi

14

(American Instrument Co., Inc., MD, US). The cell extract was then loaded to a Ni column (GEHealthcare, Illinois, US), imidazole (200 mM for SL-ME, 400 mM for E coli NADP-ME, MaeB)
was applied to strip the target protein which was further purified through size exclusion
chromatography (SEC) with a AKTA explorer FPLC system equipped with a sephadex G-200
gel filtration column (GE Healthcare, Illinois, US). After that, the product was concentrated
with ultra centrifugal filters (Merck, New Jersey, US ) and stored in TMG buffer containing 50
mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 50% (v/v) glycerol (38).

2.2.6 Enzyme assay for NADP-MEs

NADP-ME activities were measured in 1 mL reaction mixture containing 50mM Tris-HCl, pH
7.5, 50 mM L-malate, 10 mM MnCl2 and 0.2 mM NADP+ at 30 °C. A UV-Vis spectrometer(Thermo Fisher Scientific, Massachusetts, US) was used to monitor Absorbance at 340 nm inreal-time. Water bath (TAITEC, Koshigaya, Japan) were used for heat-treatment.

2.2.7 Stability measurement through circular dichroism

The thermostability of the S. livingstonensis Ac10 and E. coli NADP-ME was measured bymonitoring circular dichroism (CD) spectra using a J-820 CD spectrometer (JASCO, Tokyo,
Japan). 10 μM of protein solutions dialyzed against 20 mM sodium phosphate buffer (pH = 7.5)
were analyzed. The CD spectra from 200 to 250 nm was scanned at 25 °C in a quartz cuvetteof a path length of 1 mm. The CD ellipticity change at 222 nm was monitored from 30 to 90 °C

15with the heating rate of 1.0 °C/min. The resulting raw data were subjected to nonlinear leastsquares fitting, as described by Uchiyama et al. (39). The data points obtained were corrected
for the slope of the base lines for the native and denatured forms, and were normalized to
calculate the fraction of the denatured protein. The resulting fraction was plotted as a functionof temperature.
Table 2.1 Plasmids and strains used in this research
Strains, plasmids

Genotype or phenotype

Resource

Psychrophilic bacterium, Rifr

(40)

Cloning host deoR, supE44,

TOYOBOand primers
StrainsS. livingstonensis
Ac10
E. coli DH5α

hsdR17(rk-, mk+), phoA, recA1,
endA1, gyrA96, thi-1, relA1,
D(lacZYA-argF)U169,
f80dlacZDM15, F-, lE. coli BL21(DE3)

Suitable for high-level T7 expression

TAKARAof recombinant proteins. F– ompT
hsdSB (rB–, mB–) gal dcm (DE3)
Plasmids
pET28b (+)

Expression vector with T7 promoter,
Kanr,
16

MERCK

Table 2.1 (continued)
Primerssfri_3757_F

CATATGATGTCAGATATTCGTCAThis study

ACAAGCTCT
sfri_3757_R

CTCGAGCACTTTGTATTCAGTCThis study

GGTA
maeB_F

ATGCGAATTCATGGATGACCAG

This study

TTAAAACAAAGTG
maeB_R

ATGCCTCGAGCAGCGGTTGGGT

This study

TTGCGCTTCTACC

2.3 Results

2.3.1 Thermostability of S. livingstonensis Ac10 metabolic enzymes

Several TCA cycle relevant enzymes including lactate dehydrogenase (LDH), aconitase,pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (IDH), oxoglutarate dehydrogenase(OGDH), citrate synthase succinyl-CoA synthase (SCS) and SL-ME were tested for their
thermostabilities at 50 and 60 °C. The result was showed in Table 2.2, most of them showedreasonable limited thermostabilities and lost most of their activities after the heat treatment
except SL-ME. SL-ME kept largely active even after heat treatment at 60 °C.

17

Table 2.2: Relative activities of serval organic acid metabolic enzymes in S. livingstonensis
Ac10 after heat treatment (HT)Enzyme

SL-ME

LDH

PDH

Aconitase

IDH

OGDH

HT (°C)

CitrateSCS

synthase

50

91.0%

N.D.

0.2%

N.D.

0.02%

N.D.

2.8%

N.D.

60

72.6%

N.D.

N.D.

N.D.

N.D.

N.D.

2.4%

N.D.

2.3.2 Cloning, expression, and purification of S. livingstonensis Ac10 NADP-ME

Since SL-ME is clearly a unique enzyme in terms of thermostability, further investigation was
performed. A plasmid for over-expressing recombinant SL-ME in E. coli was constructed basedon pET-28b (+). NADP-ME gene of S. livingstonensis Ac10 was sequenced (DDBJ:
LC627056). It has a high degree of similarity to S. frigidinamrina Sfri_3757, and the aminoacid sequences of the proteins are identical. The deduced SL-ME amino acid sequence is highly
similar to other NADP-MEs in Shewanella species such as S. frigidimarina (identical), which
was same as the cases in cytochrome c5 and cytochrome c’ from S. livingstonensis and S.
frigidimarina (41, 42), S. benthica (NCBI: WP_005500301.1, 84% identity), S. violacea(NCBI: WP_013049565.1, 84% identity), S. amazonensis (NCBI: WP_011758568.1, 87%
identity), and S. oneidensis (GenBank: PZP30003.1, 84% identity). S. benthica and S. violacea
belong to Group I Shewanella species, which are usually psychrophilic and piezophilic. Later
two belong to Group II Shewanella species are mesophilic, and not piezophilic (43). Highsequence identity of SL-ME to the counterparts of other Shewanella species in both Group I
and II indicates that NADH-MEs are widely conserved among the species. SL-ME also shares18

a relatively high identities with a truncated MaeB (44) from N-terminal to A468 (52.8%
identities). A completely same MaeB putative NADP-binding domain was confirmed (193GAAGAAAIA-202) in SL-ME, but the residues in the vicinity of this domain are largely
different (Fig. 2.1)
The purification of recombinant SL-ME began with a nickel column, followed by size exclusion
chromatography. SL-ME is a 100 kD homodimer in solution, as determined by SDS–PAGE
and native PAGE (Fig. 2.2). Surprisingly, this homodimer is very stable that the loading buffer
containing SDS and 2-mercaptoethanol failed to fully denature it.

19

Fig. 2.1 Sequence alignment of SL-ME, NADP-MEs from S. benthica (WP_005500301.1), S.
violacea (WP_013049565.1), S. amazonensis (WP_011758568.1), S. oneidensis (PZP30003.1)
and MaeB (N-terminal to A468). Putative NADP-binding domain is marked by underline.
20

a
MW (kD)
250
150
100

L1

L2

b

L3

MW (kD)

L1

L2

L3

210
140

75

90
70

50

55

37

40
35

25
20

Fig. 2.2 SDS–PAGE result of purified recombinant SL-ME (a). L1, Molecular weight marker;
L2, fractions of gel filtration, treated in 4 M urea at 100 °C for 5 min before running; L3,
fractions gel filtration without any additional treatment. Native PAGE result of purified
recombinant SL-ME (b). L1, Molecular weight marker; L2, fractions of gel filtration, treatedby in 4 M urea at 100 °C for 5 min before running; L3, fractions gel filtration without any
additional treatment. Relevant lanes have been rearranged in both panels for the clarity.

2.3.3 Kinetic characteristics of SL-ME

SL-ME kinetic parameters were measured. The reactions were carried out at 30 °C with various
concentrations of L-malate and NADP+. Kcat (S-1) and Km (mM) for L-malate were 11.8 and 7.3,
respectively, while Kcat and Km for NADP+ were 10.7 and 0.2 (Fig. 2.3).

21

a
1/V (μmol-1 min mg)

1
y = 0.7722x + 0.1055
R² = 0.989

0.8
0.6
0.4
0.2
0

-1

-0.5

0

0.5

1/V (μmol-1 min mg)

1.5

1/S (mM-1)

-0.2

b

1

0.5
y = 0.0247x + 0.1156
R² = 0.9984

0.4
0.3
0.2
0.1
0

-10

-5

0

5

-0.1

10

15

1/S (mM-1)

Fig. 2.3 Kinetic parameters of SL-ME, Lineweaver-Burk plot was used to determine the Km
and Vmax of the enzyme. Error bars represent the standard deviation of the mean (n = 3).

22

2.3.4 Effects of temperatures on the activity of SL-ME

The activity of SL-ME was measured at temperatures ranging from 4 to 80 °C. As a result,though 70 °C is the ideal temperature for the reaction, the psychrophilic enzyme retains 8.9%
and 91.9% of its original activity at 4 and 80 °C, respectively (Fig. 2.4a). For comparison, theeffects of temperature on E. coli MaeB were investigated. This time, unlike the thermostability,
the psychrophilic and mesophilic NADP-ME behaved differently. It is worth noting that
although SL-ME has higher specific activities at temperatures above 70 °C, it is less active than
MaeB at temperatures below 30 °C where S. livingstonensis Ac10 grows (Fig. 2.4b).

23

a 100Relative activities (%)

90
80
70
60
50
40
30
20
10
0
0

20

40

60

80

Reaction temperatures (°C)

Specific activities (U/mg)

b 14
12
10
8
6
4
2
0

0

20
40
60
80
Reaction temperatures (ȭ)

Fig. 2.4 Effects of temperature on the activity of SL-ME and MaeB. The reaction was
performed at various temperatures ranging from 4 to 80 °C. Relative activities (a) and specificactivities (b) were showed. Circle, S. livingstonensis Ac10 NADP-ME; triangle, E. coli NADPME. Error bars represent the standard deviation of the mean (n = 3).

24

2.3.5 Thermostability of SL-ME activity

Purified enzyme solution was incubated at different temperatures ranging from 20 to 80 °C toexamine the thermostability of the recombinant SL-ME. The results indicated that the NADPME activity of S. livingstonensis Ac10 remained stable at temperatures up to 60 °C (Fig. 2.5a).
The thermostability of NADP-ME from E. coli (MaeB) was also investigated as a contrast.
Surprisingly, SL-ME had comparable thermostability to its mesophilic counterpart. The
thermostability of SL-ME at 60 °C was explored further by incubating the protein at 60 °C for
0.5, 1, 2, and 3 hours, with no activity loss observed (Fig. 2.5b).

25

Relative activities (%)

a 120
100
80
60
40
20
0
20

30

40

50

60

70

80

2.5

3

Incubation temperatures (ȭ)

Relative activities (%)

b 120
100
80
60
40
20
0
0.5

1

1.5
2
Incubation time (h)Fig. 2.5 Relative activities of SL-ME and MaeB incubated at various temperatures for 0.5 h(a), circle, SL-ME; triangle, MaeB. Thermostability of the SL-ME at 60 °C with longincubation time (b). Specific activity of enzymes kept on ice was defined as 100%.

26

2.3.6 Thermal denaturation of SL-ME

The effect of temperature on the structural stability of SL-ME and E. coli MaeB was verified
by CD measurements. Both proteins showed negative peaks at 208 and 222 nm (Fig. 2.6a),
indicating a predominantly α-helical structure. Denaturation curves for SL-ME and MaeB
were obtained by detecting the CD ellipticity at 222 nm. The transition temperature mid-point
during the thermal denaturation (Tm) of SL-ME was 71.9 ± 0.5 °C (from three independent
experiments, ± standard deviation), which was significantly higher than that of E. coli MaeB
(64.6 ± 0.4 °C). Thus, despite its origin, the psychrophilic NADP-ME has a more stable
overall structure than its mesophilic counterpart (Fig. 2.6b). The density rate of SL-ME
reached 0.14 at 69.5 °C, in contrast MaeB reached this level at 58.5 °C, which means SL-ME
started to denature at a much higher temperature than its mesophilic counterpart. Both SL-ME
and MaeB exhibited loss of reversibility at 70 °C (Fig. 2.6c). These results were consistent
with the activity measurements for both enzymes, which showed only ~20% activity when
exposed to 70 °C.

27

10[q] x 10-3 (deg cm2 dmol-1)

a

0

MaeB

-10
-20
-30
-40
200

210

220

230

240

250

Wavelength (nm)[q] x 10-3 (deg cm2 dmol-1)

10

SL-ME

0
-10
-20
-30
-40
200

210

220

230

240

250

Wavelength (nm)Fig. 2.6 Thermal denaturation analysis for SL-ME and MaeB by circular dichroism. (a)Circular dichroism scans at 25 °C.

28

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

30

40

50

60
Temperature (ºC)

MaeB

70

SL-ME

80

90

The raw data is shown by the gray lines.

29

Fig. 2.6 Thermal denaturation analysis for SL-ME and MaeB by circular dichroism. (b) Representative normalized denaturation curves for SL-ME and MaeB.

bFraction of protein denatured

30(CD) after thermal denaturation. Samples were increased to 50, 60, 70, and 80 °C at a rate of 1.0 °C/min.

Fig. 2.6 Thermal denaturation analysis for SL-ME and MaeB by circular dichroism. (c) Reversibility checks of SL-ME and MaeB using circular dichroism2.4 Discussion

Psychrophiles need cold adaptive enzymes to survive at low temperatures. Although
psychrophilic enzymes are generally considered to have relatively low functional temperatures,investigations are still needed to prove this hypothesis. It is important for PSCat to inactivate
homologous enzymes through heat treatment to achieve a high production yield. Thus, several
organic acid metabolic enzymes were tested for their thermostabilities at 50 and 60 °C. Mostof them are as heat-vulnerable as expected, except SL-ME. An NADP-ME from psychrophile(SL-ME) with abnormal thermostability was discovered in this study. The result strongly
suggested that not all the enzymes in psychrophiles need to be cold-adapted. Although this does
not change the foundation of PSCat, cautions still need to be made when introduce newpathways in S. livingstonensis Ac10 with reactions related to NADP+ and NADPH regeneration.
SL-ME shares high identities with other NADP-MEs in non-psychrophilic Shewanella species
like S. amazonensis and S. oneidensis, it is possible that SL-ME is not engaged in cold
adaptation in S. livingstonensis Ac10, but it is still important for Shewanella species to survive.
A highly conservative NADP-binding domain is confirmed in SL-ME and this may explain thereason why SL-ME cannot use NAD+ as a cofactor for its activity (data not shown) just like
MaeB. While low degree of conservation around the NADP-binding domain may have an
influence on their different temperature adaptations.
Leuenberger et al. had examined the thermostability of mesophile and thermophile on aproteomic scale (45). Thermophiles were discovered to achieve thermo adaptation by unequally
improving protein thermostabilities. For psychrophiles, a similar mechanism may be31considered.
SL-ME is stable and functioning at a wide temperature range. Although it has a greater optimal
operating temperature than its E. coli equivalent, this is unlikely to be physiologicallysignificant because S. livingstonensis Ac10 cannot survive at such high temperatures.
SL-ME appears to be involved in S. livingstonensis Ac10's organic acid metabolism, as enzyme
activity was observed in the cell extract. The role of SL-ME in S. livingstonensis Ac10
metabolism is waiting to be explored and mechanism behind its unusual thermostability is still
unclear and needs to be explored in the future.

32

Chapter 3 Efficient itaconic acid production by apsychrophile-based simple biocatalyst
3.1 Introduction

Whole-cell biocatalysts have broad applications in the chemical and pharmaceutical industry.
Compared to the purified enzymes, whole-cell biocatalysts for the production of biologicalmaterial have been researched extensively in past years. Allowing multistep reactions is one of
the advantages of whole-cell biocatalysts. Based on the results from chapter 2, an NADPdependent malic enzyme remains active after heat treatment, which means any new reaction
containing NADPH regeneration may be influenced by the SL-ME. Aiming to show thepotential of PSCats, a bioconversion process without NADP(H) involvement was selected inthis chapter as the target process of PSCat.
Itaconic acid is an unsaturated dicarboxylic acid with broad industrial applications. It is also a
potential substitute for acrylic acid or methacrylic acid (46), a component for polymer
production (47). Itaconic acid can be produced from glucose through fermentation by
Aspergillus. terreus (48). Two enzymes are crucial for this process, aconitase and cis-aconitic
acid decarboxylase (CAD). The fact that aconitase and CAD are present in different organelles(cytoplasm and mitochondria respectively) may limit the itaconic acid production by A. terreus(49). Using whole-cell biocatalyst to produce itaconic acid can be an alternative way to obtaina higher titer, purity, and productivity because all enzymes will be expressed in the cytoplasm
and metabolic flux is not vital for itaconic acid biosynthesis. Kim et al. developed a whole-cellbiocatalyst based on E. coli to produce itaconic acid (50). As mentioned in chapter 1, the cell
33

membrane of whole-cell biocatalyst often serves as a barrier to substrate influx. For example,
Pseudomonas pseudoalcaligenes cells cannot convert maleate to D-malate because the cell
membrane inhibits substrate uptake (51). Many methods have been developed to improve cell
membrane permeability, such as solvent treatment, detergent treatment, and genetic engineering
to change the constitution of cell membrane. In the research of Kim et al., detergentpermeabilized cells have achieved itaconic acid productivity of 2.19 g/L/h. However, additional
chemical reagents may increase the complexity and the cost of downstream processing.
Therefore, a simple method is required to improve the membrane permeability of whole-cellbiocatalysts. The cell membrane of psychrophiles is very flexible due to the membrane lipid
composition (52), allowing the passive transport of tiny hydrophilic molecules. Therefore,
using psychrophile as the host for whole cell catalyst could be a potential solution for
permeability issue.
In this chapter, a new approach to solve the permeability issue of whole-cell biocatalyst was
described. A whole-cell biocatalyst based on a psychrophilic bacterium, S. livingstonensis Ac10,
was constructed to produce itaconic acid from citric acid. Two enzymes vital for itaconic acidproduction from citric acid, aconitase B from E. coli and CAD from A. terreus, were expressedin S. livingstonensis Ac10 cells which were then heated at a moderate temperature to increase
permeability. The optimum temperature for heat treatment and the performance of the
biocatalyst was determined upon recycling it several times. An attempt to perform itaconic acidproduction at a high concentration of substrate was also made, aiming to obtain high titer of the
product, but it was unsuccessful probably because of the product inhibition. After that the CAD
was engineered through site-directed mutation to remove the product inhibition, no promising34

mutants have been confirmed yet.
Mechanism behind the permeability improvement caused by heat treatment was also explored.
A relative species of S. livingstonensis, S. frigidimarina was found to behave differently after
heat treatments at the same conditions, thus it may provide some clues about the permeability
improvement. Lipids composition was considered to be the key reason and both the membrane
lipid composition of S. livingstonensis and S. frigidimarina was investigated. No difference intheir lipid composition was confirmed and the possible mechanism of the permeability
improvement remained unclear.

3.2 Experimental procedures

3.2.1 Medium and reagents

See chapter 2.

3.2.2 Gene cloning and plasmid construction

Procedures for molecular cloning is the same as described in chapter 2 if not mentioned.
Aconitase B gene (acnB, EG12316) from E. coli and cis-aconitic acid decarboxylase (cadA,
ATEG_09971) gene from A. terreus were amplified through PCR using KOD plus Neopolymerase (TOYOBO, Osaka, Japan) following the procedures described in chapter 2,
Templates of PCR amplification for acnB and cadA were genomic DNA of E. coli and a plasmid
pUCIDT-AMP: 1_ATEG_09971 containing a codon-optimized chemical synthesis cadA geneof A. terreus, respectively. The PCR products of acnB and cadA were ligated into pHA12 at35

restriction sites between EcoRI and KpnI and between SmaI and HindIII, respectively. Genes,
primers and plasmids used in this research are listed in Table 3.1.
The plasmid containing acnB and cadA was first introduced into E. coli S17-1 following themethod described in chapter 2 then into S. livingstonensis Ac10 by conjugation; E. coli S17-1
cells harboring the recombinant plasmid were cultured at 37 °C overnight, and S.
livingstonensis Ac10 was cultured at 18 °C for 30 hours. They were then collected bycentrifugation at 3,300 ×g for 3 min and washed twice with LB to remove antibiotics. After
resuspending the E. coli and S. livingstonensis Ac10 cells with 250 μL LB respectively, thesuspension was mixed together and spotted on a LB plate (without any antibiotics). The plate
was then incubated at 18°C. After one day, 1 mL sterilized water was used to wash the plate,then the suspension was diluted 10, 100, and 1,000 times respectively. One hundred microlitersof each group of diluted cell suspension was spread on plates containing ampicillin andrifampicin. After incubation at 18 °C for 5 days, the colonies on the plates were picked up,
cultured and the plasmid was extracted to confirm the DNA fragments.
For expressing CAD in E. coli BL21 (DE3) for purifying, the gene was cloned following thesame procedure described in chapter 2.

3.2.3 Strains and culture conditions

For recombinant S. livingstonensis Ac10 strains, 100 mg/L ampicillin was added in the medium.
To overexpress cadA and acnB, S. livingstonensis Ac10 was transformed with plasmid pHA12cadA-acnB, and their cloned genes were incorporated into plasmid pHA12 and expressed in S.

36

livingstonensis Ac10 upon induction with 100 μM isopropyl-β-D-thiogalactopyranoside(IPTG). To generate cells overexpressing the target proteins, transformed Ac10 cells were
cultured in 4 mL of TSB medium for 36 h and then inoculated into 200 mL of TSB culture
medium (1% inoculum). The cultures were incubated in 500-mL Erlenmeyer flasks underconstant agitation at 120 rpm on a rotary shaker. After 24 h of culturing, gene expression was
induced by adding 100 μM IPTG.

3.2.4 Preparation of the cell suspension

Recombinant S. livingstonensis Ac10 cells were harvested by centrifugation at 5,000 × g for 10
min at 4 °C after culturing, washed twice, and resuspended in 50 mM phosphate buffer (PB,
pH = 6.5). The dry weight of cells was measured via dissociation at 70 °C overnight.

3.2.5 Preparation of the cell extract

The cell suspension of recombinant S. livingstonensis Ac10 was disrupted via ultrasonication,with 1-s pulse/1-s intervals 600 times, using an ultrasonic disintegrator (Digital Sonifier 450;
Branson, Connecticut, US). The supernatant was then harvested via centrifugation at 10,000 ×g for 30 min at 4 °C.

3.2.6 Itaconic acid production and organic acid analysis

The cell suspension was heat-treated at various temperatures and transferred to 1 mL reaction
mixture containing 200 mM PB (pH = 6.5) and 50 mM citric acid, final concentration of cells
in the reaction mixture was approximately 35 mg/ml. The reaction was performed at various
temperatures as described later. The reaction was terminated by heat treatment at 80 °C for 1537

min or mixed with an equal volume of phenol-chloroform and vortexed for 5 minutes. A water
bath (SM-05 N; Taitec, Saitama, Japan) was used for reactions and heat treatment. Citric acid,cis-aconitic acid, and itaconic acid were detected via high-performance liquid chromatography(HPLC; Jasco, Tokyo, Japan) system equipped with a UV detector at 210 nm and a column(Shodex RSpak KC-811; Showa Denko, Kanagawa, Japan) with a guard column (Shodex
RSpak KC-G; Showa Denko). The products were flowed at 45 °C using 0.1% (v/v) phosphoricacid as a mobile phase at a flow rate of 0.7 mL/min. Citric acid was also measured by citricacid analysis kit (R-Biopharm AG, Germany) because some metabolites in the cells have close
retention time to citric acid thus may disturb the quantification of it.

3.2.7 Protein purification

See chapter 2
Table 3.1 Strains and plasmids used in chapter 3
Strains, plasmids

Genotype or phenotype

Resource

and primers
Strains
S. firgidimarina

Host strain for whole-cell biocatalyst, Lab stock

DSM 12253

Rifr

S.

livingstonensis See chapter 2

See chapter 2

Ac10
Plasmids
pHA12

Expression vector, Ampr
38

(53)

Table 3.1 (continued)
pUCIDT-AMP:

Contains codon-optimized CAD gene This study (chemical

1_ATEG_09971

based on A. terreus CAD gene cadA

synthesis by I. D. T)

ATGCCCCGGGAGAGATGAACAa

This study

Primers
AcnB_F

GTGCTAGAAGAATACCGTAAAcnB_RATGCAAGCTTAAACCGCAGTCTThis study

GGAAAATCA
CAD_F

ATGCGAATTCGGAGAGATGAAC

This study

A ATGACTAAGCAATCAGCAGA
CAD_R

ATGCGGTACCTTATACCAGCGG

This study

CGATTTTAC
a: Sequences with underline refer to SD sequence.

3.2.8 Docking simulation

Docking simulation of the CAD and itaconic acid was performed on AutoDock (v1.5.6) – Vina(v1.1.2) using a putative 3D structure of A. terreus CAD generated by I-TASSER (54) basedon mouse CAD (PDB: 6R6T). The result was visualized with PyMol (v1.7.5.0).

3.2.9 Library construction

All polar amino acid residues 6.5 Å from itaconic acid were considered as potential binding
39

sites. Besides, putative active site of mouse CAD were also selected. All 15 residues were
subjected to site-specific saturation mutagenesis, generating all possible 285 mutants.
The mutations were prepared by amplifying full-length plasmid expressing CAD based on pET28b(+) mentioned before, primers used for mutation generating are described in Appendix,
procedures for molecular cloning were the same with those in formal chapters. All mutant genes
were expressed by E. coli BL21 (DE3).

3.2.10 Library screening

A high throughput screening method based on Mizoroki-Heck reaction to detect itaconic acidin the reaction mixture was modified to screen variants (55). Briefly, A 96-well plate containing
200 μL LB with 50 mg/L kanamycin for each well was used to cultivate all variants at 37 °C
for 2 ~ 3 hours, 200 rpm. IPTG was then added to a final concentration of 2 mM and the plate
was transferred to 18 °C, cultivated overnight.
The culture was then transferred to a 96-well PCR plate and harvest by centrifugation at 2,500
× g for 5 min. Cells were washed once by 20 mM PIPES (pH = 6.5) and lysed by Bacterial
Protein Extraction Reagent (Thermo Scientific, US) following the manufacturer procedure.
After that, first round screening was performed by mixed cell extracts (with debris) to a reaction
mixture containing 50 mM PIPES and 2 mM cis-aconitic acid. The plate was sealed andimmersed in 37 °C water bath to get a more average heat transformation (56) for 10 min. The
reaction was then stopped by adding 4 μL concentrated HCl and 10 μL supernatant wascollected after centrifugation at 4,000 × g for 5 min. For itaconic acid detecting, 10 μL

40

supernatant 10 μL, 180 mM indobenzene in DMSO, 2 μL, 4.5 mM Pd(OAc)2 was added to eachwell of a 96-well plate. The plate was then sealed and heated by water bath at 80 °C for 1 hour.
After the reaction, 2 μL HCl was added to adjust pH before adding 50 μL, 5% soluble starchand 50 μL, 5% NaNO2 to the reaction mixture. The plate was freezed at ‒20 °C to fix the color.
Then the purple color can be measured at 595 nm by a Thermo Scientific Varioskan FlashSpectral Scanning Multimode Reader (Thermo Scientific, US).
All mutants with comparable activities to the wild type were picked up for second roundscreening. In the second round screening, cell extract of each mutant was prepared as describedat chapter 2. The cell extract was then mixed with reaction mixture containing 200 mM PIPES(pH = 6.5) and 500 mM cis-aconitic acid to produce itaconic acid, which was measured by
HPLC after the reaction.

3.2.11 Extraction of total lipids and separation of phospholipids

Extraction of total lipids were performed based on methods described by Fang et al. (57). S.
livingstonensis Ac10 and S. firgidimarina DSM 12253 were cultivated at 18 °C for 72 h, Cells
were harvest through centrifugation at 5,000 × g for 10 min, washed twice by 20 mM Phosphate
Buffer (pH = 6.5), freeze dried and stored at ‒80 °C.
For total lipids extraction, freeze dried cells were mixed with extraction solvent containing
dichloromethane (DCM), methanol and 50 mM potassium phosphate buffer (pH = 7.4) at a ratioof 1:2:0.8. For 55mg of cells, 9.5 ml extraction liquid was added, shaken to mix and then stored
at 4 °C in dark place to for overnight. After that, 2.5 mL DCM and 2.5 mL potassium phosphatebuffer (pH = 7.4) were added, stored at 4 °C overnight.

41

The mixture was centrifugated at 5,000 × g for 10 min to separate layer, organic phase (lower
layer) was collected and 0.5 g Na2SO4 was added to dry the liquid (total volume about 3 mL).
A No.2 filter was used to filter the liquid. After that the total lipid was stored at ‒80 °C until
further processing.
For phospholipid separation, total lipid sample was first dried by N2 then dissolved again in 1
mL CHCl3. Total lipid was separated into 3 classes by a silicic acid column (Wakogel, C-200;
Wako, Osaka, Japan), neutral lipids, glycolipids and phospholipids. Since cell membrane is theresearch target, phospholipids were collected and applied to further experiments. The total lipid
was first loaded on to the column, 8 mL solvent was then applied to elute different kind of lipids,
CHCl3 for neutral lipids, acetone for glycolipids and methanol for phospholipids. The
phospholipid dissolved in methanol was dried by N2 stream and stored at ‒80 °C.

3.2.12 Preparation of Fatty Acid Methyl Esters (FAME) and analysis of membrane
phospholipids composition

Preparation of FAME were performed. Briefly, dried phospholipid was dissolved in 0.5 mL
methanol and toluene mixture (1:1, v/v). 0.5 ml of 0.2 M KOH in methanol was added to the
mixture. The mixture was then incubated at 37 °C for 15 min in water bath. After that, thereaction mixture was cooled down in room temperature and mixed with 2 mL CHCl3, 2 mL H2O
and 0.5 mL of 0.2 M acetic acid, centrifuged for 5 min at 3,000 × g. Organic phase (lower phase)
was collected, dried by N2 stream and stored at ‒80 °C for further experiments.

42

3.2.13 Gas Chromatography / Mass Spectrometry (GC/MS) analysis

Dried FAME was dissolved in hexane and analyzed by a GC/MS system, Aglient Technologies
7890A GC system equipped with a DB-WAX column interreacted with 7000 GC/MS Triple
Quad (Aglient Technologies, California, US). The oven temperature was programmed from
50 °C for 1 min, then to 200 °C at 25 °C/min, and to 230 °C at 3 °C/min, kept for 18 min. The
detector temperature was set at 280 °C and the injection port temperature was 250 °C.

43

3.3 Results

In this chapter, a recombinant plasmid based on a broad range host, pHA12, was constructed
and introduced into S. livingstonensis Ac10 to express CAD and AcnB (Fig. 3.1). Aconitase B
converts citric acid to cis-aconitic acid, which is then converted to itaconic acid by CAD.

Fig. 3.1 Physical map of the expression vector, pHA12-cadA-acnB.

3.3.1 Cell membrane behaves as a barrier for substrate uptake

First, to clarify if itaconic production was really blocked by cell membrane barrier, an E. coli
S17-1 strain harboring the recombinant plasmid mentioned above expressing AcnB and CAD
was tested for itaconic acid production. The cell suspension was mixed with 50 mM citric acidto produce itaconic acid at 37 °C. The cells were also disrupted by sonication to remove the cell
membrane completely for itaconic acid production. As the result showed in Fig. 3.2, the cell
extract produced 37.6 mM itaconic acid in 3 hours while the intact cells only produced 4.3 mM,
revealing a severe problem on substrate uptake. Because itaconic acid production of the cell
44

extract represents the production without any influence from cell membrane, the ration of intact
cells productivity to the cell extract productivity is considered be used as an indicator for
membrane permeability for subsequent experiments.

40
Itaconic acid (mM)

35
30
25

20
15
10
5
0
Cell
cell extract

Intactintact cells

Fig. 3.2 Itaconic acid production by cell extract and intact cells of recombinant E. coli
Error bars represent the standard deviation of the mean (n = 3).

3.3.2 Effects of heat treatment on improvement of membrane permeability

Itaconic acid productivity of recombinant S. livingstonensis Ac10 cells was measured toevaluate membrane permeability. As a comparison, itaconic acid productivity of recombinant
E. coli S17-1 constructed for conjugation was also measured at the same conditions. The
percentage productivity of intact cells to that of their cell extracts was calculated (Fig. 3.3).
When the conversion was performed at 28 °C for 1 hour without heat treatment, itaconic acidproductivities were only 2.7% and 2.6% of their cell extracts (E. coli S17-1 vs S.
livingstonensis Ac10 cells). It suggests that the cell membrane behaved as a powerful barrier
for citric acid uptake. However, the productivity ratio of recombinant S. livingstonensis cells
45

after heat treatment at 40 °C for 15 min was improved to 14.2% while only a slight increase(up to 3.7%) was detected in recombinant E. coli cells treated at the same condition.

16

Percentage productivity

14
12
10
8
6
4
2
0
E. coli

S. livingstonensis

Fig. 3.3 Percentage productivity of recombinant E. coli and S. livingstonensis Ac10 cells totheir cell extract respectively. Orange, cells without heat treatment. Blue, cells with heat
treatment (40 °C, 15 min).

3.3.3 Optimal conditions for heat treatment

Optimal conditions for heat treatment were investigated to improve the permeability of the cell
membrane and to achieve high itaconic acid productivity. S. livingstonensis cells expressing
CAD and AcnB were collected and treated at various temperatures ranging from 35 °C to 50 °C
for 15 minutes before they were mixed with citric acid to produce itaconic acid. The results
showed that heat treatment at 45 °C significantly enhanced itaconic acid productivity. As shownin Fig. 3.4a, the productivity in the cells with heat treatment improved approximately 6-fold(0.22 to 1.41 g/L/h) compared to that in the cells without heat treatment.

46

Itaconic acid productivity (g/L/h)

a 11.5
1.2
0.9
0.6
0.3
0

Control
control

35

40

45

50
Temperature (ȭ)

100

b 90
Relative activity (%)

80
70
60
50
40
30
20
10
0
Control
control

35

40

45

50

Temperature (ȭ)
Fig. 3.4 Itaconic acid productivity of recombinant S. livingstonensis Ac10 cells which weretreated at various temperatures. The control group comprises cell suspensions kept at 28 °C
before the reaction (a). Thermostability of crude enzyme treated at various temperatures, theconditions for heat treatment and reaction are the same with the experiment described before(b).
47

Further increase of heat treatment temperature provides no help for improving itaconic acidproductivity because CAD is possibly unstable at such high temperatures (58). To clarify the
influence of heat treatment on the enzyme activity, cell extracts were also tested in the same
condition. Surprisingly, only 43.85% of the enzyme activity was remained when the cell
extracts were treated at 45°C for 15 min (Fig. 3.4b).
To detect whether any enzyme leakage caused by heat treatment, the cell suspension after heat
treatment incubated at 28°C for 1 hour to simulate the process of the itaconic acid production.
After incubation, the cell suspension was separated into supernatant and sediment (cells) bycentrifugation (5,000 × g, 10 min, 4°C). Both productivities of the supernatant and the cells
were measured by mixed with 50 mM citric acid and 200 mM PB (pH = 6.5) and incubated at
28°C for 1 hour. The results (Fig. 3.5) showed that the permeability of S. livingstonensis Ac10cell membrane was indeed improved because itaconic acid productivity in the sediment of thecell suspension was significantly enhanced by heat treatment at 45 °C for 15 min comparedwith cells without heat treatment (0.21 to 0.80 g/L/h). Slight itaconic acid productivity (0.11g/L/h at 45 °C) was detected in the supernatant, indicating that a limited amount of enzymes
leaked out from the cells.

48

Itaconic acid productivity(g/L/h)

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Control
control

35

40

45

50

Temperature (ȭ)
Fig. 3.5 Itaconic acid productivity of the supernatant and the sediment (cells). Orange bars,
sediment. Blue bars, supernatant.

Optimal time for heat treatment was also investigated. Recombinant S. livingstonensis Ac10cell suspension and cell extracts were treated at 45°C for 5 to 30 minutes. Itaconic acidproduction was then performed in conditions described above. The result indicated that longheat treatment time did improve the productivity, but the improvement was limited after 10
minutes (Fig. 3.6a). While cell extracts heat-treated for 10 and 15 minutes retained 87.0% ofactivities (Fig. 3.6b), and heat treatment longer than 15 minutes damaged the enzyme activity(75.9% for 20 minutes and 70.1% for 30 minutes). Heat treatment for 15 minutes was applied
for all production experiments later considering facts that 15 minutes did help to increase theproductivity compared with shorter time period and time period longer than 15 minutes
influenced the enzyme activity but did not improve the overall productivity too much.

49

Itaconic acid productivity (g/L/h)

a 1.5
1.2
0.9
0.6

0.3
0

5

10

15

20

30

Treatment time (min)

b 100
90
Relative activity (%)

80
70
60
50

40
30
20
10
0
5

10

15

20

30

Treatment time (min)Fig. 3.6 Itaconic acid productivity of recombinant S. livingstonensis Ac10 cells treated at
45 °C for various time periods. Error bars represent the standard deviation of the mean (n = 3)(a). Relative activities of cell extracts, the conditions for heat treatment and reaction are thesame with the experiment described before (b).

50

After exploring the optimal conditions for heat treatment, itaconic acid production was
performed by this whole-cell biocatalyst heat-permeabilized at optimal conditions.
Recombinant cells were heat-treated at 45°C for 15 min and then reacted with 50 mM citricacid as substrate at 28°C (Fig. 3.7). The yield (itaconic acid production / citric acid & cisaconitic acid consumption) was 110.69%, probably because some substrates were absorbed by
the cells thus could not be observed in the reaction mixture.

Concentration (mM)

60

50
40
30
20
10
0
0

10

20

Reaction time (h)Fig. 3.7 Time-profile of itaconic acid production by heat-permeabilized S. livingstonensis Ac10
cells at 45°C for 15 min. Rectangle, citric acid; circle, itaconic acid; triangle, cis-aconitic acid.

51

3.3.4 Optimal reaction temperature for itaconic acid production

Next, Itaconic acid production was performed at higher temperatures ranging from 35 °C to
55 °C for 1 hour with 500 mM citric acid as substrate. Maximal itaconic acid productivity
appeared at 50 °C (Fig. 3.8a). The productivity declined when the reaction temperature wasover 50 °C and a middle product, cis-aconitic acid accumulated (Fig. 3.8b). This suggested
that CAD might be inactivated. The cells were also tested for recycling after the reaction at
50 °C for 6 hours (Fig. 3.9a), only 0.16 g/L/h productivity was detected while 5.70 g/L/h forthe first batch (Fig. 3.9b). Considering the poor recyclability of cells at 50 °C, itaconic acidproduction was still performed at 28 °C despite better reaction rate.

52

Itaconic acid productivity (g/L/h)

a

18
16
14

12
10
8
6
4
2
0
28

35

40

45

55

Temperature (ȭ)

b 18
cis-Aconitic acid concentration (mM)

50

16
14
12
10
8
6
4
2
0
28

35

40

45

50

55

Temperature (ºC)
Fig. 3.8 Optimal reaction temperature for itaconic production by recombinant S.
livingstonensis Ac10 cells after heat permeabilization (a). cis-Aconitic acid accumulation
after production (b). Error bars represent the standard deviation of the mean (n = 3).

53

Concentration (mM)

a 600
500
400
300
200
100
0
0

1

2

3

4

5

6

Reaction time (h)Itaconic acid productivity (g/L/h)

b 7
6
5
4
3
2
1
0
1st

2nd

Fig. 3.9 Time-profile of itaconic production at 50 ºC by recombinant S. livingstonensis Ac10
cells after heat permeabilization (a). Rectangle, citric acid; circle, itaconic acid; triangle, cisaconitic acid. Itaconic acid productivity of recycled cells (b). Error bars represent the standard
deviation of the mean (n = 3).

54

3.3.5 Recycling of cells after heat treatment

After confirmation of no severe enzyme leakage after heat treatment, the sustainability of heat-treated S. livingstonensis Ac10 cells was investigated in terms of itaconic acid production.
Recombinant cells were heated at 45 ºC for 15 min and applied for itaconic acid production at
28 °C in 15 mL tubes with 5 mL reaction mixture containing 50 mM citric acid. After a 10-h
reaction, cells were separated via centrifugation and mixed with new reaction mixture.
Concentration of itaconic acid was recorded at both start and end points of each batch so thatitaconic productivity for each batch could be calculated. The result showed that after heat
treatment, itaconic acid productivity of recombinant cells reached 0.47 g/L/h, and the
productivity remained almost stable during the first four batches (Fig. 3.10). Notably, 67.3% of
the productivity was observed in the fifth batch, revealing that the cell membrane integrity was
still preserved to some extent after reactions. In contrast, when recombinant cells were washedby 50 mM PB buffer (pH = 6.5) once after each reaction, the productivity declined dramaticallyafter the second batch suggesting that the cell membrane was fragile after heat treatment andmay be disrupted by mechanical impact of pipetting.

55

Itaconic acid productivity(g/L/h)

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1st

2nd

3th

4th

5th

Fig. 3.10 Itaconic acid production by recycled recombinant S. livingstonensis Ac10 cells after
heat permeabilization. Cells were separated from the reaction mixture every 10 hours via
centrifugation and resuspended by the reaction mixture. Orange bars, cells without washingafter each batch; Blue bars, cells washed with phosphate buffer (50 mM, pH = 6.5) after eachbatch. Error bars represent the standard deviation of the mean (n = 6).

3.3.6 Itaconic acid production was limited by product inhibition

It was estimated that costs of downstream process in itaconic acid production take up nearly
half of the whole cost (59). Therefore, if high final concentration is achieved, it will
significantly decrease the production cost. In 3.3.4, 500 mM citric acid was added to performthe itaconic acid production, but only approximately half of them were converted to itaconic
acid, suggesting that the process may be inhibited by itaconic acid. To clarify if the reaction
was inhibited by itaconic acid, cell suspension of PSCats was mixed with reaction mixture
containing 500 mM citric acid and various concentrations of itaconic acid. As showed in Fig.
3.11, itaconic acid production decreased with more itaconic acid added in advance.

56

Itaconic acid (mM)

200
150
100
50
0
0

50

100

150

Itaconic acid loading (mM)
Fig. 3.11 Itaconic acid production by PSCat at 37 °C for 6 hours with various concentrationsof itaconic acid added in advance. Error bars represent the standard deviation of the mean (n =3).
One of the crucial enzymes involved in itaconic acid production, CAD was then cloned,
expressed, and purified from recombinant E. coli to further confirm the inhibition. Product

Relative activities (%)

inhibition was also confirmed on purified CAD (Fig. 3.12).

100
90
80
70
60
50
40
30
20
10
0
0

50

75

100

150

Itaconic acid loading (mM)
Fig. 3.12 CAD activities in existence of extra itaconic acid in the reaction mixture.

57

3.3.7 Identification of residues related to itaconic acid binding

Two pockets were found in the CAD (Fig. 3.13), pocket A is a putative active site, and pocket
B is a putative itaconic acid binding site identified by docking simulation. There are 15 polaramino acid residues in these two pockets. To clarify if they are really associated with substrateor product binding, alanine substitutions were constructed and tested for enzyme activities. The
result (Table 4.1) shows that most of the alanine mutants were completely inactivated and only
S374A showed comparable activities, suggesting that these residues are indeed involved in
substrate or product binding which means they are potential targets for protein engineering.

Pocket B

Pocket A

Fig. 3.13 Two putative pockets involved in substrate or product binding in A. terreus CAD,
residues in pocket A: R54, W57, T99, S348, Q352, S374, E375; Residues in pocket B: E100,
Y104, H111, H168, K217, K288, C293, W339.

58

Table 3.2 Activities of alanine substitutions of each residue in the pockets:
Pocket A:
R54A

W57A

T99A

S348A

Q352A

S374A

E375A

ND

ND

25.6%

ND

71.1%

ND

Y104A

H111A

H168A

K217A

K288A

C293A

W339A

ND

ND

ND

ND

ND

ND

ND

Activities ND
Pocket B:
E00A
Activities ND

3.3.8 Library screening

All possible variants of 15 residues were constructed, among those which are already tested, 11of them (R54H, R54K, T99I, T99C, E100W, H168L, K217Q, W339N, S348V, S374N, E375P)
showed comparable activities to the wild type but none of them can produce more itaconic acidfrom 500 mM cis-aconitic acid than the wild type.

59

3.3.9 Shewanella frigidimarina based whole-cell biocatalyst is not permeabilized by heat
treatment

At first, a whole-cell biocatalyst based on a related species of S. livingstonensis was also
constructed. Itaconic acid productivity of S. frigidimarina based whole-cell catalyst can also be
improved by heat treatment. However, unlike S. livingstonensis, severe enzyme leakage wasobserved even without heat treatment (Fig. 3.14). This provides a clue for the mechanism of
permeability change on S. livingstonensis because the two strains are assumed to be similar as
described in chapter 2. By investigating the difference in membrane of two species, themechanism of permeability improvement may be revealed.

9

Itaconic acid(mM)

8
7
6
5
4
3
2
1
0

Supernatantsupernatant

Sediment
sediment

Fig. 3.14 Itaconic acid production by S. frigidimarina based whole-cell catalyst. Blue bar,without heat treatment, orange bar, with heat treatment at 40 °C for 5 min. Error bars represent
the standard deviation of the mean (n = 3).

60

3.3.10 FAME composition of S. frigidimarina and S. livingstonensis

Phospholipids composition of S. frigidimarina and S. livingstonensis Ac10 was analyzed. The
results are showed in Fig. 3. 15, of all five FAMEs detected by GC/MS, none of them showeda clear difference in amount between S. frigidimarina and S. livingstonensis, indicating that it
is not the difference in phospholipids that causes the different behavior of these two strains after
the heat treatment. The mechanism behind permeability improvement is therefore still unclear.

1.2E+10
Area of peaks

1E+10
8E+09
6E+09
4E+09
2E+09
0

13:0
(13:0)

14:0(14:0)

15:0(15:0)

16:0(16:0)

16:1(16:1)

carbon:unsaturation

Fig. 3.15 FAME analysis result of S. frigidimarina and S. livingstonensis. Blue bar, S.
livingstonensis, orange bar, S. firigidimarina.

61

3.4 Discussion

One of the major obstacles to the application of whole-cell biocatalyst is that cell membrane of
the whole-cell biocatalyst host often serves as a barrier for substrate influx. Present chapter
described a simple method to resolve the permeability issue by constructing whole-cellbiocatalyst based on psychrophile. The results in this chapter indicate that the cell membraneof S. livistonensis Ac10 could be permeabilized at moderate conditions. The improvement on
permeability was so effective that even the enzyme was partly inactivated, itaconic acidproductivity of the whole-cell biocatalyst still increased significantly.
Generally speaking, high membrane fluidity is required for psychrophiles to grow andreproduce at a low temperature. However, it seems that it is not high membrane fluidity thatfacilitates citric acid influx because when recombinant S. livingstonensis Ac10 cells wereintended for itaconic acid production without heat treatment, no significant difference inpermeability was observed in comparison with E. coli cells. The reason underlying theimprovement in membrane permeability in S. livingstonensis Ac10 after heat treatment
remained unexplored. Some previous studies provide evidence to support this improvement inmembrane permeability. The cell membrane of E. coli could be permeabilized at 55 °C due tomembrane lipid loss, thereby increasing the porosity (60, 61); Similar phenomenon may occur
at a lower heat treatment temperature for the psychrophilic cell membrane considering different
lipid composition. Moreover, some membrane-bound molecules in psychrophiles may also be
related to the membrane fluidity. For example, carotenoids are reportedly associated withregulating the membrane fluidity of some psychrophiles (62). A type of these pigment
molecules in the cell membrane may dissociate from the cell membrane due to heat treatment,62thereby increasing the porosity of the cell membrane and resulting in more efficient membranetranslocation of the substrate.
Based on current results, it is possible to recycle cells after heat treatment, but low reaction
temperature and avoiding furious mechanical impacts are necessary because the cell membrane
becomes vulnerable after heat treatment. The cell membrane integrity still remained, and a largepart of itaconic acid productivity was preserved after the heat treatment when the cells weretreated probably. Considering that it is difficult to recycle the cells permeabilized by surfactant
because of cell lysis, psychrophile-based biocatalyst may be more suitable for industrial
application to lower down the cost of culturing. Placing heat-treated in a more stableenvironment such as crosslinking the cells with the surface of specific materials (63) may help
to improve the performance of them. The reaction temperature is another essential factor in
influencing membrane integrity. 28 °C was chosen because S. livingstonensis Ac10 cannot growat a temperature beyond 30 °C, the reaction temperature was slightly lower than it rather thanthe optimal temperature for itaconic acid production. The mechanism underlying theimprovement in membrane permeability due to heat treatment remained unexplored, tointensify membrane tolerance to heat treatment and the reaction temperature, it is crucial tounderstand them in the future.
Product inhibition was confirmed both on the whole-cell biocatalyst and purified CAD, and it
may be the reason for underachievement of high titer production. Considering the similarity of
citric acid and itaconic acid on structure and chemical property, it may be difficult to separate
them after the reaction. An attempt to engineer CAD to remove the product inhibition throughdirected-mutation was described.
63

Protein engineering is another powerful tool to eliminate or at least alleviate product inhibition(64). Traditionally, a random mutation library of the target protein was generated through ErrorProne PCR, the mutations were then tested in a high-throughput screen method to selectdesirable features. With the development of computer science and bioinformatics, it becomes
possible to simulate interactions between enzymes and small molecules for example theirsubstrates or inhibitors and delimit the scale of residue mutations. In this way, a library
containing a small number of mutations could be constructed therefore reduce the labor for
screening significantly (65).
Since crystal structure of mouse CAD has been identified recently (66), a docking simulation
between CAD and its inhibitor, itaconic acid was performed, based on the docking result alibrary containing 285 mutants was constructed and some of them were tested for their itaconic
acid tolerance. Unfortunately, no tested mutations showed better performance than the wild
type.
Phospholipids composition of S. livingstonensis was analyzed and compared to a related species,
S. frigidimarina, which cannot be permeabilized by heat treatment but lose some enzymes
because the cell membrane is damaged. No difference in phospholipids composition was foundbetween these two species and the mechanism of heat permeabilization remained unclear.

64Chapter 4 General discussion and conclusion
Enzymes are essential components for biomass-based chemical industry, and whole-cellbiocatalyst is one of the most effective tools human beings have to introduce enzymes into
industry field. However, despite those great achievements which have been made, the fields for
enzymatic reactions to step in and replace traditional processes are still limited. Pyschrophilebased simple biocatalyst is a new approach to solve some of these problems. The present
research focuses on two limitations of whole-cell biocatalyst, side reactions caused by host
homologous enzymes and cell membrane barrier which blocks the substrate influx in the wholecell biocatalysts.
In chapter 2, an overall investigation of enzyme thermostability in S livingstonensis Ac10 was
made. An NADP-dependent malic enzyme (SL-ME) with unexpected thermostability was
found. It has operational temperatures even higher than its mesophilic counterpart, an NADPdependent malic enzyme from E. coli (MaeB). SL-ME was cloned and expressed in E. coli,
purified SL-ME was used to investigate its enzymatic characteristics including kinetic
parameters, thermostability measured both by specific activities after heat treatment and by CD
ellipticity at 222 nm, as well as its operational temperature range. The results clearly show that
SL-ME does not share any similarity in terms of thermostability and functional temperature
range with other homologous enzymes in S. livingstonensis Ac10. Based on sequence alignment
with malic enzyme genes from other Shewanella species, malic enzyme genes are highly
conserved among those species no matter in mesophilic or psychrophilic bacteria suggestingthat malic enzyme may play a crucial role in Shewanella species.

65Psychrophilic bacteria were assumed to be heat-vulnerable and that is the reason why they werechosen as the host for whole-cell biocatalysts. By all of 8 organic acid relevant enzymes
investigated, only SL-ME is thermostable and the rest of them are all inactivated at moderatetemperatures. Therefore, the concept that expressing mesophilic enzymes in psychrophile andthen inactivate host homologous enzymes is proven to be realistic. Discovery of thermostablemalic enzyme in psychrophilic bacterium suggests that there may be some reactions that could
be distracted by homologous enzymes and knowledge on the host enzyme’s thermostability will
pave the way for us to design and introduce new pathways to the host cell.
In chapter 3, a new approach was developed to solve the permeability issue of whole cellbiocatalyst. A whole-cell biocatalyst based on psychrophilic bacterium S. livingstonensis Ac10,
was constructed. The cells express two crucial enzymes, aconitase B from E. coli and cisaconitic acid decarboxylase from A. terreus, for itaconic acid production from citric acid. Cells
were heat-treated before application for itaconic acid production.
Optimal conditions for heat treatment to improve the membrane permeability and preserve the
enzymatic activity simultaneously. Optimal reaction temperature for itaconic acid production
was also explored. The optimal reaction temperature was 50 °C but the cells lost theirrecyclability after reaction at such a high temperature.
Itaconic acid productivity was improved approximately 6 folds by heat treatment at 45 °C for
15 minutes. And the cells could be recycled up to 5 times after the reaction. According to Kim
et al. (48), citric acid is difficult to be taken by E. coli based whole-cell biocatalyst. To solve
this problem, they used detergent to lyse the cells while in the meantime the membrane integrity
is lost. Considering the price of itaconic acid and its role as a basic building block for chemical
66

industry, low production is definitely desirable. Therefore, it is favorable to recycle cells after
the production.
Compared to other methods to improve membrane permeability such as surfactant treatment orsolvent treatment, heat treatment is more feasible for industrial application because it does not
introduce any additional molecules in the reaction mixture thus simplifies downstream process
for purifying the product. In this chapter, an attempt to produce a high titer of itaconic acid wasmade but failed, probably because of the product inhibition. Current chapter is aiming to
remove the product inhibition through directed mutation of CAD.
Final titer of the product is also an important parameter which need to be considered whenconstructing a real-world process because a high titer will significantly decrease thedownstream cost. Product inhibition has been the main obstacle for bioconversion (67). One of
the most well-known product inhibition cases is Acetone Butanol Ethanol (ABE) production,in which the production of multiple solvents, especially butanol (68) is strictly limited. Taking
the advantage of volatility of the solvent, a gas stripping approach has been developed toremove products from the reaction mixture simultaneously. The final titer has been increased
significantly by gas stripping, final titer of ABE production by Clostridium beijerinckii BA101
can be increased from 27-29 g/L to an astonishingly 165g/L. Since citric acid and itaconic acidare not as different as glucose and organic solvent involved in ABE production, protein
engineering was selected to remove or at least alleviate the product inhibition. Unfortunately,
no promising mutants were found indicating the catalyzing efficiency of A. terreus CAD may
already reach the optimal range. Other methods may need to be considered to improve the tierof itaconic acid such as In Situ Product Removal (ISPR) Strategies (69).
67The present study demonstrates the potential of psychrophilic bacterium S. livingstonensis
Ac10 as the host for whole-cell biocatalyst to produce itaconic acid from citric acid and thefeasibility of increasing the permeability of the cell membrane by heat treatment at a moderatecondition. Based on the present knowledge, no previous study has focused on the methods toincrease cell membrane permeability of psychrophiles.
In summary, current thesis explored the potential of S. livingstonensis Ac10 as a host for wholecell biocatalyst as well as develop a promising process for producing valuable chemicals. By
investigating several key enzymes involved in organic acid metabolism, the fact that most ofthem could be heat inactivated has been confirmed. Which supports the very basic concept of
psychrophile-based simple biocatalyst, e.g., introducing heterologous enzymes frommesophiles into psychrophiles, and then inactivate homologous enzymes in hosts to eliminate
side reactions thus improve the yield of target products. There is also an unexpected discovery
of a thermostable malic enzyme in S. livingstonensis Ac10. The enzyme was characterized but
the mechanism laying behind its irregular thermostability along with its role in metabolism isstill unclear. Itaconic acid was selected as the target product because its production is reportedly
impeded by the cell membrane barrier and no ideal solutions have been found yet. To improve
substrate permeability, cells were treated by heat and, under the optimal condition for heat
treatment and enzyme activity, productivity of itaconic could be increased significantly and thecells could be reused after the production. A better understanding of the membrane structure
and the mechanism to increase its permeability is necessary for further development of thisapproach and its industrial application.

68

Acknowledgements
This doctoral thesis is submitted to the Graduate School of Advanced Science of Matter,
Hiroshima University. The research was conducted in the Laboratory of BiomolecularTechnology, Department of Molecular Biotechnology, Graduate School of Advanced Sciencesof Matter, Hiroshima University, during the academic periods from 2018 to 2021.
I would like to express the most profound appreciation to my former advisor, Prof. Junichi Kato,and current advisor Prof . Takahisa Tajima, who have been supporting me before and throughout
my Doctoral course and thesis. Their knowledge and patience have always been surprising andencouraging me. The guidance of their intelligentsia gave me the opportunity to pursue myresearch in my own way. Their wise support conveyed the spirit of adventure in regard toresearch and teaching me. Without their trust and persistent help, this project would not havebeen possible.
My sincere thanks also go to Prof. Yoshihiro Sambongi and Assistant Professor Sotaro Fujii in
Graduate School of Integrated Sciences for Life, Hiroshima University for their valuableguidance, assistance, and technical skills. I will never forget his influential role and kindness inmaking this project a success.
I would like to thank all the staff of the Laboratory of Biomolecular Technology (Associate
Professor Makoto Fujie, Assistant Professor Takeru Kawasaki, and Assistant Professor Akiko
Hida) for their valuable discussion and encouragement.
I would also like to thank my fellow researchers, Mr. Sho Nakano, Ms. Miho Fujino and Mr.
Takumi Koda), who helped me a lot in this project. I thank Professor Tatsuo Kurihara at Kyoto

69University for providing S. livingstonensis Ac10.
I would also like to thank my dissertation committee members, Professor Yutaka Nakashimada
and Professor Akio Kuroda, who offered insightful comments.
Special thanks to my friend Dr. Mohammad Mojarrad who has been working with me in thesame place for 3 years. I’m lucky to have him suffering with me.
Finally, I want to thank my family for supporting and encouraging me to make this possible.

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80

Appendix
Primers used for mutation library construction
Primer name

Sequence (5’ - 3’)

Ala substitution
R54A_F

ATGGGTTGGCGCTGCTGTG

R54A_R

GCGCAGGCAATACCGTCTAA

W57A_FTGCCAGCTAGTGAGAAATATG

W57A_RCACGAGCGCCAACCCATGC

T99A_F

AGCTGCTGAATTAGACGATTATC

T99A_RTGAATGAATGCACTATTAG

E100A_FAGCTACCGCTTTAGACGATTATC

E100A_RTGAATGAATGCACTATTAGTCY104A_FAGACGATGCTCATAGTGAAGCAC

Y104A_R

AATTCGGTAGCTTGAATGAATGC

H111A_F

AAGCACCTTTAGCTAGTGCTAGTATH111A_RCACTATGATAATCGTCTAATTC

H168A_F

ACGGATGGGCTTGCGGAGCGGT

H168A_R

TATTAAGCAAGTCAGAGCCGTK217A_F

GTATGGTTGCTCGCGTTCAGCA

K217A_R

CTCCATATTGGGCCGACATCA

K288A_F

ATCCGTATCGCTCTTTACGCATG

K288A_RGGTAAAGGTATGCCAAAAGCT
81

Primers used for mutation library construction (continued)
C293A_F

ACGCATGTGCTGGTTTAGTCCATG

C293A_R

AAAGTTTGATACGGATGGTAAAG

W339A_F

ACATTGTGGTGCTATTCCTGAAGAGC

W339A_R GAATTAGAGGCGGTTGATAAC
S348A_F

CGATTAGCGCTATTGCCGGTCAAAT

S348A_R

GGCGCTCTTCAGGAATCCA

Q352A_F

GGTGCTATGTCAGTGGCCTAT

Q352A_R

GGCAATGCTGCTAATCGGGCG

S374A_F

CAATTTGCTGAATTTGATGATAATS374A_R

GGCTAAAAGACATTGTTGGT

E375A_F

ATTTTCAGCTTTTGATGATAAT

R54
R54F_F

GGGTTGGCGCTTTCGTGCCATGGAG

R54F_RCTCCATGGCACGAAAGCGCCAACCC

R54L_F

GGGTTGGCGCTCTGGTGCCATGGAG

R54L_RCTCCATGGCACCAGAGCGCCAACCC

R54I_F

GGGTTGGCGCTATTGTGCCATGGAG

R54I_RCTCCATGGCACAATAGCGCCAACCC

R54M_F

ATGGGTTGGCGCTATGGTG

R54S_F

GGGTTGGCGCTAGCGTGCCATGGAG

82

Primers used for mutation library construction (continued)
R54S_RCTCCATGGCACGCTAGCGCCAACCC

R54T_F

GGGTTGGCGCTACGGTGCCATGGAG

R54T_RCTCCATGGCACCGTAGCGCCAACCC

R54K_F

ATGGGTTGGCGCTAAGGTG

R54Y_F

ATGGGTTGGCGCTTATGTG

R54H_F

GGGTTGGCGCTCATGTGCCATGGAG

R54H_RCTCCATGGCACATGAGCGCCAACCC

R54Q_F

ATGGGTTGGCGCTCAGGTG

R54N_F

ATGGGTTGGCGCTAACGTG

R54D_F

GGGTTGGCGCTGACGTGCCATGGAG

R54D_RCTCCATGGCACGTCAGCGCCAACCC

R54E_F

ATGGGTTGGCGCTGAGGTG

R54W_F ATGGGTTGGCGCTTGGGTG
R54C_F

GGGTTGGCGCTTGCGTGCCATGGAG

R54C_RCTCCATGGCACGCAAGCGCCAACCC

R54P_F

GGGTTGGCGCTCCGGTGCCATGGAG

R54P_RCTCCATGGCACCGGAGCGCCAACCC

R54G_F

GGGTTGGCGCTGGCGTGCCATGGAG

R54G_RCTCCATGGCACGCCAGCGCCAACCC

R54V_F

ATGGGTTGGCGCTGTGGTG

83

Primers used for mutation library construction (continued)
W57
W57F_FTGCCATTCAGTGAGAAATATG

W57L_FTGCCACTGAGTGAGAAATATG

W57I_FTGCCAATTAGTGAGAAATATG

W57M_F TGCCAATGAGTGAGAAATATG
W57S_FTGCCAAGCAGTGAGAAATATG

W57T_F

TCGTGTGCCAACGAGTGAGAAATATW57T_R

GCGCCAACCCATGCGCAGGCAAT

W57K_FTGCCAAAGAGTGAGAAATATGW57Y_FTGCCATATAGTGAGAAATATG

W57H_FTGCCACATAGTGAGAAATATG

W57Q_FTGCCACAGAGTGAGAAATATG

W57N_FTGCCAAACAGTGAGAAATATG

W57D_FTGCCAGACAGTGAGAAATATG

W57E_FTGCCAGAGAGTGAGAAATATG

W57C_FTGCCATGCAGTGAGAAATATG

W57R_FTGCCACGTAGTGAGAAATATG

W57P_FTGCCACCGAGTGAGAAATATG

W57G_FTGCCAGGCAGTGAGAAATATG

W57V_FTGCCAGTGAGTGAGAAATATG

T99

84

Primers used for mutation library construction (continued)
T99F_F

TCATTCAAGCTTTCGAATTAGACGAT99F_RTCGTCTAATTCGAAAGCTTGAATGA

T99L_FAGCTCTGGAATTAGACGATTATC

T99I_F

TCATTCAAGCTATTGAATTAGACGA

T99I_RTCGTCTAATTCAATAGCTTGAATGA

T99M_FAGCTATGGAATTAGACGATTATC

T99S_FAGCTAGCGAATTAGACGATTATC

T99K_FAGCTAAGGAATTAGACGATTATC

T99Y_F

AGCTTATGAATTAGACGATTATC

T99H_FAGCTCATGAATTAGACGATTATC

T99Q_FAGCTCAGGAATTAGACGATTATC

T99N_FAGCTAACGAATTAGACGATTATC

T99D_F

AGCTGACGAATTAGACGATTATC

T99E_F

AGCTGAGGAATTAGACGATTATC

T99W_F AGCTTGGGAATTAGACGATTATC
T99C_F

AGCTTGCGAATTAGACGATTATC

T99R_FAGCTCGTGAATTAGACGATTATC

T99P_FAGCTCCGGAATTAGACGATTATC

T99G_F

AGCTGGCGAATTAGACGATTATC

T99V_F

TCAAGCTGTGGAATTAGACGAT

T99V_FTGAATGAATGCACTATTAG

85

Primers used for mutation library construction (continued)
E100
E100F_FAGCTACCTTCTTAGACGATTATC

E100L_FAGCTACCCTGTTAGACGATTATC

E100I_FAGCTACCATTTTAGACGATTATC

E100M_FAGCTACCATGTTAGACGATTATC

E100S_FAGCTACCAGCTTAGACGATTATC

E100T_FAGCTACCACGTTAGACGATTATC

E100K_FAGCTACCAAGTTAGACGATTATC

E100Y_FAGCTACCTATTTAGACGATTATC

E100H_FAGCTACCCATTTAGACGATTATC

E100Q_FAGCTACCCAGTTAGACGATTATC

E100N_FAGCTACCAACTTAGACGATTATC

E100D_FAGCTACCGACTTAGACGATTATC

E100W_F AGCTACCTGGTTAGACGATTATC
E100C_FAGCTACCTGCTTAGACGATTATC

E100R_FAGCTACCCGTTTAGACGATTATC

E100P_FAGCTACCCCGTTAGACGATTATC

E100G_FAGCTACCGGCTTAGACGATTATC

E100V_FAGCTACCGTGTTAGACGATTATC

Y104
Y104F_FAGACGATTTCCATAGTGAAGCAC

86

Primers used for mutation library construction (continued)
Y104L_FAGACGATCTGCATAGTGAAGCAC

Y104I_FAGACGATATTCATAGTGAAGCAC

Y104M_FAGACGATATGCATAGTGAAGCAC

Y104S_FAGACGATAGCCATAGTGAAGCAC

Y104T_FAGACGATACGCATAGTGAAGCAC

Y104K_FAGACGATAAGCATAGTGAAGCAC

Y104H_FAGACGATCATCATAGTGAAGCAC

Y104Q_FAGACGATCAGCATAGTGAAGCAC

Y104N_FAGACGATAACCATAGTGAAGCAC

Y104D_FAGACGATGACCATAGTGAAGCAC

Y104E_FAGACGATGAGCATAGTGAAGCAC

Y104W_F AGACGATTGGCATAGTGAAGCAC
Y104C_FAGACGATTGCCATAGTGAAGCAC

Y104R_FAGACGATCGTCATAGTGAAGCAC

Y104P_FAGACGATCCGCATAGTGAAGCAC

Y104G_FAGACGATGGCCATAGTGAAGCAC

Y104V_FAGACGATGTGCATAGTGAAGCAC

H111
H111F_F

AAGCACCTTTATTCAGTGCTAGTATH111F_R

ATACTAGCACTGAATAAAGGTGCTT

87

Primers used for mutation library construction (continued)
H111L_F

AAGCACCTTTACTGAGTGCTAGTATH111L_R

ATACTAGCACTCAGTAAAGGTGCTT

H111I_F

AAGCACCTTTAATTAGTGCTAGTATH111M_F

AAGCACCTTTAATGAGTGCTAGTATH111S_F

AAGCACCTTTAAGCAGTGCTAGTATH111S_R

ATACTAGCACTGCTTAAAGGTGCTT

H111T_F

AAGCACCTTTAACGAGTGCTAGTATH111T_R

ATACTAGCACTCGTTAAAGGTGCTT

H111K_F

AAGCACCTTTAAAGAGTGCTAGTATH111K_R

ATACTAGCACTCTTTAAAGGTGCTT

H111Y_F

AAGCACCTTTATATAGTGCTAGTATH111Q_F

AAGCACCTTTACAGAGTGCTAGTATH111Q_R

ATACTAGCACTCTGTAAAGGTGCTT

H111N_F

AAGCACCTTTAAACAGTGCTAGTATH111N_R

ATACTAGCACTGTTTAAAGGTGCTT

H111D_F

AAGCACCTTTAGACAGTGCTAGTATH111E_F

AAGCACCTTTAGAGAGTGCTAGTATH111W_F

AAGCACCTTTATGGAGTGCTAGTATH111W_R ATACTAGCACTCCATAAAGGTGCTT
H111C_F

AAGCACCTTTATGCAGTGCTAGTATH111R_F

AAGCACCTTTACGTAGTGCTAGTAT88

Primers used for mutation library construction (continued)
H111R_R

ATACTAGCACTACGTAAAGGTGCTT

H111P_F

AAGCACCTTTACCGAGTGCTAGTATH111P_R

ATACTAGCACTCGGTAAAGGTGCTT

H111G_F

AAGCACCTTTAGGCAGTGCTAGTATH111V_F

AAGCACCTTTAGTGAGTGCTAGTAT

H168
H168F_F

ACGGATGGTTCTGCGGAGCGGT

H168L_F

ACGGATGGCTGTGCGGAGCGGT

H168I_F

ACGGATGGATTTGCGGAGCGGT

H168M_F

ACGGATGGATGTGCGGAGCGGT

H168S_F

ACGGATGGAGCTGCGGAGCGGT

H168T_F

ACGGATGGACGTGCGGAGCGGT

H168K_F

ACGGATGGAAGTGCGGAGCGGT

H168Y_F

ACGGATGGTATTGCGGAGCGGT

H168Q_F

ACGGATGGCAGTGCGGAGCGGT

H168N_F

ACGGATGGAACTGCGGAGCGGT

H168D_F

ACGGATGGGACTGCGGAGCGGT

H168E_F

ACGGATGGGAGTGCGGAGCGGT

H168W_F ACGGATGGTGGTGCGGAGCGGT

89

Primers used for mutation library construction (continued)
H168C_F

ACGGATGGTGCTGCGGAGCGGT

H168R_F

ACGGATGGCGTTGCGGAGCGGT

H168P_F

ACGGATGGCCGTGCGGAGCGGT

H168G_F

ACGGATGGGGCTGCGGAGCGGT

H168V_F

ACGGATGGGTGTGCGGAGCGGT

K217
K217F_F

GTATGGTTTTCCGCGTTCAGCA

K217L_F

GTATGGTTCTGCGCGTTCAGCA

K217I_F

GTATGGTTATTCGCGTTCAGCA

K217M_F

GTATGGTTATGCGCGTTCAGCA

K217S_F

GTATGGTTAGCCGCGTTCAGCA

K217T_F

GTATGGTTACGCGCGTTCAGCAK217Y_F

GTATGGTTTATCGCGTTCAGCA

K217H_F

GTATGGTTCATCGCGTTCAGCA

K217Q_F

GTATGGTTCAGCGCGTTCAGCA

K217N_F

GTATGGTTAACCGCGTTCAGCA

K217D_F

GTATGGTTGACCGCGTTCAGCA

K217E_F

GTATGGTTGAGCGCGTTCAGCA

K217W_F GTATGGTTTGGCGCGTTCAGCAK217C_F

GTATGGTTTGCCGCGTTCAGCA

90

Primers used for mutation library construction (continued)
K217R_F

GTATGGTTCGTCGCGTTCAGCA

K217P_F

GTATGGTTCCGCGCGTTCAGCA

K217G_F

GTATGGTTGGCCGCGTTCAGCA

K217V_F

GTATGGTTGTGCGCGTTCAGCA

K288
K288F_F

ATCCGTATCTTCCTTTACGCATG

K288L_F

ATCCGTATCCTGCTTTACGCATG

K288I_F

ATCCGTATCATTCTTTACGCATG

K288M_F

ATCCGTATCATGCTTTACGCATG

K288S_F

ATCCGTATCAGCCTTTACGCATG

K288T_F

ATCCGTATCACGCTTTACGCATG

K288Y_F

ATCCGTATCTATCTTTACGCATG

K288H_F

ATCCGTATCCATCTTTACGCATG

K288Q_F

ATCCGTATCCAGCTTTACGCATG

K288N_F

ATCCGTATCAACCTTTACGCATG

K288D_F

ATCCGTATCGACCTTTACGCATG

K288E_F

ATCCGTATCGAGCTTTACGCATG

K288W_F ATCCGTATCTGGCTTTACGCATG
K288C_F

ATCCGTATCTGCCTTTACGCATG

K288R_F

ATCCGTATCCGTCTTTACGCATG

91

Primers used for mutation library construction (continued)
K288P_F

ATCCGTATCCCGCTTTACGCATG

K288G_F

ATCCGTATCGGCCTTTACGCATG

K288V_F

ATCCGTATCGTGCTTTACGCATG

C293
C293F_FTTTACGCATGTTTCGGTTTAGTCCA

C293F_R

TGGACTAAACCGAAACATGCGTAAA

C293L_F

ACGCATGTCTGGGTTTAGTCCATG

C293I_F

ACGCATGTATTGGTTTAGTCCATG

C293M_F

ACGCATGTATGGGTTTAGTCCATG

C293S_F

ACGCATGTAGCGGTTTAGTCCATG

C293T_F

ACGCATGTACGGGTTTAGTCCATG

C293K_F

ACGCATGTAAGGGTTTAGTCCATG

C293Y_F

ACGCATGTTATGGTTTAGTCCATG

C293H_F

ACGCATGTCATGGTTTAGTCCATG

C293Q_F

ACGCATGTCAGGGTTTAGTCCATG

C293N_F

ACGCATGTAACGGTTTAGTCCATG

C293D_F

ACGCATGTGACGGTTTAGTCCATG

C293E_F

ACGCATGTGAGGGTTTAGTCCATG

C293W_F ACGCATGTTGGGGTTTAGTCCATG
C293R_F

ACGCATGTCGTGGTTTAGTCCATG

92

Primers used for mutation library construction (continued)
C293P_F

ACGCATGTCCGGGTTTAGTCCATG

C293G_F

ACGCATGTGGCGGTTTAGTCCATG

C293V_F

ACGCATGTGTGGGTTTAGTCCATG

W339
W339F_F

ACATTGTGGTTTCATTCCTGAAGAGC

W339L_F

ACATTGTGGTCTGATTCCTGAAGAGC

W339I_F

ACATTGTGGTATTATTCCTGAAGAGC

W339M_F ACATTGTGGTATGATTCCTGAAGAGC
W339S_F

ACATTGTGGTAGCATTCCTGAAGAGC

W339T_F

ACATTGTGGTACGATTCCTGAAGAGC

W339K_F

ACATTGTGGTAAGATTCCTGAAGAGC

W339Y_F

ACATTGTGGTTATATTCCTGAAGAGC

W339H_F

ACATTGTGGTCATATTCCTGAAGAGC

W339Q_F

ACATTGTGGTCAGATTCCTGAAGAGC

W339N_F

ACATTGTGGTAACATTCCTGAAGAGC

W339D_F

ACATTGTGGTGACATTCCTGAAGAGC

W339E_F

ACATTGTGGTGAGATTCCTGAAGAGC

W339C_F

ACATTGTGGTTGCATTCCTGAAGAGC

W339R_F

ACATTGTGGTCGTATTCCTGAAGAGC

W339P_F

ACATTGTGGTCCGATTCCTGAAGAGC

93

Primers used for mutation library construction (continued)
W339G_F ACATTGTGGTGGCATTCCTGAAGAGC
W339V_F ACATTGTGGTGTGATTCCTGAAGAGC

S348
S348F_F

CGATTAGCTTCATTGCCGGTCAAAT

S348L_F

CGATTAGCCTGATTGCCGGTCAAAT

S348I_F

CGATTAGCATTATTGCCGGTCAAAT

S348M_F

CGATTAGCATGATTGCCGGTCAAAT

S348T_F

CGATTAGCACGATTGCCGGTCAAAT

S348K_F

CGATTAGCAAGATTGCCGGTCAAATS348Y_F

CGATTAGCTATATTGCCGGTCAAAT

S348H_F

CGATTAGCCATATTGCCGGTCAAATS348Q_F

CGATTAGCCAGATTGCCGGTCAAAT

S348N_F

CGATTAGCAACATTGCCGGTCAAATS348D_F

CGATTAGCGACATTGCCGGTCAAATS348E_F

CGATTAGCGAGATTGCCGGTCAAAT

S348W_F

CGATTAGCTGGATTGCCGGTCAAATS348C_F

CGATTAGCTGCATTGCCGGTCAAATS348R_F

CGATTAGCCGTATTGCCGGTCAAATS348P_F

CGATTAGCCCGATTGCCGGTCAAATGTCA

S348G_F

CGATTAGCGGCATTGCCGGTCAAAT

94

Primers used for mutation library construction (continued)
S348V_F

CGATTAGCGTGATTGCCGGTCAAAT

Q352
Q352F_F

GGTTTCATGTCAGTGGCCTAT

Q352L_F

GGTCTGATGTCAGTGGCCTAT

Q352I_F

GGTATTATGTCAGTGGCCTAT

Q352M_F

GGTATGATGTCAGTGGCCTAT

Q352S_F

GGTAGCATGTCAGTGGCCTAT

Q352T_FGGTACGATGTCAGTGGCCTAT

Q352K_FGGTAAGATGTCAGTGGCCTAT

Q352Y_F

GGTTATATGTCAGTGGCCTAT

Q352H_F

GGTCATATGTCAGTGGCCTAT

Q352N_FGGTAACATGTCAGTGGCCTAT

Q352D_F

GGTGACATGTCAGTGGCCTAT

Q352E_F

GGTGAGATGTCAGTGGCCTAT

Q352W_F GGTTGGATGTCAGTGGCCTAT
Q352C_F

GGTTGCATGTCAGTGGCCTAT

Q352R_F

GGTCGTATGTCAGTGGCCTAT

Q352P_F

GGTCCGATGTCAGTGGCCTAT

Q352G_F

GGTGGCATGTCAGTGGCCTAT

Q352V_F

GGTGTGATGTCAGTGGCCTAT95

Primers used for mutation library construction (continued)
S374
S374F_F

CAATTTTTCGAATTTGATGATAAT

S374L_F

CAATTTCTGGAATTTGATGATAAT

S374I_F

CAATTTATTGAATTTGATGATAATS374M_F

CAATTTATGGAATTTGATGATAATS374T_F

CAATTTACGGAATTTGATGATAATS374K_F

CAATTTAAGGAATTTGATGATAATS374Y_F

CAATTTTATGAATTTGATGATAATS374H_F

CAATTTCATGAATTTGATGATAATS374Q_F

CAATTTCAGGAATTTGATGATAATS374N_F

CAATTTAACGAATTTGATGATAATS374D_F

CAATTTGACGAATTTGATGATAATS374E_F

CAATTTGAGGAATTTGATGATAATS374W_F CAATTTTGGGAATTTGATGATAATS374C_F

CAATTTTGCGAATTTGATGATAATS374R_F

CAATTTCGTGAATTTGATGATAATS374P_F

CAATTTCCGGAATTTGATGATAATS374G_F

CAATTTGGCGAATTTGATGATAATS374V_F

CAATTTGTGGAATTTGATGATAAT

E375

96

Primers used for mutation library construction (continued)
E375F_F

ATTTTCATTCTTTGATGATAAT

E375L_F

ATTTTCACTGTTTGATGATAAT

E375I_F

ATTTTCAATTTTTGATGATAAT

E375M_F

ATTTTCAATGTTTGATGATAAT

E375S_F

ATTTTCAAGCTTTGATGATAAT

E375T_F

ATTTTCAACGTTTGATGATAAT

E375K_F

ATTTTCAAAGTTTGATGATAAT

E375Y_F

ATTTTCATATTTTGATGATAAT

E375H_F

ATTTTCACATTTTGATGATAAT

E375Q_F

ATTTTCACAGTTTGATGATAAT

E375N_F

ATTTTCAAACTTTGATGATAAT

E375D_F

ATTTTCAGACTTTGATGATAAT

E375W_F ATTTTCATGGTTTGATGATAAT
E375C_F

ATTTTCATGCTTTGATGATAAT

E375R_F

ATTTTCACGTTTTGATGATAAT

E375P_F

ATTTTCACCGTTTGATGATAAT

E375G_F

ATTTTCAGGCTTTGATGATAAT

E375V_F

ATTTTCAGTGTTTGATGATAAT*Residues in red are substitutions.

97

බ⾲ㄽᩥ
         (1) Accelerating itaconic acid production by increasing membrane
permeability of whole-cell biocatalyst based on a psychrophilic
bacterium Shewanella livingstonensis Ac10
Luo, G., Fujino, M., Nakano, S., Hida, A., Tajima, T., & Kato, J.
Journal of Biotechnology, 312, 56-62 (2020)(2) Unexpectedly high thermostability of an NADP-dependent malic
enzyme from a psychrophilic bacterium, Shewanella livingstonensis
Ac10Luo, G., Fujii, S., Koda, T., Tajima, T., Sambongi, Y., Hida, A., Kato, J.
Jounral of Bioscience and Bioengineering,
DOI: 10.1016/j.jbiosc.2021.07.005.