Polymerase Chain Reaction Based Cloning of Acetate Kinase in Propionibacterium Acidipropionici

Home , Acetate kinase

POLYMERASE CHAIN REACTION BASED CLONING OF

ACETATE KINASE IN PROPIONIBACTERIUM ACIDIPROPIONICI

A Thesis

Presented in Partial Fulfillment of the Requirements for

the Degree Master of Science in the

Graduate School of The Ohio State University

Yu-Hua Liao, B.S.

* * * * *

The Ohio State University

2003

Approved by Master’s Examination Committee: Dr. Shang-Tian Yang, Advisor Dr. Ahmed Yousef Dr. Hua Wang Advisor Department of Food Science and Technology

ii ABSTRACT

Currently, the U.S. market for propionate is about 400 million pounds per year,

and the demand is increasing at 3 ~ 4 % annually. Fermentation process can use

byproducts from the food industry, such as corn steep liquor and cheese whey, to produce

propionic acid. It is thus desirable to convert these low-grade byproducts to a marketable

product, such as propionic acid by fermentation.

However, propionic acid fermentation with Propionibacterium acidipropionici is

limited by the low product yield due to its heterofermetation nature. Metabolic

engineering to modify the dicarboxylic acid pathway can be an effective method to alter

carbon flow to improve propionate yield by knocking-out the acetate formation. Acetate

kinase (ACK) is a key enzyme involving acetate formation in the pathway, and was the

knockout target in this research. Integrational mutagenesis by homologous recombination

can be used to disrupt ack gene by first constructing a non-replicative plasmid with

partial ack and then transforming it into cells. Integration of the non-replicative plasmid

can result in duplicate repeats of the homologous regions flanking DNA sequences, and

thus cause mutagenesis. In this work, partial ack, encoding ACK, was first cloned and sequenced by PCR based cloning. A 477 bp of partial ack sequence was obtained.

However, low yield of genomic DNA preparation from P. acidipropionici might have attributed to failure to clone the ack gene from the genomic DNA using the degenerate

iii primers in the PCR. A selective marker, TetR, failed to ligate into plasmid with partial ack due to the difficulty of ligating two large DNA fragments. Further study to improve the cloning method is necessary to construct the plasmid needed for transformation

Dedicated to my parents

v ACKNOWLEDGMENTS

My great appreciation goes to my advisor, Dr Shang-Tian Yang, for his enormous help academically throughout the completion of my Master thesis. I am deeply grateful for his inspiring advice, warm support and encouragement, and also the flexibility he gave me in conducting and scheduling my research. Without his support and understanding, my research would not have been pleasant and fruitful. I have greatly enjoyed my staying here as his student.

I would also like to acknowledge with sincere gratitude to the members of my dissertation committee, Dr Wang and Dr Yousef. I am so grateful for their helpful suggestions, and advice.

I would like to thank Dr. Ying Zhu, Ms Supaporn Suwannakham and Ms. Xiao-

Guang Liu for their help to my research and thesis. My colleagues within my research group in the Department of Chemical Engineering offered many supports over the last two years. I benefited a lot from discussions and sharing the knowledge on my research. I have been fortunate to work with such a good team. And my home department - Food

Science and Technology, gave me many kinds of help over the past two years I deeply thank all of you to give these warm supports.

Finally, special thanks go to my parents and my family for their sustained, the warmest and strongest supports.

vi VITA

June 15, 1978 ...………………………………… Born – Taipei, Taiwan July 2000 ...…………………………………… B.S. Animal Science National Taiwan University, Taipei, Taiwan

FIELD OF STUDY Major Field: Metabolic engineering

vii TABLE OF CONTENTS

Page

ABSTRACT ....…...……………………………………………………….…….….……ii DEDICATION ..…..…...………………………………………………….……....….…iv

ACKNOWLEDGEMENT ....….……………………………………………….…….…..v VITA ...... ………………...…………………………………………………………....…vi LIST OF TABLES.….…………………………….……….…………...………………..x LIST OF FIGURES …...…………………………………..……………………………xi Chapters:

1. Introduction ...………….…………………………………………………..…….1

1.1 Interests of thesis. ……………………………………………………………1 1.2 Approaches and research objectives……….……………………….....……3 1.2.1 Preparation of genomic DNA with P. acidipropionici …………...... …..3 1.2.2 PCR (Polymerase Chain Reaction)…………………………….……..…4 1.2.3 TA cloning………..……....……………………………………..…....…4 1.2.4 Free cell fermentation ..………..…….…………………………….……5 1.3 Thesis organization…… ……………...……………………………………..6 1.3.1 Propionic acid fermentation …..……………………...….…….....……..6 1.3.2 Metabolic Engineering in P. acidipropionici….……………………..…6 1.3.3 PCR based cloning…………………………………………….……..…6 1.3.4 Conclusions and recommendations…………………………………..…7

2. Propionic acid Fermentation…………………………………………………..……8

2.1 Propionic acid…………………………………..….………..……………………………....…8 2.2 Microorganisms……………..………………..…………………….……....10 2.3 Metabolic Pathway in P acidipropionici………..…………………..…...... 12 2.4 Fermentation Process…...………….……………..………………….……16 2.5 Free cell fermentation…………………….……………………………..….18 2.5.1 Method………………………………………………………………...19 2.5.2 Results and discussion…...……………………………………………20

viii 3. Metabolic engineering in P. acidipropionici……………………..………..…..24

3.1 Genetic engineering in P. acidipropionici…..…………………..…...……24 3.1.1Acetic acid formation during propionic acid fermentation………...... …24 3.1.2 Acetic acid formation pathway genes and enzymes………..….……… 25 3.1.3 Molecular studies of propionibacteria………………..………....…...... 27 3.1.3.1 Plasmids in propionibacteria………..…………………………27 3.1.3.2 Shuttle vectors construction and transformation…..…………..29 3.1.3.3 Strategies of gene manipulation………………………………..32 3.2 Objective of gene disruption in acetate kinase of P. acidipropionici…….33 3.3 The concept of gene disruption in P. acidipropionici.…………..…...……34 3.3.1 PCR based gene manipulation…….…...…………...……………...... …34 3.3.2 Integrational mutagenesis by homologous recombination.…….……....35

4. PCR based cloning………………………………………………………...... 38

4.1 Introduction…..………………………..……………………………………38 4.2 Materials and Methods…………...……....…………………………...……39 4.2.1 Bacterial strain and Plasmid..…………….………..………….……..…39 4.2.2 Genomic DNA isolation……………………….. ……….……………..40 4.2.3 PCR amplification and TA cloning………………….………………....40 4.2.4 Construction of non-replicative plasmid with partial ack………..….…46 4.3 Results and Discussion....……………………….………………..…...……46 4.3.1 Genomic DNA isolation…………..………….. ….…….…...…………46 4.3.2 PCR amplification.…………………..…………..…….………………46 4.3.3 TA cloning and sequence analysis……..……………….……………..48 4.3.4 Ligation……………………………………..…………………………49

5. Conclusions and recommendations………..…………….………………….57

5.1 Conclusions..….………..…………………….……….……….……...……57 5.2 Recommendations…….…………………………………………………..58

Reference……………..…………………………………..………………………..……60

Appendices………………………………………………..………………………..……70

Appendix A Medium compositions. ………………………….…...…….....…70 A1 Medium compositions for Propionibacterium acidipropionici ……..70 A2 Medium compositions for E. coli –Luria-Bertani (LB) medium....….70 ix Appendix B Analatical Methods ……………….…………….………...…….71 B1 Cell Concentration …………….………….……...……….....…..…..70 B2 High Performance Liquid Chromatography ...…….….….………….70

Appendix C Protocols of Genetic Experiments …………….………….……72 C1 Preparation of Genomic DNA from P. acidipropionici with QIAGEN Genomic DNA kit ….………….…………..………..72 C2 Preparation of Plasmid DNA by QIAprep Spin Miniprep Kit ...…...73 C3 DNA Electrophoresis ..………………….…………………………..75 C4 PCR Amplification of ack from P. acidipropionici…………………….……………………………..76 C5 TA Cloning Protocol (Invitrogen Original TA Cloning Kit-TOP10.………..…………….78 C6 DNA/Plasmid Digestion and Ligation …..………...……………..…80 C7 DNA Purification byQIAquick Gel extraction Kit …....…………....81

Appendix D Reagents and Buffers …...……………..………...………...……82

x LIST OF TABLES

Table Page 3.1 Plasmids in Propionibacterium strains ………………………………….…………..28 4.1 Bacterial strains and plasmids used in this work……………...…………………….43

xi LIST OF FIGURES

Figure Page

2.1 Schematic diagram of the dicarboxylic acid pathway in Propionibacterium acidipropionici……………..…..…………………………….13 2.2 The free-cell bioreactor system used for propionic acid fermentation………....……21 2.3 The cell growth of free-cell fermentation with wild-type of P. acidipropionici…………………..………………….………….22 2.4 Acid production and glucose consumption by wild-type of P. acidipropionici fermentation at pH 6.5 and 32 ℃……..…………....23 3.1 Construction of non-replicative plasmid……………………………...……………..36 3.2 The concept of integrational gene into chromosome to cause integrational homologous mutagenesis and disrupt ack gene fuction……………………….……37 4.1 The protein alignment of ACK enzymes among Bacillus subtilis, M. thermophila, E. coli and P. acidipropionici………..…………………….……..44 4.2 TA cloning method……………………………………………………………….…45 4.3 Gel electrophoresis of P. acidipropionici genomic DNA……..…………....……….51 4.4 PCR amplification by using primers of Ack-F and Ack-R from propionibacteria genomic DNA………………………………………………….....52 4.5 PCR amplification by using primers of AK-forward and AK-reverse with pack-21 as template DNA…………………………….…………………………….53 4.6 The DNA sequence of partial ack (749 bp) from Dr. Yan Huang (1998)…………………….…………………………………..54 4.7 The DNA sequence of partial ack (477 bp) from primers AK-reverse and AK-forward………….……………………………..55 4.8 The restriction map in the partial ack (477 bp) sequence…………………………...56

xii

CHAPTER 1

INTRODUCTION

1.1 Interest of thesis

Propionibacterium acidipropionici ATCC 4875 is a gram-positive, rod-shaped,

non-spore forming, and facultative anaerobic bacterium. It is used as a starter in Swiss cheese fermentation to develop the characteristic of eye formation and its unique flavor.

It also has been studied as a potential candidate to produce propionic acid and vitamin

B12 via fermentation (Blac and Goma 1987; Lewis and Yang 1992). Propionic acid is an

important factor to produce the unique flavor in cheese making; moreover, it can be used for manufacturing pharmaceutical products, herbicides, and chemical intermediates. In the food industry, propionic acid is used widely in food preservation, artificial fruit flavors, animal feed and grain feed.

Traditionally, propionic acid can be produced from petrochemical processes.

However, unlike traditional petrochemical process, fermentation process is an alternative method to produce propionic acid by using the byproducts from food processing such as corn steep liquor and cheese whey. Byproducts from the food processing industry are

always a challenge for the food industry, which must solve the waste disposal problem

generated from processing. Therefore, fermentation process using wastes to produce

propionic acid and to solve the waste disposal problem is an interest to the food industry.

Propionic acid fermentation with Propionibacterium acidipropionici is mainly by

the dicarboxylic acid pathway, which has the potential to yield two or more products.

Based on the dicarboxylic acid pathway (Figure 2.1), the theoretical ratio of propionic

acid to acetic acid is 2.0 (mol/ mol), and the fermentation yields from glucose are

approximately 55 % (wt/wt) for propionic acid, 22 % (wt/wt) for acetic acid, and 17 %

(wt/wt) for carbon dioxide (Playne, 1985). However, propionate production by P.

acidipropionici must be enhanced in the fermentation process for industrial applications.

Propionic acid production can be increased if production of acetate, a main byproduct of

dicarboxylic acid pathway can be eliminated by knocking out key enzyme in the acetate

formation pathway in Propionibacterium acidipropionici. More carbons can be used for

propionic acid formation by reducing the carbons to the acetate formation pathway.

Therefore, a higher fermentation yield of propionate with Propionibacterium

acidipropionici can be expected. Also, a reduced formation of acetic acid in the propionic

acid fermentation can reduce product recovery and purification cost contributing to a low-cost fermentation product.

1.2 Research objectives and approaches

The metabolic pathway in propionic acid fermentation has heterofermetation

nature. To enhance propionic acid yield, an effective method to alter the metabolic flux is

to change carbon flow or flux distribution by using molecular genetic tools. In this study,

PCR (polymerase chain reaction) and partial ack cloning to construct a non-replicative

plasmid were performed.

Overall goal of this study was to use PCR technique to obtain partial ack fragment

and clone it into a non-replicative plasmid. The preparation of genomic DNA with P.

acidipropionici was the first step. Primers were designed based on the amino acid

sequence (Huang, 1998) using primer design software. Furthermore, batch fermentation

with free cells was carried out to characterize the wild type of P. acidipropionici. There

are four specific objectives proposed in this work, which are described as follows.

1.2.1 Preparation of genomic DNA with P. acidipropionici

Currently, genomic map of P. acidipropionici has not been investigated. However,

ack genes have been cloned and sequenced in several microorganisms, such as E. coli

(Matsugaya, et al., 1989; Kukada, et al., 1994), Methanosarcina thermophila (Katimer

and Ferry et al., 1993), Bacillus subtilis (Grundy, et al., 1993), and Clostridium acetobutylicum ATCC 824 (Zhuang, et al., 1996). The DNA sequences of ack from

Mycoplasma genitalium, M. capricolum, and Haemophilus influenza are also available in the genomic database, but only defined by sequence homology. Protein alignment of

homologous genes can be a suitable method to design primers for PCR amplifications to

solve this problem of undefined genome.

Genomic DNA is a critical factor affecting the efficiency of PCR and cloning. P.

acidipropionici is a gram-positive bacteria, which has a thick cell wall compared with

gram-negative bacteria such as E. coli. Lysozome and Proteinase K were used in the lysis

of cells. Obtaining pure and high concentration of genomic DNA was the objective for

further experiments.

1.2.2 PCR (Polymerase Chain Reaction)

Polymerase chain reaction has been used a lot in cloning and other molecular

purposes. In this study, PCR was used to obtain the partial ack fragment by designed

primers. Primers, two short oligonucleotides, were designed based on the amino acid

sequence of partial ack from the dissertation of Dr. Yan Huang, The Ohio State

University, 1998. Partial ack was used for further cloning of a non-replicative plasmid.

1.2.3 TA cloning

Partial ack gene was ligated into a commercial vector, pCR2.1 TOPO (Invitrogen)

to construct a non-replicative plasmid. TA cloning was the method to ligate partial ack and vector together. Adenine bases, which are on two ends of partial ack sequence can anneal into Thymine bases, which are on two ends of pCR2.1 TOPO vector by ligation.

This is called “T-A cloning”.

1.2.4 Free cell fermentation

Free cell fermentation ranges from simple stirred tank reactors to more complicated systems. Fermentation process is a common method to characterize the acid productivities in propionic acid fermentation with P. acidipropionici. In this study, a simple stirred tank bioreactor was used to observe the acid productions by the wild type of P. acidipropionici. Fermenting samples were taken by a regular interval, 6 hours in this study. All samples were analyzed for cell density, glucose concentration, and acid productions, including propionic acid, acetic acid, succinic acid, and lactic acid. Cell density was measured as optical density of cell suspension at O. D. 600 nm with a spectrophotometer. Glucose consumption and acid production were measured by HPLC, compared with the standard solution.

1.3 Thesis organization

1.3.1 Propionic acid fermentation

Chapter 2 provides a literature review on propionic acid production and fermentation. The characteristics of P. acidipropionici and its potential for propionic acid production are discussed. Fermentation results with wild type of P. acidipropionici, including glucose consumption, cell growth, and acid production, are presented and discussed in details as well.

1.3.2 Metabolic engineering in P. acidipropionici

To understand the idea of gene manipulation in P. acidipropionici, Chapter 3

provides a description of metabolic engineering in P. acidipropionici to enhance the

production of propionic acid. Literatures related to acetate formation and genetic studies

in P. acidipropionici are described in Chapter 3 to provide the necessary background.

Overall objective and the concept of metabolic engineering in P. acidipropionici are also

presented in this chapter.

1.3.3 PCR based cloning

The experimental design, results and discussion of PCR based cloning are

provided in Chapter 4. First, Genomic DNA with P. acidipropionici was prepared by

using a commercial protocol. And then, PCR amplification was performed by using a

thermal cycler and analyzed by running gel electrophoresis; results are shown and

analyzed in this chapter. Furthermore, obtained ack fragment was ligated into a commercial vector by TA cloning.

1.3.4 Conclusions and Recommendations

In Chapter 5, it concludes this study with an overall discussion of the results and

ways to improve the project. Finally, recommendations for future work are also provided.

CHAPTER 2

PROPIONIC ACID FERMENTATION

2.1 Propionic Acid

Propionic acid (C3H6O2) and its salts have been used in many processes including in the manufacture of herbicides, chemical intermediates, artificial fruit flavors

(citronellal propionate and geranyl propionate), pharmaceuticals, cellulose acetate propionate, and preservatives for food, animal feed, and grain (Playne, 1985). In animal therapy, sodium propionate has been used in dermatoses, wound infections, anti-arthritic drugs, and conjunctives. In the food industry, propionic acid and its sodium, calcium and potassium salts are incorporated to suppress the growth of mold, which can cause ripeness in cheeses and breads. Several of the propionic esters have pleasant aromas and can be used in the preparation of synthetic flavors and perfumes. Moreover, it is also strongly recommended to have a good effect by using the association of lactic acid and propionic acid (Piveteau et al., 1995). In the cheese making industry, propionic acid has also been used to improve the unique flavor of Swiss cheese and its eye formation for a long time. Lactate in cheese ingredients produce propionate, acetate, and CO2 during the extension ripening (Hettinga and Reinbold, 1972; Langsrud and Reinbold, 1993). CO2 is

responsible for eye formation, a unique characteristic in Swiss cheese, while propionic acid contributes to the development of the characteristic nutty flavor (Gautier et al., 1993).

Currently, the U.S. market for propionate is about 400 million pounds per year, and the demand is increasing at 3 ~ 4 % annually (Yang and Silva, 1995). Presently, propionic acid is produced mainly by petrochemical processes, with a market price of $

0.46/lb. Only a small amount of propionic acid is produced by fermentation, and used as a natural ingredient in food and pharmaceutical industries. However, the price of propionic acid from fermentation can be as high as $ 2.0/lb, which is much higher than that from petrochemical routes. The market share of propionate from fermentation processes should be improved and increased, because of increasing demand of propionate in the U.S. market. Since fermentation processes can use low-grade biomass as feedstock and be considered as environmentally benign, propionic acid from fermentation processes would have a great potential market for competitive and economic production. Propionic acid can be produced by anaerobic fermentation of a variety of carbon sources using propionibacteria. Several carbon sources, such as glucose (Emde and Schink 1990; Lewis and Yang, 1992), xylose (Hendricks et al., 1986; Carrondo et al., 1988), maltose

(Babuchowski et al., 1993), sucrose (Quesada-Chanto et al., 1994), lactic acid

(Babuchowski et al., 1993), and whey lactose (Lewis and Yang, 1992; Colomban et al.,

1993), have been studied for this fermentation.

2.2 Microorganisms

Several genera bacteria, Propionibacterium, Rhodospirillum, Micrococcus,

Rhizobium, Mycobacterium, as well as the protozoan, Ochromonas malhamensis (Wegner et al., 1968; Haase et al., 1984) are known for producing propionic acid. Only in propionibacteria, such as Propionibacterium freudenreichii subsp. shermanii and P. acidipropionici, it serves as the main metabolic pathway for energy generation; while in other species, it is just another way of living. In this study, P. acidipropionici was the investigated microorganism.

Propionibacteria are gram-positive, non-motile, non-sporeforming, rod-like and facultative anaerobic microorganisms. The G+C content of their DNA is in the range of

53-67 % (Sneath, et al., 1986). The genus of Propionibacterium is classified into two groups, the dairy group and acne group. Dairy propionibacteria are also classified into six species: P. freudenreichii, P. theonii, P. jensenii, P. acidipropionici, P. coccoides, and P. cyclohexanicum (Vorobjeva, L.I., 1999). In the cheese-making industry, dairy propionibacteria have been used as starters to produce the unique characteristic of eye formation in Swiss cheese, because concomitant production of CO2 can assist in hole- forming in the cheese body. Moreover, they can produce many flavor precursors and flavor compounds to give special flavors and textures to Swiss cheese. Organic acid production, including propionic acid and succinic acid, proline accumulation (Quelen et al., 1995) and metabolites from amino acid catabolism are key factors in flavor formation

(Hettinga and Reinbold, 1972a, 1992b, and 1992c). These are the most important applications of propionibacteria in the food industry.

In the fermentation process with propionibacteria, several parameters should be controlled and adjusted to obtain the optimum acid production, including nutrients, temperature, and pH value. The nutrient requirements and growth conditions for propionibacteria have been investigated extensively (Playne, 1985). Propionibacteria can utilize a broad range of carbon source, such as glucose, fructose, lactic acid, and lactose, to produce the propionic acid. The optimum pH for propionibacteria cell growth is ranged from pH 6.0 ~ 7.0. There was practically no cell growth when the pH was below 4.5

(Playne, 1985; Hsu and Yang, 1991). The optimum temperature for propionibacteria growth is 32℃ under anaerobic conditions.

To date, P. acidipropionici is the most commonly used species for industrial production of propionic acid (Colomban et al., 1993) by fermentation; because it has a better ability to produce propionic acid than other propionibacteria species. Besides producing propionic acid, propionibacteria has been also studied to produce bacteriocin against some pathogenic microorganisms in food and other applications (Grinstead and

Barefoot, 1992; Lyon and Glatz, 1991; Faye et al., 2000).

2.3 Metabolic Pathway in P. acidipropionici

In 1981, Wood published a review of the metabolism in dairy propionibacteria

(Wood, 1981). He compiled all the hypotheses made, all the mechanisms proposed or

confirmed over years, and listed many mechanisms that needed to be confirmed or

remained obscure. According to the isolation and identification of the intermediates of

glucose fermentation, verification of the expected end products (Wood et al., 1937) and

analysis of the distribution of labeled products (Wood and Leaver, et al., 1953), it showed that glycolysis is the main pathway of glucose utilization by propionic acid bacteria.

Glucose is phosphorylated, forming hexose monophosphate (van Neil, 1928), and hexose diphosphate (Pett and Wynne, 1993). Transcarboxylase, hexosephosphate isomerase

(Wood et al., 1963), fructose diphosphate aldolase (Sibley and Lehninger, 1949; Wood et al., 1963) and triosephosphate dehydrogenase activities were found in propionibacterium cells (van Demark and Fukui, 1956).

The dicarboxylic acid pathway with Propionibacterium acidipropionici is shown

in Figure 2.1.

1.5 Glucose

+ 3ADP 3NAD EMP Pathway

3ATP 3NADH

3PEP

3NADH 3NAD+

3 Lactate 3Pyruvate CoA+NAD+

CO2+NADH

Acetyl-CoA 2 Methylmalonyl-CoA Pi 2 Oxaloacetate 2NADH Phosphotransacetylase 2NAH+ 2 Malate CoA 2NADH+2ADP+2FP Acetylphosphate 2Propionyl-CoA 2 Succinyl-CoA ADP 2NAD++2ATP+ 2 Fumarate Acetate kinase 2FPH2 2FPH ATP 2

Acetate 2FP 2 Succinate 2 Propionate

Figure 2.1 Schematic diagram of the dicarboxylic acid pathway in Propionibacterium acidipropionici. Propionate (propionic acid) is the desired product in this fermentation. Two main byproducts can also be produced by this fermentation, acetate and succinate. Acetate formation pathway is highlighted in the left by a bold box. In this study, acetate kinase (ack) is the enzyme to be manipulated.

In the dicarboxylic acid pathway, 1.5 moles of glucose can be fermented into 2 moles of propionic acid, 1 mole of acetic acid, and 1 mole of CO2, respectively. Initial work on propionic acid fermentation resulted in the formulation of the Fitz equation:

3 Lactic acid 2 Propionic acid + 1 Acetic acid + 1 CO2 + 1 H2O (2.1)

1.5 Glucose 2 Propionic acid + 1 Acetic acid + 1 CO2 + 1 H2O (2.2)

Following transport into cytoplasma, glucose is metabolized to pyruvate via the

Embden-Meyerhof-Parnas (EMP) pathway. Two moles of ATP and two moles of NADH are produced by one mole of glucose via the EMP pathway. Then, one pyruvate (triose) is oxidized to CO2 and acetylphosphate,

It was once suggested that propionate was formed by propionic acid bacteria by the reaction below (Kaziro and Ochoa, 1964):

Propionyl-CoA + CO2 + ATP Methylmalonyl-CoA +ADP + Pi Propionyl carboxylase

However, the reverse rate of this reaction is very slow and does not correlate with the high rates of propionate production in propionic acid bacteria. After several investigations, and the metabolic pathway of propionic acid fermentation was confirmed by estimating the carbon balance to the production of propionic acid in propionibacteria.

Reactions leading to the production of propionic acid in propionibacteria can be represented by the following sequences:

Transcarboxylase Pyruvate + D-Methylmalonyl-CoA Oxaloacetate + Propionyl-CoA (2.3)

Oxaloacetate + 4 H+ Succinate (2.4)

CoA Transferase Succinate + Propionyl-CoA Succinyl-CoA + Propionate (2.5)

Methylmalonyl isomerase Succinyl-CoA L-Methylmalonyl-CoA (2.6)

Methylmalonyl racemase L-Methylmalonyl-CoA D- Methylmalonyl-CoA (2.7)

Summary Pyruvate + 4H+ Propionate (2.8)

The conversion of oxaloacetate to succinate is catalyzed by the enzymes of the citric acid cycle: malate dehydrogenase, fumarase, and succinate dehydrogenase, isolated from P. shermanii cells (Allen et al., 1964). The reductive steps in the formation of propionate, including the reduction of oxaloacetate and fumarate, are coupled with the oxidation of NADH produced from phosphoglyceraldehyde oxidation and oxidative decarboxylation of pyruvate.

The metabolism of acetate is of major importance in the physiology of both

Bacteria and Archaea domains. Acetate is an end product of energy-yielding metabolism in most strictly and facultative anaerobes. It is formed as a result of oxidative decarboxylation of pyruvate in following equations:

Pyruvate dehydrogenase + + Pyruvate + NAD + CoA Acetyl-CoA + H + NADH +CO2 (2.9)

Phosphotransacetylase Acetyl-CoA + Pi Acetyl-Phosphate + CoA (2.10)

Acetate Kinase Acetyl-Phosphate + ADP Acetate + ATP (2.11)

2.4 Fermentation process

Several chemical processes exist for the production of propionic acid, including

reaction between carbon monoxide and steam, Reppe process from ethylene, and the

Larson process from ethanol and carbon monoxide using boron trifluoride as a catalyst.

Today, the chemical process, Oxo process, is the most common way to produce propionic

acid in industries. However, it is very interesting to produce propionic acid by using

novel fermentation processes instead of traditional chemical processes. Fermentation is

not only a method to produce propionic acid by using byproducts from food industry as

inexpensive media, but also has a higher efficiency than petrochemical processes.

Moreover, the increasing consumer demand for organic natural products, and fewer

supplies and higher cost of oil provide a good opportunity for microbial fermentation

processes to be economically attractive.

According to the dicarboxylic acid pathway, 1.5 moles of glucose can be

converted into two moles of propionic acid and one mole of acetic acid, one mole of CO2,

and one mole of H2O. Theoretical maximum yields of this fermentation are 54.8% (w/w) for propionic acid, 22% (w/w) for acetic acid, and 17% (w/w) for CO2. However, the actual propionic acid yield from glucose is much less than 50% (w/w) in a typical batch fermentation. Propionic acid yield was commonly observed in the range from 25 % to

40% (w/w) (Lewis and Yang, 1992; Babuchowski, et al., 1993; Obaya, et al., 1994).

Lower yields of propionic acid may be caused by significant cell growth during the fermentation process.

However, propionic acid is also a factor to inhibit the process of propionic acid fermentation and cell growth. It leads to lower consumption and production rates in fermentation. Conventional fermentation processes for propionic acid production from glucose and lactose are limited by several restrictions: (1) low reactor productivity (<1 g/L．h), (2) low product yield (<50 % w/w), and (3) low propionic acid concentration

(<40 g/L). Two alternative systems are reported to correct these limitations of conventional fermentation process, immobilized and extractive fermentation systems. It has been reported that a higher reaction rate of propionic acid fermentation can be obtained by immobilized cell systems (Lewis and Yang, 1992; Woskow et al., 1991; Gu et al., 1998; and Huang et al., 2002). The advantages of using immobilized cell system in propionic acid fermentation also include the ability to use a higher dilution rate to achieve a higher productivity without culture washout, and to reduce the cost of the product recovery (Brodelius et al., 1987). Compared to conventional batch fermentation, higher productivity ( ~1 g/L．h), higher propionic acid production (up to 0.66 g/g), higher final product concentration (75 g/L) and higher purity (~90 %) were obtained. In Extractive fermentation, it improved the fermentation performance by reducing product inhibition 16

and also resulted in a metabolic pathway shift to favor more propionic acid and less

byproducts formation (Jin, and Yang 1998).

2.5 Free cell fermentation

2.5.1. Method

Batch fermentation of P. acidipropionici was performed in a 5-liter stirred-tank

fermentor containing 2 liters of propionibacterial growth medium supplemented with

glucose (30 g/L). The bioreactor set up is presented in Figure 2.2. Anaerobic condition

was achieved by purging nitrogen gas into medium for 30 minutes before starting a batch.

The pH was adjusted to 6.5 with 6 N HCl before sterilizing the medium. Medium

sterilization was carried out at 18 psi, 121℃ for 30 minutes. The inoculation of P.

acidipropionici cell suspension (100 ml) was prepared in a serum bottle, which was incubated for 2 ~ 3 days to reach a suitable O.D. value of 2.0 before use to start the batch fermentation. The experiment was carried out at 32 ℃, and the pH was controlled at a value of 6.5 by adding NH4OH. Samples were taken at every 6 hours from the

fermentation broth for analyzing cell growth, acid production, and glucose consumption.

The fermentation was ended when glucose was completely exhausted and acids

production stopped. Samples taken from the fermentor were separated by centrifugation.

Cell concentrations were analyzed to examine the growth of bacteria. The O.D. value was

measured with a spectrophotometer (Sequoia-Turner, Model 340) at the wavelength of

620 nm. The O.D. value of the original medium without cells was used as the background.

Furthermore, cell free broth was kept for HPLC analysis.

2.5.2. Results and discussion

Free cell fermentation ranges from simple stirred tank reactors to more complicated systems, such as hollow fiber membrane reactors. Fermentation process is a common method to characterize acid productivities in propionic acid fermentation with P. acidipropionici. In this study, a simple stirred tank bioreactor was used to observe acid production from glucose. Autoclaved reactor, medium, and other apparatus were prepared before inoculating propionibacteria. Fermenting samples were taken at a regular interval, 6 hours in this study. All samples were analyzed by cell density, glucose concentration, and acid production, including propionic acid, acetic acid, succinic acid, and lactic acid. Cell density was measured as optical density of the suspension at O.D.

600 nm with a spectrophotometer. Substrate and acid concentration were analyzed with

HPLC.

Acid production, substrate consumption and cell growth of wild type of

Propionibacterium acidipropionici were analyzed in batch fermentation. The result of wild type cell growth is shown in Figure 2.3. Four acid products were analyzed in P. acidipropionici fermentation, including propionic acid, acetic acid, succinic acid, and lactic acid (Figure 2.4). Substrate, glucose in this study, was dramatically decreased when cells grew in the exponential phase. Propionic acid production also had a significant increase when cell grew in the exponential phase. Its final concentration was around 9 g/L. Two main byproducts, acetic acid and succinic acid, had almost the same productivity in the fermentation, and these final concentrations were less than 2 g/L.

However, lactic acid production was high in this propionic acid fermentation. Lactic acid decreased significantly after glucose was exhausted. Wild type of P. acidipropionici 18

began to utilize lactate as the substrate to continue the fermentation. Referring to the metabolic pathway, P. acidipropionici can utilize lactate to convert into pyruvate, and then transferring carbon flow into acid formation pathways. Therefore, lactic acid concentration began to decrease, and other acid productions still increased after glucose was exhausted. Propionic acid yield was around 30 % (w/w). This was not the best result as compared to these reported in the literature (Lewis and Yang, 1992). One reason of less productivity from this study is a different type of fermentation used. Free cell fermentation has a lower productivity than immobilized cell system. Fibrous-bed bioreactor (FBB) has been reported as an effective system in propionic acid fermentation

(Lewis and Yang, 1992). Immobilized cells have a higher ability to tolerate acid as compared to free cells. These cells were immobilized on a spirally wound fibrous matrix packed in a fibrous-bed bioreactor developed for multi-phase biological reactions or fermentation and adapted to be acid-tolerant. The low propionic acid production was resulted by product inhibition, which inhibited the bacteria growing and producing acid.

The adapted cells in the immobilized cell system had a higher acid tolerant ability, and then could continually produce acid without acid inhibition.

pH meter

6..5

pH Probe

Base Agitator

Pump Sample

Inoculating

Batch Fermentor

Figure 2.2. The free-cell bioreactor system used for propionic acid fermentation

O.D. value P. acidipropionici wild type cell growing

6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

time (hours)

Figure 2.3. The cell growth of free cell fermentation with wild type of P. acidipropionici

concentration(g/L) 30 27 24 21 18 15 12 9 6 3 0 0 102030405060708090 Hours Glucose Succinic Lactic Acetic Propionic

Figure 2.4. Acid productions and glucose consumption by wild type of P. acidipropionici fermentation at pH 6.5 and 32 ℃.

CHAPTER 3

MATABOLIC ENGEERING IN P. ACIDIPROPIONICI

3.1 Genetic engineering in P. acidipropionici

3.1.1 Acetic acid formation during propionic acid fermentation

Propionibacteria have branched pathways, resulting in the formation of propionate, acetate, CO2, and succinate. Acetate (CH3COOH) is the main byproduct in propionic acid fermentation. As a result, a part of the carbon flow is directed into acetate formation, which significantly reduces the propionic acid yield. To enhance propionic acid production, reducing or eliminating acetate formation by genetically modifying acetic acid formation pathway in P. acidipropionici can be an effective way.

Acid formation pathway always involves energy-yield in most facultative and strictly anaerobic microorganisms, including propionibacteria. It has been reported that, in ack/pta genes inactivated mutant of Clostridium acetobutylicum ATCC 824, lack of

ATP generation from acetic acid formation pathway might lead to a lower cell growth than that in wild type, but reached higher biomass concentrations (Green et al., 1996). As a result, disrupting gene functions of acetate forming enzymes may cause a metabolic burden in cells. Therefore, increasing energy flux from the other ATP generating route, 23

which could be propionic acid formation pathway, can be a feasible way to balance

energy demands in cells.

3.1.2 Acetic acid formation pathway genes and enzymes

During acid fermentation, the enzymes involved in acetate formation are acetate kinase (ACK, EC 2.7.2.1) and phosphotransacetylase (PTA, EC 2.3.1.8). In the acetate formation pathway, acetyl-CoA is an important intermediate with the production of ATP as an important energy source. Acetyl-CoA is converted into acetylphosphate by the enzyme, phosphotransacetylase, and acetate kinase catalyzes acetylphosphate to acetate, resulting in ATP generation through this pathway.

P CoA ADP ATP i CH COSCoA 2- CH COOH 3 CH3CO2PO3 3 Phosphotransacetylase Acetate kinase

However, only few studies investigated the molecular biology of ACK and PTA enzymes, even though they are important in the carbon energy cycling and energy metabolism in both aerobes and anaerobes. Ack gene, which encodes ACK, and pta gene, which encodes PTA, are involved in synthesizing these enzymes. The ack gene has been cloned, sequenced, and characterized only in Escherichia coli (Matsuyama et al., 1989),

Methanosarcina thermophila (Latimer et al., 1993), Bacillus subtilis (Grundy et al., 1993)

and Clostridium acetobutylicum (Zhuang et al., 1996). The PTA-encoding gene, pta, has

also been cloned, sequenced and characterized in M. thermophila (Latimer et al., 1993) and E. coli (Yamamoto-Otake et al., 1990; Kakuda et al., 1994a and 1994b; Matsuyama et al., 1994). Furthermore, ACK enzymes have been purified from M. thermophila, C. thermoaceticum, Salmonella typhimurium, and E. coli. ACK enzymes from S. typhimurium and E. coli are homodimers with the same subunit molecular mass of 40 kDa (Roseman et al., 1986). ACK from C. thermoaceticum has a subunit molecular mass of 60 kDa (Schaupp and Ljungdahl, 1974). And it was found that a homodimer in M.

thermophila has a molecular mass of 94 kDa (Singh-Wissmann et al., 1988). Several

PTA enzymes are isolated and partially characterized as well from C. thermoaceticum, C.

acidiurici, and C. kluyveri with a molecular mass of 88 kDa, 63 ~75 kDa, and 60 kDa,

respectively (Wood et al., 1981; Robinson and Sagers, 1972; Stadtman 1955). In the anaerobic archaebacterium, M. thermophila, PTA exists as a monomer, and its molecular mass was found in the range from 42 to 52 kDa (Ferry et al., 1988).

A single copy of ack gene was identified in the microorganisms studied. It was reported that ack gene and pta gene are contiguous as one operon in the chromosome of studied microorganisms. In Corynebacterium glutamicum, a potential microorganism to produce amino acid in a larger-scale, the functional acetate kinase/phosphotransacetylase were cloned, sequenced, and inactivated (Reinscheid et al., 1999). In C. glutamicum, the sequence analysis of 3657 base pairs DNA revealed that pta gene is upstream of this operon and the last nucleotide of pta stop codon (TAA) is overlapping the first of the ack start codon (ATG). In Methanosarcina thermophila, ack and pta also were cloned and sequenced. The analysis result showed that they are arranged on the chromosome with

pta upstream of ack (Litimer et al., 1993; Singh-Wissmann and Ferry, 1995). As a result, ack and pta are considered to be co-transcribed in the cell.

3.1.3 Molecular biology studies of Propionibacteria

3.1.3.1 Plasmids in propionibacteria

To date, not many genetic manipulation studies have been published for propionibacteria. This may be caused by several reasons, including the high GC content of this bacterium, lack of information on plasmid sequence determination, and lack of available plasmid vector for gene transformation. To develop a suitable molecular genetic system in bacteria, it is important to construct efficient shuttle cloning vectors. Replicon and selective marker are two critical components for vector construction. Several endogenous plasmids in propionibacteria have been isolated and characterized (Table 3.1).

Species Strains Plasmids Size (Mda) P. acidipropionici E17 pRGO1 4.4 E214 pRGO1 ATCC4875 pRGO1 ATCC14072 pRGO1

P. freudenreichii PS18 pGRO1 4.4 PS49 pGRO1 93 pGRO3 6.3 93 pGRO7 25 5932,F32 pGRO4 30 5932,F32 pGRO6 5.6

P. jensenii PP798, 13 pGRO1 4.4 E1.1.1,PJ154 pGRO2 6.3 13 pGRO5 35

Table 3.1 Plasmids in Propionibacterium strains (Rehberger et al, 1990)

Several different propionibacteria strains were investigated in this field listed above; mainly, they are among the species of P. acidipropionici, P. freudenreichii and P. jensenii. About 20 % of these propionibacteria were found to carry one or two plasmids and distinguished according to a restriction map and size. These plasmids are named pRGO1 through pROGO7. P. freudenreichii strain contains the most diverse of plasmid profiles; on the other hand, P. acidipropionici has only one plasmid, pRGO1, which is also commonly found in strains of P. acidipropionici, P. freudenreichii, and P. jensenii.

Plasmids pLME106 (6.9 kb) and pLME108 (3.6 kb) were also found in P. jensenii and P. freudenreichii, respectively (Miescher et al., 2000). Small plasmids, such as p545 and p546, were found in P. freudenreichii LMG 16545 and P. freudenreichii subsp.

Freudenreichii LMG16546, respectively. These small plasmids have the same size, 3.6 kb, and are related.

3.1.3.2 Shuttle vectors construction and transformation

In propionibacteria, several antibiotic resistant genes were tested for construction of efficient shuttle vectors. The hygromycin B resistant gene (hygBr) was tested to be a selective marker in both E. coli and propionibacteria (Kiatpapan, et al., 2000). This resistant gene was isolated from Streptomyces hygroscopicus and was reported to be expressed in several gram-positive bacteria containing high GC content, such as mycobacterium species (Garbe et al., 1994), Streptomyces sp. and E. coli (Blondelet-

Rouaul et al., 1997; Pulido et al., 1988). On the other hand, another antibiotic resistant gene, tetracyclin resistant gene (Tet r) was also reported as a suitable marker in gram- positive bacteria. In this study, the tetracycline resistant gene was used as the selection marker in Propionibacterium acidipropionici.

Two general techniques developed to transform gene-carrying vector into bacteria are protoplast transformation and vegetative cell transformation. Protoplast transformation is a method to introduce DNA into cell protoplasts with wall-less, and then regenerate into walled vegetative cells. On the other hand, vegetative cell transformations are usually carried out by either chemical permeabilization or

electroporation. Electroporation is considered as a useful transformation technique for gram-positive bacteria. Electroporation is a process where low-voltage of electricity is used to make pores in the cell wall, through which foreign DNA molecules are transported into cells. It not only involves the introduction of DNA into cells, but also has been used in several applications, such as (1) loading cells with molecules for drug delivery, (2) introduction of probes, enzymes and antibodies for research, and (3) transporting molecules into/out of tissues for therapeutic application. In this study, electroporation (electropermeabilization) was the process used for transforming plasmid

DNA into an individual cell.

Since gram-positive bacteria have strong cell walls as compared to gram-negative bacteria, one would expect the transformation of the shuttle plasmids by chemical transformation to be difficult. However, in early studies, transformation in propionibacteria used protoplast transformation and electroporation (Luchasky et al.,

1988) but had low transformation efficiencies in protoplast transformation. Therefore, electroporation is recommended for transforming DNA into gram-positive bacteria in genetic studies. For optimum transformation efficiency, several factors are involved, such as strain differences, cell growth medium, the phase of cell growth, pH, choices of electroporation buffer, and the concentration of plasmid. Speaking of the growth phase of cells, several studies recommended that higher transformation efficiency could be obtained by using bacteria in the exponential phase (Allen and Blaschek, 1988; Oultram et al., 1988; Scoot and Rood, 1989). Others have also found that better transformation

rates could be obtained from the late exponential bacteria (Allen and Blaschek, 1990;

Kim and Blaschek, 1993) or early exponential bacteria (Phillips-Jones, 1990;

Mermelstein et al., 1992).

Electroporation protocol in propionibacteria was developed using competent cells,

prepared in 10 % glycerol medium. A high efficiency of transformation rate, 106 to 107

cfu/µg DNA, was found in P. freudenreichii HUT8606, P. freudenreichii subsp. shermanii IFO12426, and HUT8612, and P. freudenreichii ATCC 4915. Those transformations by electroporation were prepared with the shuttle plasmid of pPK705 from P. freudenreichii IFO12426. Some studies showed that a higher transformation rate by electroporation was achieved in gram-positive bacteria with growing in the early exponential phase and treating by lysozyme (Dunny et al., 1991; Holo and Nes, 1989;

Powell et al., 1988). However, it has been reported that lysozyme treatment was not effective for propionibacteria because many species of propionibacterium were resistant to lysozyme (Johnson and Cummins, 1972). Without lysozyme treatment of propionibacterium competent cells, transformation rate was observed about 32 cells/µg

DNA. But a higher transformation rate, 105 cfu/µg of DNA, was obtained when cells

were grown in the medium containing glycine. It was reported that glycin was

incorporated into cell wall component and resulted in reduced cross-link in the cell wall

(Gautier et al., 1995). In addition, strong cell wall in propionibacteria is a very critical

factor for cell transformation by electroporation. It is known that a lower efficiency of

electroporation can be obtained in gram-positive bacteria than that in gram-negative bacteria, although some developed electroporation techniques were modified to obtain a higher efficiency of electroporation in gram-positive bacteria, such as Bacillus,

Streptococcus, and Lactococcus (Dunny, et al., 1991; Holo and Nes, 1989; Powell et al.,

1988). These studies reported that transformation rates ranged from 104 to 107 cells/µg of

DNA among Bacillus, Streptococcus, and Lactococcus. Transformation rates (cells/µg of

DNA) were variable from species.

3.1.3.3 Strategies of gene manipulation in propionibacteria

Manipulating gene by using genetic techniques are mainly done by two methods, gene inactivation and overexpression. However, most studies of genetic manipulation in propionibacteria are only focused on the construction of shuttle plasmids (Kiatpapan et el., 2000), biosynthetic pathway of vitamin B12 (Hashimoto et al., 1996; Murakami et al.,

1993) and their probiotic effects (Faye et al., 2000). Not many studies investigated the metabolic network by using genetic manipulation to enhance or block out the acid production. To date, no study about genetic manipulation of acid formation pathway in propionibacteria has been published, and no genomic sequence of P. acidipropionici has been determined or published in any public database.

Genetic manipulation of acid formation pathway was investigated in Clostridium acetobutylicum ATCC 824. Clostridial phosphotransacetylase (pta) and acetate kinase

(ack) were cloned, sequenced and expressed to investigate the acetate formation in C. acetobutylicum ATCC 824, and characterized by studying their enzyme activities and acetate product yield (Green et al., 1996; Zhuang et al., 1996). In C. acetobutylicum

ATCC 824, gene inactivation and overexpression were accomplished by non-replicative and replicative plasmids, respectively. For the purpose of disrupting the enzyme function, gene inactivation is a useful molecular tool. A selective gene in the chromosome was

inactivated and integrated into a non-replicative plasmid by genetic engineering techniques. This integrational plasmid, which contains a selective gene from the host chromosome, also carries a suitable marker for selection. Integration is occurred by homologous recombination involving Campbell-like mechanism (Campbell, 1962)

(Figure 3.2). After transforming into cells, plasmid integration can result in duplicate repeats of the homologous regions flanking DNA sequences. The mutagenesis will occur if the homologous DNA with plasmid is internal in the transcriptional unit, it results in the disruption of the unit and function (Piggot et al., 1984). On the other hand, if the homologous DNA fragment on the integrational plasmid contains the entire gene, it can lead a stable gene amplification, called overexpression (Young, 1984; Leenhouts et al.,

1989). Integrational mutagenesis is described in Figure 3.2. This genetic technique, a construction of non-replicative integrational plasmid, have been used in advanced genetic studies of gram-positive bacteria, but also limited by the difficulty of transformation in gram-positive bacteria (Perego et al., 1993; Kieser and Hopwood, 1991; Chassy and

Murphy, 1993). As a result, a proper transformation can help the construction of mutant by integration technique.

3.2 Objective of gene disruption in acetate kinase of P. acidipropionici

In the metabolic pathway of P. acidipropionici, carbon flow can be directed into two different acid productions, propionic acid and acetic acid (Figure 2.1). To enhance the desired propionic acid production, alternation of carbon flow to block acetic acid formation is considered as an effective way by using advanced genetic engineering

techniques. Gene disruption in key enzymes involving acetate formation is the main idea

of blocking acetate formation pathway in P. acidipropionici. Since acetate kinase (ACK)

is the enzyme in the pathway catalyzing acetyl-phosphate to acetate. Disrupting gene

encoding ack enzyme, ack, is expected to increase propionic acid production.

3.3 The concept of gene disruption in P. acidipropionici

3.3.1 PCR-based gene manipulation

PCR-based gene manipulation is an effective method to clone partial ack gene in

the genome of P. acidipropionici. Although genomic map of P. acidipropionici is not

available, protein alignment of ack from other microorganisms can provide useful

information for the design of degenerate primers for PCR. The partial ack sequence can

be cloned from PCR by TA cloning and then integrated with a non-replicative plasmid.

Gene manipulation would be accomplished by integration of plasmid with a

selective marker. A non-replicative plasmid can be constructed to contain partial ack gene and an antibiotic resistant gene for being a selection marker (Figure 3.1). In this

study, tetracycline resistant gene (Tetr) was inserted to the plasmid since wild type of P.

acidipropionici is sensitive to tetracyclin. After transforming this non-replicative

integrational plasmid with partial ack gene into P. acidipropionici cells, a mutant can be

produced through homologous recombination, resulting in the integrational mutagenesis

as illustrated in Figure 3.1.

3.2.2 Integrational mutagenesis by homologous recombination

Non-replicative plasmids have been used to advanced genetic studies in gram- positive bacteria. Such plasmids need a DNA fragment from the host and a genetic marker for further selection. After transforming into competent cells, integration may be established by inserting DNA into homologous region on the host replicon in a

Campbell-like fashion (Campbell, 1962), and then result in two direct repeats of homologous region flanking the plasmid DNA. Insertion can be mutagenic if the homologous DNA fragment with the integrational plasmid lies entirely within a gene or transcription unit (Piggot et al., 1984). Once insertion in chromosome DNA results in the disruption of the gene and loss of function, mutant phenotype can be produced. This is called “integrational mutagenesis” (Figure 3.2).

EcoRI EcoRI Partial Ack

SphI

f1 ori pUC ori PCR2.1 TOPO 3.9kb Km’

Amp’ Deletion ECE98 SphI

XbaI ApaI

Ack XbaI TetR 1.8kb

pUC ori pAck 1 Insertion 2.9kb ApaI

Amp’

Ack XbaI

pUC ori pAcK-Tet R 4.7kb Tet

ApaI

Amp’

Fig 3.1 Construction of non-replicative plasmid

oriE CAM Partial ack

P T

P CAM oriE T

Figure 3.2 The concept of integrational gene into chromosome to cause integrational mutagenesis and disrupt ack gene function.

CHAPTER 4

PCR BASED CLONING

4.1 Introduction

Polymerase chain reaction (PCR) is a technique used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested. To date, PCR based approach has been applied in many ways of genetic research, such as identifying with a very high-probability, disease-causing viruses and bacteria. By using two short oligonucleotides, target DNA fragments can be searched and amplified dramatically. It also had been applied in understanding certain DNA functions or manipulating genes to alter biological activities in organisms. Either for DNA function analysis or manipulating gene, targeted gene alternation is an important step for evaluating efficiencies in organisms. Therefore, PCR is one of the most critical steps in gene manipulation.

For unknown genome of microorganism, such as P. acidipropionici, degenerate

PCR is considered as a suitable method to target desired gene within the genome.

Degenerate PCR is a method to use degenerate primers, which have a number of options at several positions in the sequence to allow annealing and amplifying a variety of related sequences. It has been used for searching specific genes in a genome, without a known 37

genomic map. By using degenerate primers, conserved sequences of a gene or genes from

the genome of an organism can be amplified to get the entire nucleotide sequence.

Furthermore, this amplified PCR product can be cloned into a non-replicative plasmid by

an efficient method, TA cloning, for gene disrupting.

4.2. Materials and methods

4.2.1 Bacterial strains and Plasmids

The bacteria strains and plasmids used in this study are listed in Table 4.1. These

materials were obtained from Invitrogen, ATCC, and Bacillus genetic stock center at The

Ohio State University.

P. acidipropionici ATCC 4875 was grown anaerobically at 32 ℃ in

propionibacteria growth medium. The composition of the medium is described in

Appendix A1. P. acidipropionici colonies were obtained from plates with the same medium after incubating for 2 days at 32℃. E. coli , TOP 10 competent cells, was

obtained from Invitrogen and grown at 37 ℃ in Luria-Bertani (LB) (Appendix A2)

medium containing Ampicillin (100 µg/ml) and tetracycline (15 µg/ml) as required.

4.2.2 Genomic DNA isolation

Genomic DNA was isolated and purified by using QIAGEN DNasyTM Tissue kit

(Appendix C1). PCR amplification was undertaken by Tag DNA polymerase (cloned,

Amersham biosciences) (Appendix C4).

4.2.3 PCR amplification and TA cloning

Isolation of plasmids DNA from E. coli was undertaken by using QIAprep Spin

Miniprep kit (Qiagen) (Appendix C2). Restriction enzymes, including EcoRI, XbaI, ApaI,

and SphI (Amersham biosciences), and T4 ligase (Invitrogen) were used in accordance

with the supplier’s instruction. Degenerate oligonucleotides (primers) of Ack-F and Ack-

R were derived based on the homologous region of the ack protein sequence among E.

coli, M. thermophila, and B. bacillus (Figure 4.1). AK-forward and AK-reverse primers

were designed based on the DNA sequence of partial ack (749 bp) reported by Dr. Yan

Huang’s dissertation (1998) (Figure 4.7). These custom primers were obtained from

Invitrogen.

The DNA sequences of primers for ack gene used in this study were:

Degenerate primers:

Ack-F: 5’-AAG GAT CCA YMG IGT IGT ICA YGG IGG 3’ Ack-R: 5’-AAG GAT CCT CIC CDA TIC CIS CIG TRA A 3’

Specific primers AK-forward: 5’- GAT CCG CAA ATA CGG GTT TC -3’ AK-reverse: 5’- GCG GTG AAA ATA ATC GCA TC -3’

The PCR conditions used for thermal cycling in this study were:

I. For degenerate primers:

Step Temperature Time 1. Denaturing 94℃ 3 minutes 2. Denaturing 94℃ 50 seconds 3. Annealing 42℃ 50 seconds 4. Extension 72℃ 50 seconds 5. Repeat step2 to step 4 for 15 times 6. Denaturing 94℃ 50 seconds 7. Annealing 50℃ 50 seconds 8. Extension 72℃ 50 seconds 9. Repeat step 6 to step 8 for 30 times 10. Extension 72℃ 5 minutes

II. For specific primers

Step Temperature Time 1. Denaturing 94℃ 3 minutes 2. Denaturing 94℃ 50 seconds 3. Annealing 50℃ 50 seconds 4. Extension 72℃ 50 seconds 5. Repeat step 3 to step 4 for 30 times 6. Extension 72℃ 10 minutes The compositions of mixture for PCR are given in Appendix C4.

Thermal cycling was performed in a DNA engine machine (MJ Research). TA cloning

(Figure 4.2) was performed using TOPO TA cloning kit (Invitrogen). PCR product was amplified and cloned into a commercial vector, pCR 2.1 TOPO, 3.9 kb of TOPO TA cloning (Invitrogen) (Appendix C5), and then sequenced by DNA sequencing center at

The Ohio State University.

4.2.4 Construction of non-replicative plasmid with partial ack

A 1.5 kb of DNA fragment was deleted by restriction enzyme, SphI, from plasmid pCR-Ack (4.4 kb), which contained partial ack gene (~ 500 bp). SphI-deleted plasmid was re-ligated to form pCR-Ack1 (2.9 kb). The other plasmid, ECE98, was obtained from

Bacillus center, the Ohio State University, containing a TetR cassette. TetR cassette, 1.9 kb, was removed from ECE98 by two restriction enzymes digesting, XbaI and ApaI. The

1.9 kb of TetR cassette was ligated into XbaI-ApaI digested pCR-Ack1, resulting in the plasmid, pAck-Tet (4.8 kb) (see Figure 3.2).

Strains / Plasmids Relevant Source / reference characteristics

P. acidipropionici ATCC ATCC 4875 E. coli TOP 10 Ampr, Kmr Invitrogen

Plasmid pCR2.1 TOPO Ampr Invitrogen pCR-Ack Ampr This study pCR-Ack1 Ampr This study ECE 98 Tetr Bacillus genetic stock center at the Ohio State University

Table 4.1 Bacterial Strains and Plasmids used in this work

Footnotes ack-: the deletion of ack tetr : tetracycline resistant gene Ampr: ampicillin resistant gene Kmr: kanamycin resistant gene

E. coli 1 MSSKLVLVLNCGSSSLKFAIIDAVNGEEYLSGLAECFHLPEARIKWKMDGNKQEAALGAG 60 M. thermophila 1 ---MKILVINCGSSSLKYQLIESKDGNVLAKGLAERIGINDSLLTHNANGEKIKIKKDM- 56 B. subtilis 1 --MSKIIAINAGSSSLKFQLFEMPSETVLTKGLVERIGIADSVFTISVNGEKNTEVTDI- 57 P. acidipropionici 1 ------1

E. coli 61 AAHSEALNFIVNTILAQKP---ELSAQLTAIGHRIVHGGEKYTSSVVIDESVIQGIKDAA 117 M. thermophila 57 KDHKDAIKLVLDALVNSDYGVIKDMGEIDAVGHRVVHGGEYFTSSVLITDDVLKAITDCI 116 B. subtilis 58 PDHAVAVKMLLNKLT--EFGIIKDLNEIDGIGHRVVHGGEKFSDSVLLTDETIKEIEDIS 115 P. acidipropionici 1 ------HRVVHGDEIFNDSAVVNDQVLAQIEDLA 28

E. coli 118 SFAPLHNPAHLIGIEEALKSFPQLKDKNVAVFDTAFHQTMPEESYLYALPYNLYKEHGIR 177 M. thermophila 117 ELAPLHNPANIEGIKACYQIMPDVP--MVAVFDTAFHQTMPDYAYLYPIPYEYYTKYKYR 174 B. subtilis 116 ELAPLHNPANIVGIKAFKEVLPNVP--AVAVFDTAFHQTMPEQSYLYSLPYEYYEKFGIR 173 P. acidipropionici 29 ELAPLHNRANATGIRAFRAVLPDVV--QVAVFDTAFHQTMPESAFLYSLYPAYYEKYRIR 86

E. coli 178 RYGAHGTSHFYVTQEAAKMLNKPVEELNIITCHLGNGGSVSAIRNGKCVDTSMGLTPLEG 237 M. thermophila 175 KYGFHGTSHKYVSQRAAEILNKPIESLKIITCHLGNGSSIAAVKNGKSIDTSMGFTPLEG 234 B. subtilis 174 KYGFHGTSHKYVTERAAELLGRPLKDLRLISCHLGNGASIAAVEGGKSIDTSMGFTPLAG 233 P. acidipropionici 87 KYGFGHTSHKYVAMRAAELLGRPIEXLRLISCHLGNGSAIAAIQGGRSXNTXMGXTPLAG 146

E. coli 238 LVMGTRSGDIDPAIIFHLHDTLGMSVDAINKLLTKESGLLGLTEVTSDCRYV--EDNYAT 295 M. thermophila 235 LAMGTRCGSIDPSIISYLMEKENISAEEVVNILNKKSGVYGISGISSDFRDLEDAAFKNG 294 B. subtilis 234 VAMGTRSGNIDPALIPYIMEKTGQTADEVLNTLNKKSGLLGISGFSSDLRDI-VEATKEG 292 P. acidipropionici 147 VTXGTXSGNIXPALIPFIMK-TGKTAEEVLEVLXKESGLXGISGVSSDXRDIQVAAELER 205

E. coli 296 KEDAKRAMDVYCHRLAKYIGAYTALMDGRLDAVVFTGGIGENAAMVRELSLGKLGVLGFE 355 M. thermophila 295 DKRAQLALNVFAYRVKT-IGSYAAAMGG-VDVIVFTAGIGENGPEIREFILDGLEFLGFK 352 B. subtilis 293 NERAETALEVFASRIHKYIGSYAARMSG-VDAIIFTAGIGENSVEVRERVLRGLEFMGVY 351 P. acidipropionici 206 NKRAELALDIFASRIHKYIGSYAAKMAS-VDAIIFTAGIGED------246

E. coli 356 VDHERNLAARFGKSGFIN--KEGTRPAVVIPTNEELVIAQDASRLTA------400 M. thermophila 353 LDKEKNKVR--GEEAIISTAPDAKVRVFVIPTNEELAIARETKEIVETEVKLRSSIPV 408 B. subtilis 352 WDPALNNVR--GEEAFIS-YPHSPVKVMIIPTDEEVMIARDVVRLAK------395 P. acidipropionici 246 ------246

Figure 4.1 The protein alignment of ACK enzymes among Bacillus subtilis, M. thermophila, E. coli and P. acidipropionici.

Tyr-274 Topoisomerase

P OH

CCCTT A AGGG PCR Product GGGA A TTCCC OH P

Tyr-274 Topoisomeras

Insertion

Figure 4.2 TA cloning method (www. Invitrogen.com). Two Adenine bases overhanging on the two ends of PCR fragment can anneal to two Thymine bases overhaning on the two ends of vector, pCR-TOPO by Topoisomerase catalyzing.

4.3 Results and discussion

4.3.1 Genomic DNA isolation

Genomic DNA preparation is a critical step for PCR amplification and further

gene manipulation. For PCR amplification, enough amount of genomic DNA can help

primer annealing and extension. A higher final PCR product can also be expected by

using a higher amount of genomic DNA. However, it was not easy to obtain a high

concentration of genomic DNA from P. acidipropionici ATCC 4875 (Figure 4.4). The

low DNA yield from propionibacteria might be caused by incomplete cell lysis. P. acidipropionici is a gram-positive bacterium and has a thicker cell wall as compared to gram-negative bacteria. Lysozyme and Proteinase A were used to lysis cells. It seemed that lysozyme and Proteinase A were not very sufficient to breakdown cell walls of gram- positive bacteria. Although it has been reported that lysozyme treatment could help cell lysis in gram-positive bacteria, such as Bacillus and Streptococcus, lysozyme was not consistently effective to all gram-positive bacteria. It also was reported that many propionibacteria strain were resistant to lysozome treatment (Johnson and Cummins,

1972). In this study, it showed that lysozome treatment was not very effective and the amount of isolated genomic DNA from P. acidipropionici was low.

4.3.2 PCR Amplification

Several different primers were used for ack amplification in this study (Appendix

C4). Degenerate primers were designed based on protein alignment (Figure 4.1). On the

other hand, two specific custom primers, designed based on the partial ack sequence obtained by Dr. Yan Huang (Dissertation, 1998) were also used. Primers were designed by several factors, including primer length, specificity, melting temperatures (Tm), G/C content, GC clamp, Max 3’ self complementary and primer length. Melting temperature is one of critical factors to affect the annealing temperature of template DNA during PCR amplification. Different designed primers, according to primer length and GC content, require different melting temperature. Generally speaking, longer primer length with 50

% of GC content needs a high Tm. In this study, even though melting temperatures were calculated; several annealing temperatures were also tested to obtain the optimum PCR result. A range of annealing temperature was chosen from 52 to 55℃. Speaking of GC content, it is considered that the base composition of primers should be between 45 % and 55 % of GC content. However, propionibacteria genome is considered to have a higher GC content, around 60-70 %. Therefore, a higher GC content in degenerate primers may be necessary to obtain a higher efficiency of PCR.

For degenerate primers Ack-F (1) and Ack-R (2) designed from homologous region of protein alignment, it resulted in a desired size of 749 bp of band after PCR amplification, as shown in the DNA gel electrophoresis (Figure 4.4). The other primers used in this study were AK-forward (3) and AK-reverse (4). These specific primers were used to do PCR with pAck21 plasmid as the template DNA. The first time of PCR by using AK-forward and AK-reverse primers with genomic DNA obtained an expected but weak band. However, it failed when doing cloning. The same condition and primer were used to perform PCR for obtaining previous result. Several times of PCR were tried by using propionibacteria genomic DNA, but the results would not be repeated. This 46

problem might be caused by different preparations of genomic DNA. Different

preparations of genomic DNA can result in differences in DNA concentrations and

purities. Moreover, longer storage time of genomic DNA may reduce the stability of

DNA and cause degradation of DNA. For further construction of a non-replicative

plasmid, pAck21 plasmid, which contains partial ack (749 bp), was used as the template

DNA. The annealing temperature in this PCR reaction was used at 52 ℃ by using

primers with Tm of 45 ℃. Resulted size of 477 bp of ack fragment was shown in Figure

4.5. In this PCR amplification, the template DNA used was the plasmid containing partial

ack sequence, which was constructed by Dr. Yan Huang (1998). The optimal annealing

temperature was 50 ℃; lower annealing temperatures, 42 and 45 ℃, were also used, but resulted in a weaker band.

4.3.3 TA cloning and sequence analysis

The sequencing result of 749 bp showed too many stop codens during sequence.

This was considered as a negative result compared with Dr. Yan Huang’s dissertation by

using the same primers, Ack-F and Ack-R. The reasons can be: (1) using different

thermal cycler, the thermal cycler used in this study was from MJ Research. The

sensitivity of thermal adjustment during PCR affects DNA denaturing, annealing, and

extension. (2) Different PCR mixture and ratio of components in PCR mixture also affect

the PCR result. For example, PCR mixture contains MgCl2. The relationship between

MgCl2 and dNTPs concentration affects Taq DNA polymerase activity and final product

yield. The activity of Taq DNA polymerase requires free magnesium. Therefore, small

increases in the dNTP concentration can rapidly inhibit the PCR reaction because free

magnesium gets trapped. In this study, MgCl2 concentration was 15 mM, which was

original concentration in supplied 10 × buffer, compared to adding additional 5 µl of

MgCl2 solution (25 mM). (3) Different preparation of genomic DNA was used. As

discussed earlier, genomic DNA is a very critical factor to affect PCR amplification.

Therefore, different genomic DNA preparation may lead to different results of PCR, even

using the same PCR condition (temperatures). The bacterial culture used in this study was

newly obtained from ATCC, not an old stock. Different culture source may also result in

different genomic DNA preparations due to cell activity, growth and purity, if

contaminated.

On the other hand, the 477 bp of PCR amplification DNA sequence is identical to

the partial ack sequence from Dr. Yan Huang (Figure 4.6 and 4.7). This is considered as a positive result based on the comparison of protein sequence. The restriction sites of DNA sequence are shown in Figure 4.8. This 477 bp of PCR product was used in further cloning to construct a non-replicative plasmid.

4.3.4 Ligation

After cloning, a genetic selection marker, tetracyclin resistant gene, was ligated

into the plasmid with the partial ack fragment. Two restriction enzymes, XbaI and ApaI,

were used to remove the Tetr fragment, ~1.8 kb. However, tetracycline resistant gene is a

large DNA fragment (~1.8 kb). Although a deletion was done to shorten the size of 48

pCR2.1-Ack, it was difficult to ligate two large fragments together. A longer incubation time (18 hours to 24 hours) at 25 ℃ was used to expect a higher efficiency of ligation.

Transformation of competent cells is an important test to a ligation reaction because not all of the high molecular weight created in a ligation reaction will perform efficiently.

Supercoiled vector DNA can be formed after ligation. By transforming into competent cells, only successfully ligated DNA fragments can replicate in cells. In this study, ligation with two large fragments were failed after many tries. The reasons leading to failed ligation might include (1) large fragment were not easy to ligate together; (2) impropriate ligation buffer used, (3) failure or low efficiency of transformation after ligation reaction.

Genomic DNA

3kb 2kb 1kb

Figure 4.3. Gel electrophoresis of P. acidipropionici genomic DNA (8 µl); M was 1 kb marker (Bio-Rad)

123

1000 bp 700 bp

750 bp 500 bp 200 bp

100 bp

Figure 4.4. PCR amplification using degenerate primers, Ack-F and Ack-R and propionibacterium genomic DNA. Lane 1 is precision marker; Lane 2 and Lane 3 are the PCR products, which had an expected size of 750 bp.

123

1000 bp 700 bp

477 bp 500 bp 200 bp 100 bp

Figure 4.5. PCR result by using primers AK-forward and AK-reverse with pack-21 as template DNA. Lane 1 from the left is precision marker (Bio-Rad). Lane 2 and lane 3 are PCR product with ~500 bp.

File : Yan Huang’s ack gene Range 1 – 749 bp

9 18 27 36 45 54 5' TGG ATC CAT AGG GTG GTG CAC GGG GAC GAA ATT TTC AAT GAT TCT GCT GTC GTC W I H R V V H G D E I F N D S A V V

63 72 81 90 99 108 AAT GAC CAG GTG CTC GCG CAA ATT GAA GAT TTG GCG GAA CTC GCG CCG CTT CAT N D Q V L A Q I E D L A E L A P L H

117 126 135 144 153 162 AAC CGG GCG AAC GCA ACG GGG ATC CGG GCG TTC CGT GCC GTG CTG CCG GAT GTG N R A N A T G I R A F R A V L P D V

171 180 189 198 207 216 GTT CAA GTG GCC GTG TTT GAT ACC GCT TTC CAC CAA ACG ATG CCG GAA AGT GCT V Q V A V F D T A F H Q T M P E S A

225 234 243 252 261 270 TTC TTA TAC AGC CTC CCC TAT GCA TAC TAC GAA AAA TAC CGG ATC CGC AAA TAC F L Y S L P Y A Y Y E K Y R I R K Y

279 288 297 306 315 324 GGG TTT CAT GGC ACT TCC CAT AAA TAT GTG GCG ATG CGT GCC GCT GAG CTG CTC G F H G T S H K Y V A M R A A E L L

333 342 351 360 369 378 GGC AGG CCA ATT GAA CAG CTG CGC CTG ATT TCA TGC CAT TTG GGC AAC GGG GCA G R P I E Q L R L I S C H L G N G A

387 396 405 414 423 432 AGC ATT GCG GCG ATC CAG GGC GGC CGG TCA ATC GAT ACG TCC ATG GGC TTT ACG S I A A I Q G G R S I D T S M G F T

441 450 459 468 477 486 CCA TTG GCC GGC GTG ACG ATG GGC ACG CGC TCC GGC AAT ATC GAC CCC GCA CTG P L A G V T M G T R S G N I D P A L

495 504 513 522 531 540 ATC CCG TTT ATT ATG GAG AAG ACA GGC AAA ACG GCA GAA GAA GTG CTC GAA GTG I P F I M E K T G K T A E E V L E V

549 558 567 576 585 594 TTA AAC AAA GAA TCC GGG CTT CTC GGC ATT TCG GGC GTT TCC AGC GAT TTG CGC L N K E S G L L G I S G V S S D L R

603 612 621 630 639 648 GAT ATC CAG GTG GCG GCG GAA CTC GAG CGG AAC AAG CGG GCT GAA CTG GCG CTT D I Q V A A E L E R N K R A E L A L

657 666 675 684 693 702 GAC ATT TTT GCA AGC CGC ATC CAT AAA TAC ATC GGT TCG TAT GCG GCA AAA ATG D I F A S R I H K Y I G S Y A A K M

711 720 729 738 747 GCC AGC GTC GAT GCG ATT ATT TTC ACC GCC GGC ATT GGC GAG GAT CC 3' A S V D A I I F T A G I G E D

Figure 4.6 The DNA sequence of partial ack (749 bp) from Yan Huang (1998).

File : Ack gene Range : 1 - 477

9 18 27 36 45 54 5' TTG ATC CGC AAA TAC GGG TTT CAT GGC ACT TCC CAT AAA TAT GTG GCG ATG CGT L I R K Y G F H G T S H K Y V A M R

63 72 81 90 99 108 GCC GCT GAG CTG CTC GGC AGG CCA ATT GAA CAG CTG CGC CTG ATT TCA TGC CAT A A E L L G R P I E Q L R L I S C H

117 126 135 144 153 162 TTG GGC AAC GGG GCA AGC ATT GCG GCG ATC CAG GGC GGC CGG TCA ATC GAT ACG L G N G A S I A A I Q G G R S I D T

171 180 189 198 207 216 TCC ATG GGC TTT ACG CCA TTG GCC GGC GTG ACG ATG GGC ACG CGC TCC GGC AAT S M G F T P L A G V T M G T R S G N

225 234 243 252 261 270 ATC GAC CCC GCA CTG ATC CCG TTT ATT ATG GAG AAG ACA GGC AAA ACG GCA GAA I D P A L I P F I M E K T G K T A E

279 288 297 306 315 324 GAA GTG CTC GAA GTG TTA AAC AAA GAA TCC GGG CTT CTC GGC ATT TCG GGC GTT E V L E V L N K E S G L L G I S G V

333 342 351 360 369 378 TCC AGC GAT TTG CGC GAT ATC CAG GTG GCG GCG GAA CTC GAG CGG AAC AAG CGG S S D L R D I Q V A A E L E R N K R

387 396 405 414 423 432 GCT GAA CTG GCG CTT GAC ATT TTT GCA AGC CGC ATC CAT AAA TAC ATC GGT TCG A E L A L D I F A S R I H K Y I G S

441 450 459 468 477 TAT GCG GCA AAA ATG GCC AGC GTC GAT GCG ATT ATT TTC ACC G Y A A K M A S V D A I I F T

Figure 4.7 The DNA sequence of partial ack (477 bp) from primers Ack-R and Ack-F.

Figure 4.8. The restriction map in the partial ack (477 bp) sequence

CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

5.1. Conclusions The main idea of this research was using advanced genetic tools to manipulate genes involving acetate formation in Propionibacterium acidipropionici and to alter the carbon flow in the metabolic pathway for improving propionic acid yield. The work done in this study includes the preparation of genomic DNA from P. acidipropionici, PCR amplification, TA cloning, and free cell fermentation with wild type of P. acidipropionici.

Results are summarized below.

Genomic DNA with P. acidipropionici had a low yield. It could be the reason

leading a low efficiency of PCR amplification.

Ack fragment obtained in this study was 477 bp in length by two primers, AK-

forward and AK-reverse.

Batch fermentation with free cells of wild type P. acidipropionici produced a high

lactic acid concentration, 12 g/L, and propionic acid concentration, 9 g/L. Succinic

acid and propionic acid were about 2 g/L, with an initial glucose concentration of 30

g/L.

5.2. Recommendations Although the work of manipulating gene in this research has not been done, some

future works can be continued and performed. Construction of a non-replicative plasmid

containing partial ack and a selective marker was not completed, due to difficulty in ligation and transformation. The following works are recommended for future research.

Exploring an efficient method to obtain a higher yield of genomic DNA from P.

acidipropionici cells should be done first in order to increase the performance of

PCR amplification and further transformation.

Ligation of non-replicative plasmids with partial ack fragment and tetracyclin

resistant gene as the selective genetic marker should be done. An integrational

plasmid is needed in order to produce mutagenesis in P. acidipropionici cells after

introducing plasmids into cells by transformation. However, large fragments are not

easy to ligate together. Therefore, future work in ligation of exploring the efficient

method would be advantageous for constructing the integrational plasmids.

Transformation of a non-replicative integrational plasmid into P. acidipropionici

cells should be performed by electroporation. However, the efficiency of

electroporation is dependent on the electric resistant and other parameters. Therefore,

a proper protocol affecting electroporation should be explored.

Future study on fermentation analysis of ack-deficient mutant should be done to

explore the effects of mutant strain for acid production and cell growth. A higher

propionic acid yield and lower acetic acid production are expected for the ack-

deficient mutant. However, it was reported that acetyl-phosphate could function as a

global regulator in a metabolic pathway (McCleary, et al., 1993). Global

phosphorlation can lead to acetate formation from acetyl-phosphate (Wanner and

Willimes-Riesenberg, 1992). And, acetate may be formed as long as acetyl-

phosphate is accumulated in the cells; therefore, pta gene, encoding

phosphotransacetylase in the acetate formation pathway, should also be disrupted to

further reduce or eliminate acetate formation.

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APPENDICES

Appendix A Medium compositions

A1 Medium compositions for Propionibacterium acidipropionici

Medium contained (per liter):0. 25 g of K2HPO4, 0.05 g of MnSO4, 10 g of yeast

extract, and 5 g of tryptone. Glucose, 500 mg/ml, was prepared separately in a serum

bottle. Medium was adjusted to pH 6.5 and prepared in serum tubes in a 3 ml volume.

Both medium and glucose solution were purged with nitrogen gas to assure an anaerobic condition. Autoclaving medium and glucose was performed at 121 ℃ for 20 minutes at

18 psi. After cooling down, add 2-3 drops of prepared glucose by injecting with syringe aseptically.

A2 Medium composition for E. coli –Luria-Bertani (LB) medium

Medium contained (per liter): 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride (NaCl). The pH was adjusted to 7.0. Medium was autoclaved at 121 ℃ for 20 minutes at 18 psi. Add antibiotic, 100 mg/ml of ampicillin, in autoclaved medium after cooling down to 55 ℃. To prepare agar plates, add 15 g/L before autoclaving.

Appendix B Analytical Methods

B1 Cell concentration Cell concentration was described as OD value in this study. The OD value was

measured with a spectrophotometer (Sequoia-Turner, Model 340) at the wavelength of

600 nm. Fermentation samples were taken at a regular interval. Both suspended samples

and cell free (centrifuged) samples were diluted with proper ratio, 1/5~1/20, and then

pipetting into a cuvee. Two different OD values were obtained from each sample. The

cell concentration was calculated by the equation below:

Cell concentration = OD600nm of cell suspension sample － OD600nm of cell free sample

B2. High Performance Liquid Chromatography (HPLC)

The high performance liquid chromatograph consisted of an automatic injector

(Shimadzu SIL-10Ai), a pump (Shimadzu LC-10Ai), an organic acid analysis column

(Bio-Rad HPX-87H), a column oven at 45 ℃ (Shimadzu CTO-10A), and a reflective index detector (Shimadzu RID-10A). The eluent was 0.01 M H2SO4 prepared by sonicating for 30 minutes. The eluent was set at a flow rate of 0.6 ml/min. Cell-free samples were prepared with two different dilutions, 1/5 and 1/15, by adding distilled water. Samples with a volume of 15 µl were injected by the auto-sampler. The running time was set for 25 minutes. The AUX RANGE parameter of RI detector was set at 2.

A standard solution containing all the components at 2 g/L each was prepared.

Peak heights of samples were used to calculate the concentration of each component based on the analysis of standard mixtures. 70

Appendix C Protocols of Genetic Engineering Experiments

C1 Preparation of Genomic DNA from P. acidipropionici with QIAGEN

Genomic DNA kit

1. Harvest cell in a 50 ml tube with active P. acidipropionici culture anaerobically

and grow at 32 ℃ overnight to reach a O.D. around 2.0

2. Pellet down cells (max 2 x 109 cells) by centrifugation for 10 minutes at 7,500

rpm (5,000 x g), and discard the supernatant.

3. Prepare lysozyme solution-lysis buffer, at a concentration of 100 mg/ml with

autoclaved distilled water.

4. Suspend cell pellet in 180 µl of lysis buffer, and incubate for at least 30 minutes at

37 ℃.

5. Add 25 µl of Proteinase K buffer and 200 µl of Buffer AL. Mix it by vortexing.

Do not add Proteinase K into Buffer AL directly.

6. Incubate at 70 ℃ for 30 minutes.

7. Add 200 µl of ethanol (96-100%) to the sample, and mix it thoroughly by

vortexing. A homogenous solution would be yielded.

8. Apply the mixture from step 7 into the DNeasy mini column sitting in a 2-ml

collection tube provided inside the kit. Centrifuge at ≧ 8,000 rpm (6,000 x g) for

1 minute. Discard the flow-though solution and collection tube.

9. Place the DNeasy mini column into a new 2-ml collection tube (provided), and

add 500 µl of Buffer AW1. Centrifuge for 1 minute at a speed ≧ 8,000 rpm

(6,000 x g). Discard the flow-though solution and collection tube. 71

10. Place the DNeasy mini column into a new 2-ml collection tube (provided), and

add 500 µl of Buffer AW2. Centrifuge for 3 minutes at a full speed (13,200 rpm)

to dry the DNeasy membrane. Discard the flow-though solution and collection

tube.

11. Place the DNeasy column in a clean, autoclaved 1.5 ml of microcentrifuge tube,

and pipette 200 µl of Buffer AE onto the center of DNeasy membrane. Incubate at

room temperature for 1 minute, and then centrifuge for 1 minute at a speed ≧

8,000 rpm (6,000 x g).

Note: If wanting a higher concentration of DNA, elute 100 µl of Buffer AE,

instead of 200 µl, but also resulting in a lower overall DNA yield.

C2 Preparation of Plasmid DNA by QIAprep Spin Miniprep kit (QIAGEN)

1. E. coli culture was harvested for 1-5 ml in LB medium overnight after doing

transformation. Pellet down cells by centrifugation for 5 minutes at a full speed,

13,200 rpm. Discard the supernatant.

2. Add the provided RNase A solution into Buffer P1, and mix well. Add ethanol

(96-100 %) into Buffer PE before use.

3. Resuspend pelleted bacterial cells in 250 µl of Buffer P1 added with RNase A

solution. Transfer this mixture into a 1.5 ml microcentrifuge tube.

4. Add 250 µl of Buffer P2 and gently invert the tube 4 ~ 6 times to mix well. Do

not let the lysis reaction to proceed more than 5 minutes. 72

5. Add 350 µl of Buffer N3 and invert this mixture immediately and gently 4 ~ 6

times.

6. Centrifuge for at least 10 minutes at a maximum speed in a tabletop

microcentrifuge.

7. Apply the supernatant from step 6 into the 2-ml QIAprep mini column by

decanting or pipetting.

8. Centrifuge for 1 minute and discard the flow-through.

9. Add 500 µl of Buffer PB to wash the QIAprep column and centrifuge for 1

minute. Discard the flow-through.

10. Wash QIAprep mini spin column by adding 750 µl of Buffer PE and centrifuge

for 1 minute. Discard the flow-through.

11. Centrifuge for an additional 1 min to remove residual wash buffer remaining on

the membrane.

12. Place the spin column into a 1.5 ml of clean, autoclaved centrifuge tube, and then

adding 30-50 µl of EB (10nM Tris-Cl, pH 8.5), or distilled water to the center of

each QIAprep spin column. Let stand for 1 min, and centrifuge for 1 min.

Note: The obtained plasmids usually have a high concentration of 50 ~ 300 ng/µl.

C3 DNA electrophoresis

1. Prepare a proper volume of electrophoresis buffer TAE to fill the electrophoresis

tank and to prepare the DNA-grade agarose gel.

2. Add the desired amount of agarose into a proper volume of TAE buffer sufficient

for constructing 0.8 %~1.0 % gel for DNA separation. Melt the agarose with TAE

buffer by heat and swirling to mix it thoroughly. Let it cool down to 50~60 ℃.

3. Set the gel-casting platform by tape, pour the agarose mixture, and then insert the

gel comb.

4. Wait the gel until completely solidified. Remove the tape and withdraw the gel

comb.

5. Place this gel-casting platform with a solidified gel in the electrophoresis tank.

Add a sufficient TAE buffer to cover the gel to the depth of 1 mm properly.

6. DNA samples were prepared by mixing with a proper volume of loading dye

(Bio-Rad) in a ratio of 4 to 1, respectively.

7. Load a proper volume (10 µl) of prepared DNA samples into wells by pipetting.

8. Turn on the power supply of Bio-Rad PowerPac300 and set the voltage to 50 V

for Bio-Rad Mini Sub-Cell.

9. When the Bromophenol Blue dye has moved to about 2/3 of the length of the gel,

turn off the power supply.

10. Stain the gel in the buffer with 0.5 µg/ml of ethidium bromide (EB) for 5 minutes.

11. Place the stained gel in the distilled water to un-stain for 5 minutes. Using gel

documentation system, Bio-Rad Gel Doc 2000, the DNA bands can be observed

easily. 74

C4 PCR amplification of ack from P. acidipropionici

1. In a autoclaved clean 500 µl of PCR tube, add reagents listed below

Reagent Volume (µl)

10 X PCR buffer with 25mM MgCl2 (Amersham Biosciences) 5 25mM dNTPs (Amersham Biosciences) 1 Tag DNA polymerase, 5Units/µl (cloned, Amersham Biosciences) 0.5 Forward Primer 10 µM 2 Reverse Primer 10 µM 2 Template DNA 5 Sterilized water 34.5 Total volume 50

2. Load the reaction tubes in a thermal cycler (MJ Research).

3. PCR programs were set as follows:

(1) For degenerate primers:

(2) For specific primers

4. Analyze 8 µl of PCR product for each sample by using DNA electrophoresis.

5. For continuous TA cloning, PCR product can be used directly or purified from the

gel by QIAGEN gel extraction kit, and then doing ligation to T-vector

(Invitrogen).

6. Primers used in this study are listed below:

(1) Ack-F: 5’- AAG GAT CCA YMG IGT IGT ICA YGG IGG 3’

(2) Ack-R: 5’- AAG GAT CCT CIC CDA TIC CUS CUG TRA A 3’

(3) AK-forward: 5’- GAT CCG CAA ATA CGG GTT TC -3’

(4) AK-reverse: 5’- GCG GTG AAA ATA ATC GCA TC -3’

C5 TA cloning protocol (Invitrogen TA cloning Kit-TOP 10)

1. Spin down the vial of pCR 2.1 TOPO vector by centrifuging several seconds to

collect all the liquid in the tube.

2. The table below is to describe how to set up the TOPO® Cloning reaction, 6 µl in

total, for transformation into TOP 10 One Shot chemically competent E. coli cells.

Reagents Volume (µl) Fresh PCR product 0.5 ~ 4 µl Salt solution 1 µl Sterile water Add to a total volume of 5 µl TOPO® vector 1 µl Final volume 6 µl

3. Mix up the reagents gently and incubate at room temperature (22 ~ 23 ℃) for 5

minutes. If needed, extend the ligation time up to 30 minutes, depending on the

size of PCR product. Larger PCR product (> 1 kb) will get more colonies if

extending reaction time.

4. Place the reaction mixture on ice and proceed to Transforming One Shot

competent cells or stored at –20 ℃ overnight.

5. Thaw one vial of 50 µl of frozen (-80 ℃ in storage) One Shot competent cell for

each transformation.

6. Add 2 µl of the TOPO® Cloning reaction from step 3 into one vial of TOP 10

chemically Competent E. coli and mix by stirring very gently with the tip. Do not

mix by pipetting up and down, or even vortexing.

7. Incubate on ice for 5 ~ 30 minutes.

8. Heat-shock the cells for exactly 30 seconds at 42℃ without shaking, and then

immediately transfer tube to ice.

9. Add 250 µl of SOC medium, room temperature, to each tube.

10. Cap the tube tightly and shake the tube horizontally at 37 ℃ at 225 rpm for 1 hour

in an incubator.

11. Spread 40 µl of 40 mg/ml X-gal stock solution onto each LB plate and let dry for

15-20 minutes and pre-warm at 37 ℃ in an incubator.

12. Spread 10-50 µl from each transformation on pre-warmed selective LB plates

containing 100 mg/ml of ampicillin and 40 mg/ml of X-gal. Incubate plates. To

ensure evenly spreading of cells, 20 µl of SOC medium was added with

transformed cells.

13. Invert these plates and place them in an incubator without shaking at 37 ℃

overnight. After incubation, move plates from incubator to 4 ℃ for 2-3 hours for

color development.

14. Pick up a sufficient number of white colonies and inoculate into the LB medium

containing 100 mg/ml of ampicillin at 37 ℃ overnight with shaking at 225 rpm.

15. Isolate plasmids from cells obtained from step 14 by using QIA prep Miniprep kit.

C6 DNA/Plasmid Digestion and Ligation

1. Plasmid DNA digestion was performed in a 10 µl volume, containing:

Reagents Volume (µl) 10 X digestion buffer (Amersham) 1 Restriction Enzyme (Amersham) 1 Plasmid DNA 4 Sterile water 4 Final Volume 10

2. DNA ligation (cohesive ends) was performed in a 20 µl volume, containing:

Reagents Volume (µl) 5 X ligation buffer (Invitrogen) 4 T4 DNA ligase, 1 U/µl (Invitrogen) 1 Vector DNA X Insert DNA 3X Sterile water 15 - 4X Final Volume 20

The volume ratio of vector and insert DNA fragment was 3:1, based on the manufacture’s instruction. Incubate this ligation reaction at 25 ℃ for at least 3 hours.

Due to the supercoiled vector would perform after DNA ligation; transformation would be the ultimate test of a ligation reaction. Perform a transformation reaction following the instruction of manufacture, and pick up several colonies formed on plates.

Check by electrophoresis after isolating plasmids and restriction enzyme digestion.

C7 DNA Purification by QIAquick Gel extraction kit

1. Run the gel electrophoresis, and excise the DNA fragment from the agarose gel

with a clean, sharp scalpel. Try to minimize the size of gel slice by removing

extra agarose.

2. Add 3 volume of Buffer QG to 1 volume of gel in a 1.5 ml of microcentrifuge

tube. The maximum of gel slice is 400 mg.

3. Incubate at 50 ℃ for 10 minutes or until the gel slice has completely dissolved.

Mix the gel and buffer for 2 ~ 3 times by inverting or vortexing during incubation.

4. After gel has completely dissolved, add 1 gel volume of isopropanol to the

samples and mix it by inverting or vortexing.

5. Place a QIAquick spin column in a provided 2-ml collection tube. And apply the

sample mixture to column. Centrifuge for 1 min.

6. Discard the flow-through and place the column back in the collection tube.

7. Add 500 µl of Buffer QG to column, and centrifuge for 1 min.

8. Wash the column by adding 750 µl of Buffer PE. Let it stand for 2-3 minutes and

centrifuge for 1 min.

9. Discard the flow-through; place the column back in the collection tube.

Centrifuge an additional 1 min at full speed to dry the column membrane

10. Place the column in a clean, sterile 1.5 ml of microcentrifuge tube. Add 50 µl of

Buffer EB (10mM Tris-Cl, pH 8.5) or distilled water to the center of the

membrane. Let it stand for 1 min, and then centrifuge for 1 minute to elute the

DNA. Alternatively, to increase the concentration of DNA, add only 30 µl of

elution buffer or water to column instead of 50 µl. 80

Appendix D Reagents and Buffers

EDTA (pH 8.0), 0.5 M

Dissolve 18.61 g of EDTA in 80 ml distilled water and adjust pH to 8.0 by adding

solid NaOH. Mix by stirring and add distilled water to 100 ml.

Ethidium Bromide (EB), 1000X

Dissolve 0.05 g of ethidium bromide in 100 ml distilled water. Mix it well and store it in the dark. 1 x of EB buffer is diluted from 1000x of EB for staining gel after DNA electrophoresis.

TE Buffer

10 mM Tris-HCl with adjusted pH of 8.0 and 1 mM EDTA (pH 8.0).

TAE Buffer, 50X

Dissolve 242.28 g Tris, 18.61 g EDTA and 34.02 g CH3COONa · 3 H2O in 50 ml distilled water and adjust the pH to 7.6 with acetic acid. Mix and add distilled water to 1 liter.