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A Threonine and Methionine Auxotroph of C. glutamicum Avoids Concerted Feedback Inhibition and Enables Industrial Lysine Fermentations

A Threonine and Methionine Auxotroph of C. glutamicum Avoids Concerted Feedback Inhibition and Enables Industrial Lysine Fermentations

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AMINO ACIDS



311



is unique to the bacteria Corynebacterium glutamicum and Brevibacterium flavum.

C. glutamicum also differs from other bacteria since lysine does not inhibit its own

pathway after the branch point. The first enzyme in the common pathway for

methionine production in E. coli and Salmonella typhimurium consists of three

forms of aspartokinase, with each form inhibited by a different end product. While

the first step of the lysine pathway in C. glutamicum is catalyzed by a single enzyme,

it is subject to multivalent inhibition by lysine and threonine (Fig. 11.12).

An auxotroph that requires threonine and methionine for growth was selected

as a candidate for lysine production since it lacks homoserine dehydrogenase at the

branch point for aspartate semialdehyde (Fig. 11.12) (Aiba et al. 1973; Wang et al.

1979). The mutant, grown on a nutrient medium containing low levels of threonine

and methionine, accumulates large amounts of lysine since the concerted inhibition

of the first enzyme in the pathway, aspartokinase, is bypassed, and the metabolic

intermediates are shunted toward lysine. Both the first enzyme and the second

enzyme (dihydrodipicolinate) of the lysine branch are neither inhibited nor repressed

by lysine. l-Lysine decarboxylase is absent from the end of the pathway. These



Aspartate

Aspartokinase

Aspartyl phosphate



Aspartate semi-aldehyde

Dihydropicolinate

synthetase



Homoserine

dehydrogenase

Homoserine



Threonine



Methionine



Lysine



Isoleucine



Figure 11.12. Auxotrophs for producing threonine and methionine [from Wang et al.

(1979), Fig. 2.3, p. 17].



312



METABOLIC PATHWAYS



Parent II (AECrCCLrMLrFP5Ala–)



50



40



0.5

20



0

0 20 40 60 80 100



AECrDecr XMr



0



Pusant



(c)



1.2 × 10–5



Lys

100



50



1.0



60

40



0.5



Incubation time (h)



20



Parent I (DecrXMr)



(b)



60



50



40



0.5

20



0



0 20 40 60 80 100



0 20 40 60 80 100

Lys.HCI (g/l)



1.0

O.D.



100



0



0



80

R.S. (g/l)



80

Lys.HCI (g/l)



60



O.D.



1.0

O.D.



R.S. (g/l)



100



80



R.S. (g/l)



Lys



Lys.HCI (g/l)



(a)



Incubation time (h)



0



Incubation time (h)



Figure 11.13. Cell fusion for developing lysine-producing microorganism. Slow-growing

parent that produces lysine (A) is fused with fast-growing nonlysine producer (B). The

resulting fusion product is both fast-growing and high-lysine-producing (C). [Ajinomoto,

Inc. (1990)].



characteristics enabled C. glutamicum mutants to produce over 50 g of lysine/L

fermentation broth by 1972.6



Cell (Protoplast) Fusion Is a Method for Breeding Amino Acid Producers that

Incorporate Superior Characteristics of Each Parent (Lysine Fermentation)

Protoplast fusion is a versatile technique for inducing genetic recombination in both

prokaryotes and eukaryotes. This method was significant in obtaining genetically

altered organisms prior to the introduction of genetic engineering, and may continue

to find use for some applications of industrial strain development (Matsushima and

Blatz 1986). Hence it is summarized by an example presented here. The benefit of

cell fusion is illustrated in Fig. 11.13. A slow-growing lysine producer fused with a

fast-growing and vigorous carbohydrate mutant that produces little lysine gave a

fast-growing lysine producer. The lysine-producing organism required 100 h to

6



The 1995 global market for fermentation-derived lysine was estimated at about $600

million/year. The economic importance of the lysine market is suggested by the discovery

and prosecution of a worldwide lysine cartel by the US Justice Department (Kilman

1998a,b).



AMINO ACIDS



313



achieve about 70 g/L of lysine, while only 40 h were needed for the organism based

on cell fusion of the two mutants.

Protoplasts are formed by treating cells with lytic enzymes that lyse the cell

wall in the presence of a hypertonic solution7 that stabilizes the osmotic environment

of the protoplasts. Protoplasts are induced to fuse and form transient diploids in

the presence of an agent such as polyethylene glycol (PEG) that promotes fusion.

Prokaryotic bacilli and streptomyces recombine their genomes at high frequency

since the chromosomes are readily accessible in the cytoplasm. However, nuclear

fusion must follow protoplast fusion in eukaryotes since the DNA is segregated

from the cytoplasm by a nuclear membrane. The last step is the regeneration of

viable cells from the fused protoplasts. Specific techniques are given by Matsushima

and Blatz (1986).

The ability to form, fuse, and regenerate protoplasts varies greatly from organism to organism. PEG-induced fusion of cells (protoplasts) was achieved for plants

in 1974, and has subsequently been applied to fuse animal, filamentous fungal,

yeast, Bacillus spp., Streptomyces, Brevibacterium, Streptosporangium, and Staphylococcus protoplasts. Matsushima and Blatz (1986) describe and review protocols

for protoplast fusion for microbial species found to be generally applicable for

industrial strain development. “Easily scorable genetic markers” (e.g., nutrition,

antibiotic resistance, morphology) are needed to assess the efficiency of protoplast

fusion. For example, if two strains that are auxotrophic for different markers are

used to prepare protoplasts, the auxotrophy of the resulting cell can be used to differentiate between regenerated cells that have genetic information from one auxotroph rather than the other.



Amino Acid Fermentations Represent Mature Technologies

In 1993, a review of the outlook of one of the leading companies in the field of

amino acid production (Ajinomoto of Japan) indicated that amino acid production

is a mature technology. At Ajinomoto, glutamic acid and glutamine were produced

by wild-type microorganisms; lysine, by auxotrophic and regulatory mutants and

microorganisms from cell fusion; threonine, by regulatory mutants and recombinant

microorganisms; threonine, by regulatory mutants; and cystine, by enzyme catalysis

(Ladisch and Goodman 1992). Another major amino acid producer, Kyowa Hakko

Kogyo Company, Ltd., used recombinant technology to increase the production of

tryptophan from 30 to 60 g/L by cloning and expressing many of the genes for the

tryptophan pathway (Wang 1992). Ajinomoto’s culture collection has at least 21,000

strains, of which about 5000 are bacteria, 6000 are yeast, and 10,000 are unknown.

It is likely that other major manufacturers have significant culture collections. Consequently, there is a significant and diverse reserve of microorganisms for potential

use in the future, with improvements that are likely to be incremental rather than

revolutionary. Nonetheless, the continuous improvement of amino acid–producing

7



Base components of the osmotic stabilizing solution consist of 103 g sucrose, 0.25 g K2SO4,

and 2.03 g MgCl2 · 6H2O in 700 mL. This is then mixed with KH2 PO4 (to give 0.5 g/L),

CaCl2 · 2H2O (27.8 g/L), and TES buffer to keep the pH near 7.2 [TES = N-tris(hydroxymethyl)

methyl-2-amino ethyl sulfonic acid]. These and other reagents are given by Matsushima and

Blatz (1986).



314



METABOLIC PATHWAYS



strains and of manufacturing technologies will be an important factor in maintaining the cost-competitiveness of amino acids. Recombinant technology will play a

continuing role in this process.



ANTIBIOTICS

The antibacterial property of sulfanilamide, formed in vivo in mice from

Prontosil, was shown to be effective in protecting mice infected with Streptococcus

haemolyticus. According to Evans (1965), this discovery, made in 1935, marked

the beginning of a new era in the treatment of systemic microbial infections. The

first commercial antibiotics were on the market within 10 years. Prior to that, other

antibacterial drugs were known but were too toxic for oral or parenteral administration, or were not effectively adsorbed into the bloodstream.

Antibiotics were defined by Waksman as “chemical substances that are produced by micro-organisms and that have the capacity to inhibit and even to destroy

other microorganisms.” These differ from antibacterial drugs since antibacterials

are obtained through chemical synthesis. A brief history by Evans (1965), paraphrased below, illustrates the role of long-term research on the discovery of practical

methods for antibiotic production and applies to modern products derived through

modern biotechnology.

The existence of antibiotics was recognized in 1877 by Pasteur and Jobert,

who observed that anthrax were killed when contaminated by some other bacteria.

Gosio found, in 1896, that myophenolic acid from Penicillium brevicompactum

inhibited Bacillus anthracis, while Grieg-Smith showed actinomycetes produced

antibacterial compounds in 1917. Fleming published his observations on the inhibition of staphylococcus culture by growing colonies of P. notatum in 1929. When

Florey and Chain reexamined penicillin about 10 years later, culminating in development of methods to isolate penicillin and clinical trials in 1941, they found it to

be effective and “virtually devoid of toxicity [to humans].”

Commercial manufacture was facilitated when a strain of Penicillium chrysogenum was discovered to be capable of growing and producing penicillin in largescale, aerated submerged culture in 1943, as described in Chapter 1 ([see also

Langlyhke (1998) and Mateles (1998)]. The metabolism of microbial production of

antibiotics in the context of secondary metabolite (Type III) fermentation is summarized in the next section. A technical history of penicillin is given in a volume

edited by Mateles and describes the microbiological and industrial production

aspects of penicillin (Mateles 1998).



Secondary Metabolites Formed During Idiophase Are Subject to Catabolite

Repression and Feedback Regulation (Penicillin and Streptomycin)

Secondary metabolites are produced by a narrow spectrum of organisms and have

no general function in life processes. They are not produced during the cell’s rapid

growth phase (known as the trophophase). Rather, they are formed during a subsequent phase—the idiophase. The fermentation is Type III since the generation of

product is separate from the growth phase and independent of the cell’s energy



ANTIBIOTICS



315



α –ketoglutarate + acetyl – CoA



Homocitrate

cis – homoaconitate

Homoisocitrate

α –ketoadipate

α –aminoadipate (α-AA)

L – α – AA – L – CYS

δ – adenyl – α – AA

L – α – AA – L – CYS – D – VAL

δ – adenyl – α – AA

δ – semialdehyde

α – AA – δ – semialdehyde

Saccharopine



Lysine



L – α AAA – 6APA

(Isopenicillin N)



Phenylacetyl–6APA

(Benzylpenicillin)



Figure 11.14. Metabolic pathway for the production of penicillin from amino acid

precursors in Penicillium chrysogenum with feedback inhibition by lysine of homocitrate

synthetase [from Wang et al. (1979), Fig. 3.3, p. 31, Wiley-Interscience].



metabolism. Antibiotics, alkaloids, and mycotoxins are secondary metabolites

(Demain 1971).

The production of the antibiotic penicillin from amino acid precursors in

Penicillium chrysogenum occurs through a branched pathway, in which lysine

causes feedback inhibition of homocitrate synthetase (Fig. 11.14). The inhibition by

lysine of the first enzyme in its own biosynthesis limits production of intermediates,

including the α-aminodipate that is transformed to benzylpenicillin by the right

branch in Fig. 11.14. Consequently, production of penicillin (structure in Fig. 11.15)

is also inhibited unless α-aminodipate is added to the fermentation broth. A similar

mechanism in Streptomyces griseus limits production of candicidin. Tyrosine, tryptophan, and phenlalanine are end products of a pathway with the candicidin intermediate, p-aminobenzoic acid, occurring at the branch point (Wang et al. 1979).

The production of antibiotics derived from sugars is regulated by the

repression of phosphatase enzymes by phosphates at concentrations that do not

inhibit growth. The biosynthesis of streptomycin by Streptomyces griseus involves

at least three phosphatases. The intermediate is streptomycin phosphate. The



316



METABOLIC PATHWAYS



O

CH2



C



NH



H

C



H

C



C



N



CH3



S



CH3

C



OH



O



O



Figure 11.15. Benzyl penicillin is synthesized from two amino acids [from Evans (1965)].

NH

OH

H



H

OH



Ch2OH

O



NH



H



CH3NH



HO



NH



NH CNH2 H



O



C



NH2



HO

H

H



O

H



OH



HO

CH3

OHC



O



H



Figure 11.16. Streptomycin is synthesized from sugars [from Evans (1965)].



transformation of the biologically inactive streptomycin phosphate to the desired

streptomycin8 (structure in Fig. 11.16) is inhibited by phosphate (Wang et al. 1979;

Evans 1965). Control of phosphate concentration is of obvious importance.

Catabolic repression occurs when the substrate is metabolized to intermediates

that repress synthesis of enzymes that lead to formation of the secondary product.

Penicillin fermentation is subject to catabolic repression when glucose is rapidly

utilized during the tropophase. A mixture of glucose and lactose, which is the basis

of the Jarvis and Johnson media, was found to remove repression of penicillin synthesis. On glucose exhaustion and rapid growth phase, repression of lactose utilization in P. chrysogenum is eliminated. The fermentation timecourse in Fig. 11.17

shows how the increase in cell mass follows the depletion of sugar up to about 40 h,

during the tropophase, when enzymes for penicillin production are induced.

The idiophase and penicillin production begins at about 50 h (Fig. 11.17).

Although one possible explanation was that lactose served as a precursor or inducer

8



Streptomycin was discovered in 1944 by Shatz, Bugie, and Waksman after a series of disappointments in screening studies on the antimicrobial properties of metabolites from actinomycetes. Other antibiotics had previously been discovered that were effective but they were

too toxic for human use. Streptomycin from S. griseus was found to be active against both

Gram-negative and Gram-positive bacteria and inhibited Mycobacterium tuberculosis, and

was soon used to treat tuberculosis. The development of resistance to this antibiotic was

known, but its concomitant administration with other therapeutic agents was found to be

effective (Evans 1965).



ANTIBIOTICS



317



Figure 11.17. Fermentation timecourse for penicillin production. Timecourse of penicillum

(aerated) fermentation in chemically defined Jarvis–Johnson medium containing glucose

and lactose. Glucose is utilized during tropophase and lactose during idiophase. [From

Wang et al. (1979), Fig. 3.4, p. 32.]



for penicillin, further research showed that the slow hydrolysis of lactose resulting

in a slow release of glucose gave a much lower intracellular concentration of catabolites than would otherwise be present. This relieves repression of penicillin synthesis.

Slow feeding of glucose has the same effect as using lactose in the medium. Hence,

slow feeding of less expensive glucose replaced the use of lactose for derepression

of penicillin synthesis during industrial-scale production.

From the perspective of a process scientist, lactose was initially used because

its rate of consumption corresponded to maintaining the pH between 6.5 and 7.5,

which is optimal for penicillin production. When a simple sugar such as glucose

was used instead, it was rapidly consumed, causing the pH to increase to 8.5, where

penicillin synthesis ceased. The slow feeding of glucose, such as in molasses, avoided

this result (Langlyhke 1998).



The Production of Antibiotics Was Viewed as a Mature Field

Until Antibiotic-Resistant Bacteria Began to Appear

The production of antibiotics was viewed as a mature field with 50% of antibiotics

manufacturers in the United States and Japan curtailing development efforts between

about 1975 and 1990, although there were significant efforts to modify antibiotics

in order to overcome bacterial resistance (Gross and Carey 1996). This resulted in

about 160 variations of 16 basic compounds (Tanouye 1996a,b).

The unrecognized yearly cost of antibiotic resistance was estimated to be about

$100 million prior to 1991, according to Walsh (1993). While this may seem small

relative to the value of other types of biopharmaceuticals whose markets are in the

billion-dollar range, the cost of the infections, estimated to be $4.5 billion and

affecting 2 million patients in 1992 (Williams and Heymann 1998), has greatly

increased since then. The perception that antibiotics represent a mature business

is changing because of the increase in antibiotic-resistant microorganisms, with

measures to provide incentives for new antibiotics being proposed. Drug-resistant



318



METABOLIC PATHWAYS



Staphylococcus auerus is spreading with 126,000 hospitalizations reported (Chase

2006).



Bacteria Retain Antibiotic Resistance Even When Use of

the Antibiotic Has Ceased for Thousands of Generations

An example was reported for E. coli that carried a streptomycin resistance gene

(abbreviated rpsL) (Morell 1997). The E. coli was kept in an antibiotic-free environment for 20,000 generations over a 10-year period. Although the gene is “known

to markedly reduce the bacteria’s fitness,” and a single base change in the gene

would revert the E. coli to an antibiotic-susceptible state, the E. coli retained its

antibiotic resistance. Further research indicated that the development of antibiotic

resistance was accompanied by a mutation that compensated for a loss of fitness

caused by the first. This leads to the hypothesis that antibiotics that have lost their

effectiveness are unlikely to become powerful medicines again.



Antibiotic Resistance Involves Many Genes (Vancomycin Example)

Vancomycin is a glycopeptide antibiotic that was isolated from S. orientalis and

reported by Higgins et al. (1957) [cited in Evans (1965)]. It is used against Grampositive9 bacterial infections that are resistant to β-lactam antibiotics (such as peni9



The Gram strain was developed by Danish physician Hans Christian Joachim Gram in 1884,

and further modified by pathologist Carl Weigert in Germany. The procedure consists of

placing gentian violet over a “dried smear” of cells, washing it with water, and then adding

potassium triiodide to fix the dye, if it has reacted with the cells. The cells are washed with

ethanol, and then stained with safranine. Cells are Gram-positive if they take on the purple

color of gentian violet and Gram-negative if they retain the red color of the safranine. The

dyes have the following structures (Stinson 1996):

CAS 548-62-9

N



C1-



C6H5

N

+



NH2



NH2

Cl-



N+



N



CH3

Gentian violet



N



CH3



Safranine



Gram-positive cells have thick cell walls of crosslinked polysaccharide that react with

gentian violet, while Gram-negative cells have thin polysaccharide walls coated with a

lipid layer that reacts with safranine but resists staining with gentian violet. Antibacterial

drugs that act against both Gram-positive and Gram-negative bacteria are referred to as being

wide-spectrum.



ANTIBIOTICS



319



cillin), or for which penicillin cannot be used because of patient allergies. Vancomycin

binds strongly to peptidoglycan strands in the bacterial wall that terminate in the

amino acids d-Ala–d-Ala. This interferes with crosslinking of newly formed peptidoglycan strands, causing a reduction in the tensile strength of the cell wall. The

cells are then susceptible to lysis, due to osmotic pressure difference between the

internal part of the cell and the exterior fluid. Vancomycin can be effective in fighting infections when other antibiotics fail. However, by the late 1980s vancomycinresistant bacterial strains of Enterococcus began to appear, and in 1996 a strain of

Enterococcus faecium that requires vancomycin for inducing synthesis of cell wall

components was reported (News Item 1996).

Gene cloning and sequencing has delineated the mechanism by which the cell

overcomes the action of this antibiotic. The metabolic pathway and the expression

of resistance involves nine genes. A transmembrane protein is synthesized by the

cell that detects vancomycin and sends a chemical signal to a regulatory protein

that, in turn, activates transcription of genes. This leads to synthesis of peptidoglycans where d-Ala–d-Ala is replaced by d-Ala–d-lactate. The depsipeptide d-Ala–dlactate does not strongly bind vancomycin, which itself is a heptapeptide (Walsh

1993). This neutralizes the activity of the antibiotic and makes the bacterium resistant to vancomycin.

The potential impact of vancomycin resistance is likely to be significantly

greater if the genetic basis for this resistance in Enterococci10 is transferred to other

microorganisms (Tanouye 1996a,b). This is particularly true since vancomycin is

used to treat streptococcyl or staphylococcal strains that are resistant to β-lactam

antibiotics such as penicillin (Walsh 1993). Antibiotic-resistant, group A streptococcus can cause toxic shock11 and secrete a protein, pyrogenic exotoxin A, that triggers

an immune system response and causes cells that line blood vessels to be damaged,

so that fluid leaks out, blood flow dwindles, and tissues die from lack of oxygen

(Nowak 1994). Another organism, Staphylococcus aureus, is also treated with

vancomycin. The first case of a partial vancomycin resistance of a staph infection

was reported in Japan in 1996 (Gentry 1997).12

10



Bacteria are found in three forms: cocci (spheres), bacilli (rods), and spirilla (spirals). The

physical appearance of these 1–5-μm structures are schematically illustrated below:



Cocci (spheres)



Bacilli (rods)



Spirilla (spirals)



Growing bacteria are in a vegetative state. Bacteria can form endospores under adverse conditions (heat, radiation, and toxins), which germinate back into vegetative form when placed

in an environment that is conducive for growth. Hence, sterilization must be harsh enough

to kill spores as well as vegetative cells.

11



The sudden death of Muppets creator Jin Henson due to a virulent strain of streptococcus

A helped to renew attention on drug-resistant bacterial infections (Wright 1990).



12



There are 260,000 cases of staph infection per year in US hospitals. US sales of vancomycin

were more than $108 million in 1996. Eli Lilly’s sales of vancomycin (sold as Vancocin) were



320



METABOLIC PATHWAYS



Large quantities of antibiotics have been used in agricultural, animalproducing facilities, as well as for the treatment of human illness. About half of the

antibiotics produced in the United States are used for farm animals. Animal health

applications often utilize the antibiotic at subtherapeutic doses that allow resistant

microbial stains to survive (Wright 1990). This form of selective pressure and the

ability of resistant bacteria to use plasmids to pass on information on antibiotic

resistance to nonresistant cells of different species that can also proliferate is believed

by some to have helped promote antibiotic resistance.

An example of the change in viewpoint on use of antibiotics in agriculture and

heightened concern about drug resistance is given by a fluoroquinolone (sacrofloxacin) approved by the US Food and Drug Administration (FDA) for use in treating

bacterial infections in chickens in 1995. This antibiotic was used to fight E. coli

infections in chickens in order to improve productivity and thereby reduce production costs for the $15 billion chicken–turkey industry (1995 dollars). Cost savings

to the consumer were estimated at 1–2%. The debate surrounding the approval for

this application was due to the special status of fluoroquinolones as an antibiotic

of last resort for treatment of antibiotic-resistant forms of dysentery and typhoid

fever. The concern was that bacteria in poultry that become resistant will pass on

this trait to other bacteria that cause dysentery. Consequently, the use of the drug

was tightly controlled, with only 38 veterinarians in the United States allowed to

prescribe the drug for use in chickens. Anticipated use was for 150 million chickens,

or about 2% of the annual US production (Hensen 1995). The use of the antibiotic

is monitored closely by the FDA working together with the US Center for Disease

Control and Prevention (CDC) and the US Department of Agriculture (USDA).



REFERENCES

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Press, New York, 1973, pp. 28–32, 56–91, 195–345.

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Chuo-ku, Tokyo 104, Japan, 1990, pp. 1–15.

Atkinson, B. and F. Mavituna, Biochemical Engineering and Biotechnology Handbook,

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Bailey, J. E., “Toward a Science of Metabolic Engineering,” Science 252(5013), 1668–1675

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Behm, G., D. Dressler, G. Gaus, H. Herrmann, K. Küther, and H. Tanner, “Amino Acids in

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Bonn, 1988, pp. 46–52.

$250 million worldwide (Gentry 1997). While guidelines for vancomycin’s use restricts it to

treatment of antibiotic-resistant infections, or for patients with allergy to penicillin, culture

techniques that are used to identify the microbe (and therefore an alternate antibiotic) require

up to 72 h. Rather than risk patient death in the meantime, physicians prescribe vancomycin.

The concern was that widespread use (135,000 prescriptions per year) would accelerate

development of resistance to vancomycin (Gentry 1997). This has occurred.



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