Part I Introduction

This group focuses on fundamental and applied aspects of industrial microbiology, especially in amino acid production. Research works include investigation on the mechanism of mutation of amino acid-producing mutants, strain improvement by genetic engineering and metabolic engineering, and investigation on the metabolic pathway of cyclic imide in microorganism.

This group has 4 staff members, including one associate professor, two research associate and one research assistant. Besides, there are 5 MSc students. The principal investigator, Jiuyuan Ding, associate professor, was graduated from Peking University and has been working in the Institute of Microbiology, CAS, after his graduation.

Part II Background and Significance

1. Amino acid production

At present, the amino acid industry has come to occupy an important role in world chemical industries. China is a agricultural country and has a very large population. Anuual demand for amino acids used in feed additives and pharmaceutical products is huge. Most of amino acids are currently manufactured in China. But the industrial production process has not been set up for a few limited kinds of amino acids such as L-tryptophan, L-histidine and L-arginine. The extraction method is still an industrial process for L-cysteine. However, the extraction method depends on the availability of natural protein-rich resources such as hair keratin, feather and the production process is not environmental friendly as unpleasant odors produced and problems of waste treatment.. So there is an urgent need for establishing the production process in order to meet the demand for these amino acids.

The fermentation method is being applied to industrial production of most L-amino acids. Coryneform bacteria has played a principle role in the progress of amino acid fermentation industry. However, the precise genetic and physiological changes

resulting in increased overproduction of amino acids in various coryneform bacterium strains have remained unknown. Success in attempts to further increase the productivities and yields of already highly productive strains will depend on the availability of detailed information on the metabolic pathways, their regulation, and mutations.

Corynebacterium crenatum and Corynebacterim pekinese are used in amino acid production process in China as well as Corynebacterium glutamicum. But the mechanism of amino acids accumulation of those mutants has not been extensively investigated. So far the strain improvement has mainly been carried out by an iterative process of mutagenesis and screening. It is difficult to increase the production yield furtherly by these methods. Also there is an urgent need for strain improvement by genetic engineering and metabolic engineering.

2. Studies on the metabolic pathway of cyclic imide in microorganism

Cyclic imide is a kind of cyclic amide compoud. The metabolism of cyclic imide in microorganism differs from that in mammalian. It was found that two enzymes, imidase and half-amidase, involved in the metabolic pathway. Only nonsubstituted cyclic imide such as succinimide, glutarimide and maleimide could be acted as substrates for imidase. But all of cyclic imide compounds existed naturally have molecular structure with various substituted groups. No evidence has been given to prove succinimide to be a natural metabolite till now. The physiological significance of metabolic pathway of cyclic imide in microorganism was not elucidated. The purpose of this study is to investigate the enzymes and the genes encoding those enzymes.

Part III Major achievements

1. Microbial production of L-tryptophan

A microorganism with amino acid racemase activity was screend and identified as Pseudomonas putida. The amino acid racemase has low substrate specificity. It has activity toward most aliphatic amino acids, but acromatic amino acids. A technique of L-tryptophan production was established by coupling the reactions catalyzed by

amino acid racemase and tryptophan synthase. Optimization of cell cultivation and transformation conditions was carried out. In addition, two distinct amino acid racemase genes from P. putida were cloned. Only alanine racemase activity was found in recombinan E. coli TG1 containing DadX gene. Another amino acid racemase encoded by alr gene showed low substrate specificity.

2. Enzymatic synthesis of L-cysteine

DL-ATC is a precursor of L-cysteine synthesis. A microorganism having ability utilizing DL-ATC as a sole carbon source and nitrogen source and accumulating L-cysteine was screened and identified as Pseudomonas sp. A 6 Kb DNA fragment isolated from the genome DNA of Pseudomonas sp was shown to be involved in conversion of DL-ATC. Sequence analysis showed that this DNA fragment contained genes encoding ATC hydrolase and N-carbomoyl-L-cystenine hydrolase. The construction of engineering strain is ongoing.

3. Metabolic engineering for C. crenatum

1) Aspartokinase (AK) gene, phosphenolpyruvate carboxylase (PPC) gene and pyruvate carboxylase (PYC) gene from wild type C. crenatum and AEC-resistant mutant strain were cloned and sequenced. Comparision of the two AK gene sequences showed that there happened a single pointmutation L80P in the β subunit of AK of AEC-resistant mutant. This mutation resulted in antifeedback regulation of the enzyme activity. Overexpressions of those three genes in C. crenatum were investigated. The simultaneous amplification of the activities of both AKfbr and PYC resulted in growth further increased and yielded about 50% increase of L-lysine production in the middle phase and 18% increase in the late phase.

2) N-acetylglutamate kinase genes from wild type strain of C. crenatum and L-arginine-producing mutant was cloned and sequenced. The accumulation of L-arginine of the mutant was resulted from the increase of the enzyme activity. Overexpression of argB yieded about 25% increase of L-arginine production.

4. Studies on the metabolic pathway of cyclic imide in Alcalgenes eutrophus

A hydantoin-cleaving microorganism 112R4 was screened and identified to be Alcalgenes eutrophus. The A. eutrophus can utilize succinimide as a sole carbon

source and nitrogen source, which indicated that a complete transformation pathway of succinimide existed and imidase and half-amidase were suggested to be involved in the metabolic pathway. A 6 Kb EcoRI-EcoRI fragment isolated from the genome DNA of A. eutrophus 112R4 was shown to be involved in the transformation of succinimide. Sequence analysis showed 5 continuous open reading frames(ORF) existed in this fragment. The functions of the 5 ORFs were confirmed by deletion analysis, functional analysis and homology search. Those encoded half-amidase, amide transport protein, imidase and two dehydrogenases subunits, respectively. The extensive investigatation of imidase and half-amidase as well as those two encoding genes was carried out. Recently, another 6 Kb DNA fragment also involved in succinimide hydrolysis was cloned. These two 6 Kb fragments have short overlap sequences, which indicated that other metabolic pathway of imide existed in this bacteria.

Publications :

1. Wang Yu, Zhang Yingzi, Ding Jiuyuan, Liu Yangjian, Wang Jiang, Yu Zhihua Cloning, Sequence Analysis of Imidase Gene from Alcaligenes eutrophus and Its Expression in E. coli Acta Microbioloica Sinica 2002 42(2) :153-162

2. Liu Yangjian, Zhang Yingzi, Wang Jiang, Wang Yu, Yu Zhihua, Ding Jiuyuan Cloning and Sequence Analysis of Aspartokinase Genes from Corynebacterium crenatum Acta Microbioloica Sinica 2002 42(4):395-399

3. Zhang Yingzi, Wang Yu, Yu Zhihua, Liu Yangjian, Wang Jiang, Ding Jiuyuan

A Dicarboxylate Monmoamide Amidohydrolase (Half-Amidase) From Alcaligenes eutrophus 112R4 Acta Microbioloica Sinica 2003 431):88-93

4Wang Jiang, Liu Yangjian, Wang Yu, Zhang Yingzi, Yu Zhihua, Ding Jiuyuan

Cloning, Sequence Analysis and Expression of Pyruvate Carboxylase Gene in Corynebacterium crenatum CD945 Acta Microbioloica Sinica 2003 432):214-219

5. Zhao Zhi , Liu Yangjian, Wang Yu, Zhang Yingzi, Ding Jiuyuan

Expression of Feedback-resistant Aspartate Kinase Gene in Corynebacterium crenatum Acta Microbioloica Sinica 2005 45(4):530-533

6. Cao Qin, Zhao Zhi, Zhang Yingzi, Wang Yu, Ding Jiuyuan Cloning, Sequence Analysis and Expression of Alanine Racemase Gene in Pseudomonas putida Acta Microbioloica Sinica accepted

7. Hao Ning, Zhao Zhi, Wang Yu, Zhang Yingzi, Ding Jiuyuan

Cloning, Sequence Analysis and Expression of N-Acetylglutamate Kinase Gene in

Corynebacterium crenatum accepted

Part IV Future research plan

1. Microbial production of amino acids

Improve the technique of L-tryptophan production in bench works and scale-up this process in pilot plan.

Investigate the enzymes and reactions involved in L-cysteine synthesis. Construct engineering strain by gene amplification and gene disruption. Establish a technique of L-Cysteine production.

2. Metabolic engineering for C. crenatum and C. pekinese

The target strains are L-lysine-, L-arginine- and L-tryptophan-producing mutants. Investigate the nods and key enzymes of metabolic pathways and improve strain properties by gene manipulation with the help of genomic data from C. glutamicum.

Construct stable Coryneform-E. coli shuttle vector in order to solve the instability of plasmid.

3. Studies on metabolic pathway of imide in A. eutrophus

Do purification and characterization of the known enzymes. Find other enzymes or proteins involved in imide metabolism and identify the unknown function of ORFs. In addition, acquirement of the upstream sequence of half-amidase gene is necessary for understanding the whole gene organization.

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