Masako Suda

Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli

A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.

Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

ABSTRACT Production of l-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the l-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall l-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of l-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for l-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD+ ratio significantly decreased, and glucose consumption and l-valine production drastically improved. Moreover, l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for l-valine production under oxygen deprivation conditions. The l-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM l-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and the l-valine productivity reached 1,940 mM after 48 h.

Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions

ABSTRACT We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159–165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD+ ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.

Regulation of the Expression of Genes Involved in NAD De Novo Biosynthesis in Corynebacterium glutamicum

ABSTRACT Three genes, nadA, nadB, and nadC, involved in NAD de novo biosynthesis are broadly conserved in the genomes of numerous bacterial species. In the genome of Corynebacterium glutamicum, nadA and nadC but not nadB are annotated. The nadA and nadC genes are located in a gene cluster containing two other genes, designated ndnR and nadS herein. ndnR encodes a member of the Nudix-related transcriptional regulator (NrtR) family. nadS encodes a homologue of cysteine desulfurase involved in Fe-S cluster assembly. The gene cluster ndnR-nadA-nadC-nadS is genetically characterized herein. Mutant strains deficient in nadA, nadC, or nadS required exogenous nicotinate for growth, and the nicotinate auxotrophy was complemented by introduction of the corresponding gene in trans, indicating that each of these genes is essential for growth in the absence of an exogenous source of NAD biosynthesis. The results of reverse transcriptase PCR analyses and ndnR promoter-lacZ expression analyses revealed that the expression of ndnR, nadA, nadC, and nadS genes was markedly and coordinately repressed by nicotinate. The expression of these genes was enhanced by the disruption of ndnR, resulting in the loss of the nicotinate-responsive regulation of gene expression. These results suggest that NdnR acts as a transcriptional repressor of NAD de novo biosynthesis genes and plays an essential role in the regulation of the response to nicotinate.

Transcriptional regulation of Corynebacterium glutamicum methionine biosynthesis genes in response to methionine supplementation under oxygen deprivation

Expression at the mRNA level of six methionine biosynthesis genes in Corynebacterium glutamicum cells under oxygen-deprived conditions was repressed by supplementation of medium with methionine. The repression was not observed in a mutant deficient in the TetR-type transcriptional repressor McbR. Analysis of transcriptional start sites of the methionine biosynthesis genes confirmed that McbR binding motifs exist in the promoter regions of all genes repressed by methionine supplementation. Furthermore, electrophoretic mobility shift assays revealed that not only S-adenosylhomocysteine but also S-adenosylmethionine affects binding of McbR to the promoter region of metY, suggesting that both of these methionine metabolites are involved in the regulation of methionine biosynthesis genes.

Analyses of the acetate-producing pathways in Corynebacterium glutamicum

The original version of this article unfortunately contained a mistake. The presentation of Fig. 3 was published with an error. The correct version is given below.