DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome. A total of 111 E. coli genes, including those in central metabolism, key biosyntheses, and some regulatory functions, were cloned, amplified, and used as probes for detecting the level of transcripts. An E. coli strain was grown in glucose, acetate, and glycerol media, and the transcript levels of the selected genes were detected. Despite extensive studies on E. coli physiology, many new features were found in the regulation of these genes. For example, several genes were unexpectedly up-regulated, such as pps, ilvG, aroF, secA, and dsbA in acetate and asnA and asnB in glycerol, or down-regulated, such as ackA, pta, and adhE in acetate. These genes were regulated with no apparent reasons by unknown mechanisms. Meanwhile, many genes were regulated for apparent purposes but by unknown mechanisms. For example, the glucose transport genes (ptsHI, ptsG, crr) in both acetate and glycerol media were down-regulated, and the ppc, glycolytic, and biosynthetic genes in acetate were also down-regulated because of the reduced fluxes. However, their molecular mechanisms remain to be elucidated. Furthermore, a group of genes were regulated by known mechanisms, but the physiological roles of such regulation remain unclear. This group includes pckA and aspA, which are up-regulated in glycerol, and gnd and aspA, which are down-and up-regulated, respectively, in acetate. The DNA microarray technology demonstrated here is a powerful yet economical tool for characterizing gene regulation and will prove to be useful for strain improvement and bioprocess development. IntroductionUnderstanding how genes work at the genomic scale is essential for biotechnological research and application. Achieving this goal involves genome sequencing and determining the role of each gene in the cell. Recent development of DNA or oligonucleotide microarray technology has accelerated the investigation of gene regulation. By hybridization with labeled mRNA, cDNA, or chromosomal DNA, arrayed PCR products or oligonucleotides on a substrate, such as membranes, glass slides, or silicon chips, have been successfully used for monitoring transcript levels (1), single nucleotide polymorphism (2), or genomic variations between different strains (3). One of the most significant applications of this technique is the gene expression profiling at the whole genomic scale. The expression levels of the whole 6400 genes in the Saccharomyces cerevisiae genome have been detected successfully with either DNA or oligonucleotide microarray technology (4-7). The technique has also been used to investigate physiological changes of human cells, such as the aging process (8), or the response of fibloblasts to serum stimulation (9).The impact of this technology in biotechnology would be tremendous, because most problems in this field call for knowledge of global regulation, which can be studied efficiently using this technology. As illustrated in S. cerev...

The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate‐controlling steps in GGPP production, the IDI‐GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 235–241, 1999.

We have previously introduced a reconstructed isoprenoid pathway into Escherichia coli that exhibits amplified biosynthetic flux to geranylgeranyl diphosphate (GGPP), a common isoprenoid precursor. It was shown that GGPP synthase is an important rate-controlling enzyme in this reconstructed isoprenoid pathway. In this investigation, we applied directed evolution to GGPP synthase from Archaeoglobus fulgidus to enable the enhanced production of carotenoids in metabolically engineered E. coli. Eight mutants were isolated, and the best one increased lycopene production by 100%. Among the mutants that were isolated, mutation points were clustered in four "hot regions". The "hottest" region is located in the sequence upstream of the coding region, which presumably improves the expression level of the enzyme. The other three are within the coding sequence and are believed to improve the enzyme-specific activity in E coli. These results demonstrate that modulating both enzymatic expression and specific activity are important for optimizing the metabolic flux distribution.

Butyrate pathway was constructed in recombinant Escherichia coli using the genes from Clostridium acetobutylicum and Treponema denticola. However, the pathway constructed from exogenous enzymes did not efficiently convert carbon flux to butyrate. Three steps of the productivity enhancement were attempted in this study. First, pathway engineering to delete metabolic pathways to by-products successfully improved the butyrate production. Second, synthetic scaffold protein that spatially co-localizes enzymes was introduced to improve the efficiency of the heterologous pathway enzymes, resulting in threefold improvement in butyrate production. Finally, further optimizations of inducer concentrations and pH adjustment were tried. The final titer of butyrate was 4.3 and 7.2 g/L under batch and fed-batch cultivation, respectively. This study demonstrated the importance of synthetic scaffold protein as a useful tool for optimization of heterologous butyrate pathway in E. coli.

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Hexanoic acid production by a bacterium using sucrose as an economic carbon source was studied under conditions in which hexanoic acid was continuously extracted by liquid-liquid extraction. Megasphaera elsdenii NCIMB 702410, selected from five M. elsdenii strains, produced 4.69 g l⁻¹ hexanoic acid in a basal medium containing sucrose. Production increased to 8.19 g l⁻¹ when the medium was supplemented by 5 g l⁻¹ sodium butyrate. A biphasic liquid-liquid extraction system with 10 % (v/v) alamine 336 in oleyl alcohol as a solvent was evaluated in a continuous stirred-tank reactor held at pH 6. Over 90 % (w/w) of the hexanoic acid in a 0.5 M aqueous solution was transferred to the extraction solvent within 10 h. Cell growth was not significantly inhibited by direct contact of the fermentation broth with the extraction solvent. The system produced 28.42 g l⁻¹ of hexanoic acid from 54.85 g l⁻¹ of sucrose during 144 h of culture, and 26.52 and 1.90 g l⁻¹ of hexanoic acid was accumulated in the extraction solvent and the aqueous fermentation broth, respectively. The productivity and yield of hexanoic acid were 0.20 g l⁻¹ h⁻¹ and 0.50 g g⁻¹ sucrose, respectively.

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