Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.     

Effect of heating rate on pDNA production by E. coli

The effects of heating rate (HR) on the performance of two-phase (batch followed by fed-batch) high cell-density cultivations (HCDC) of E. coli DH5α for the production of plasmid DNA (pDNA) were investigated. Optimal temperatures for the HCDC, as selected from shake flask experiments at constant temperatures between 30 and 45 °C, were 35 °C for biomass accumulation in the batch phase and 42 °C for inducing pDNA replication during the fed-batch. In HCDC the temperature was increased at HR of 0.025, 0.05, 0.10 and 0.25 °C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 °C). Compared to constant 35 °C, heat-induced HCDC accumulated up to 50% less biomass within the same cultivation time and acetate and glucose accumulated to high concentrations. The overall specific productivity (Q_P) and average pDNA yield (Y_p/x) in HCDC at 35 °C were 0.22 ± 0.02 mg/g h and 5.3 ± 0.00 mg/g, respectively. Such parameters were maximum at a HR of 0.05 °C/min, reaching 0.56 ± 0.06 mg/g h and 9.3 ± 0.6 mg/g, respectively. At HR above 0.5 °C/min, Y_p/x remained relatively constant, whereas Q_P tended to decrease. The supercoiled pDNA fraction remained around 80% at all HR. Bioreactors were equipped with a capacitance/conductivity probe. In all cases biomass concentration correlated closely with the capacitance signal and acetate and glucose accumulation was accompanied by an increase in the conductivity signal. Thus, it was possible to calculate acetate and biomass concentrations, as well as μ, from online capacitance and conductivity signals using estimators. Altogether, in this study it was shown that it is possible to maximize pDNA productivity by choosing an appropriate HR and that relevant parameters can be estimated by capacitance/conductivity signals, which are useful for better process control and development.     

Fatty acid from the renewable sources: A promising feedstock for the production of biofuels and biobased chemicals

With the depletion of the nonrenewable petrochemical resources and the increasing concerns of environmental pollution globally, biofuels and biobased chemicals produced from the renewable resources appear to be of great strategic significance. The present review described the progress in the biosynthesis of fatty acid and its derivatives from renewable biomass and emphasized the importance of fatty acid serving as the platform chemical and feedstock for a variety of chemicals. Due to the low efficient conversions of lignocellulosic biomass or carbon dioxide to fatty acid, we also put forward that rational strategies for the production of fatty acid and its derivatives should further derive from the consideration of whole bioprocess (pretreatment, saccharification, fermentation, separation), multiscale analysis and interdisciplinary combinations (omics, kinetics, metabolic engineering, synthetic biology, fermentation and so on).     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.     

Assessment of bacterial acyltransferases for an efficient lipid production in metabolically engineered strains of E. coli

Microbially produced lipids like triacylglycerols or fatty acid ethyl esters are currently of great interest as fuel replacements or other industrially relevant compounds. They can even be produced by non-oleaginous microbes, like Escherichia coli, upon metabolic engineering. However, there is still much room for improvement regarding the yield for a competitive microbial production of lipids or biofuels. We genetically engineered E. coli by expressing fadD, fadR, pgpB, plsB and ‘tesA in combination with atfA from Acinetobacter baylyi. A total fatty acid contents of up to 16% (w/w) was obtained on complex media, corresponding to approximately 9% (w/w) triacylglycerols and representing the highest titers of fatty acids and triacylglycerols obtained in E. coli under comparable cultivation conditions, so far. To evaluate further possibilities for an optimization of lipid production, ten promising bacterial wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferases were tested and compared. While highest triacylglycerol storage was achieved with AtfA, the mutated variant AtfA-G355I turned out to be most suitable for fatty acid ethyl ester biosynthesis and enabled an accumulation of approx. 500 mg/L without external ethanol supplementation.     

Improved methylation in E. coli via an efficient methyl supply system driven by betaine

Methylation reactions are involved in the biosynthesis of various natural molecules, in which S-adenosyl-L-methionine (SAM) acts as the principal biological methyl donor. The limited availability of SAM often affects the biosynthesis of methylated metabolites in cells, especially when heterologous SAM-mediated methyltransferases are employed. To solve this problem, a methyl supply system driven by betaine was developed in this study to enhance SAM availability in cells. A reconstructed methionine cycle was designed in E. coli using betaine as the methyl source by introducing betaine-homocysteine methyltransferase. Ferulic acid served as a model product was used to test the efficiency of methyl supply system. ATP is a co-factor for SAM biosynthesis and a pathway for ATP regeneration from adenosine was introduced to maintain the stability of the adenylate pool. After testing two different S-adenosyl-L-homocysteine (SAH) hydrolysis pathways, the optimized SAHase pathway was adopted for converting SAH back to homocysteine (Hcy). Thus, a methyl supply system was developed which increased SAM availability and therefore improved the titer and productivity of ferulic acid by 12.6-fold and 15.9-fold, respectively. The system was also applied successfully for other methyltransferase-catalyzed reactions. This work provides an efficient methyl supply system for enhanced production of methylated chemicals using betaine as the methyl source.     

党参多糖对双歧杆菌和大肠埃希菌体外生长的影响

目的探讨党参多糖体外对双歧杆菌和大肠埃希菌生长的影响。方法每隔12 h采用分光光度法测600 nm细菌培养液A值,气相色谱法测培养48 h后的双歧杆菌培养液中乙酸含量。结果党参多糖体外对大肠埃希菌没有促进或抑制生长的作用,对双歧杆菌有促进生长的作用,在中药作用下,双歧杆菌代谢的乙酸含量与其数量呈正相关关系。结论党参多糖能够通过促进双歧杆菌的生长,从而增加乙酸的代谢,增强双歧杆菌的定植抗力,对肠道一些致病菌发挥生物拮抗作用。     

Construction of a carbon-conserving pathway for glycolate production by synergetic utilization of acetate and glucose in Escherichia coli

Glycolate is a bulk chemical which has been widely used in textile, food processing, and pharmaceutical industries. Glycolate can be produced from sugars by microbial fermentation. However, when using glucose as the sole carbon source, the theoretical maximum carbon molar yield of glycolate is 0.67 mol/mol due to the loss of carbon as CO₂. In this study, a synergetic system for simultaneous utilization of acetate and glucose was designed to increase the carbon yield. The main function of glucose is to provide NADPH while acetate to provide the main carbon backbone for glycolate production. Theoretically, 1 glucose and 5 acetate can produce 6 glycolate, and the carbon molar yield can be increased to 0.75 mol/mol. The whole synthetic pathway was divided into two modules, one for converting acetate to glycolate and another to utilize glucose to provide NADPH. After engineering module I through activation of acs, gltA, aceA and ycdW, glycolate titer increased from 0.07 to 2.16 g/L while glycolate yields increased from 0.04 to 0.35 mol/mol-acetate and from 0.03 to 1.04 mol/mol-glucose. Module II was then engineered to increase NADPH supply. Through deletion of pfkA, pfkB, ptsI and sthA genes as well as upregulating zwf, pgl and tktA, glycolate titer increased from 2.16 to 4.86 g/L while glycolate yields increased from 0.35 to 0.82 mol/mol-acetate and from 1.04 to 6.03 mol/mol-glucose. The activities of AceA and YcdW were further increased to pull the carbon flux to glycolate, which increased glycolate yield from 0.82 to 0.92 mol/mol-acetate. Fed-batch fermentation of the final strain NZ-Gly303 produced 73.3 g/L glycolate with a productivity of 1.04 g/(L·h). The acetate to glycolate yield was 0.85 mol/mol (1.08 g/g), while glucose to glycolate yield was 6.1 mol/mol (2.58 g/g). The total carbon molar yield was 0.60 mol/mol, which reached 80% of the theoretical value.     

Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.     

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP⁺ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.     

大气溴甲烷的释放与控制研究

大气溴甲烷是破坏大气臭氧层的主要化合物之一,既有人为释放,也有自然释放,目前还存在着巨大的未知源。了解大气溴甲烷释放规律和控制措施,不仅对保护臭氧层具有重要意义,而且是大气溴甲烷含量的历史追溯和未来预测的重要基础,是全球变化研究热点。全面介绍了大气溴甲烷排放的途径和机制以及调控排放通量的主要措施,并分析了近期的优先研究领域。     

Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase,an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate,is not involved in floral scent production in snapdragon flowers

Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus. The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively). Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis. SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers. Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.     

Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli

For metabolic engineering it is advantageous in terms of stability, genetic regulation, and metabolic burden to modulate expression of relevant genes on the chromosome rather than relying on over-expression of the genes on multi-copy vectors. Here we have increased the production of beta-carotene in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (P(T5)). We recombined PCR fragments with the lambda-Red recombinase to effect chromosomal promoter replacement, which allows direct integration of a promoter along with a selectable marker that can subsequently be excised by the Flp/FRT site-specific recombination system. The resulting promoter-engineered isoprenoid genes were combined by serial P1 transductions into a host strain harboring a reporter plasmid containing beta-carotene biosynthesis genes allowing a visual screen for yellow color indicative of beta-carotene accumulation. Construction of an E. coli P(T5)-dxs P(T5)-ispDispF P(T5)-idi P(T5)-ispB strain resulted in producing high titers (6mg/g dry cell weight) of beta-carotene. Surprisingly, over-expression of the ispB gene, which was expected to divert carbon flow from the isoprenoid pathway to quinone biosynthesis, resulted in increased beta-carotene production. We thus demonstrated that chromosomal promoter engineering of the endogenous isoprenoid pathway yielded high levels of beta-carotene in a non-carotenogenic E. coli. The high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes.     

Physical studies on a nucleoprotein from the ribosome of E. coli

Bacterial 5S RNA and its cognate proteins constitute an attractive system to study nucleoprotein interactions. The molecular weights of the components involved are modest and they can be prepared in the quantities necessary to permit the application of material-intensive techniques like NMR and X-ray crystallography. 5S RNA is being examined by proton NMR at 500 MHz with special attention paid to the downfield NH proton region. A substantial number of assignments can be suggested in this region based on nuclear Overhauser results. The binding of protein L25 (E. coli) to the RNA gives rise to a highly characteristic set of perturbations in the spectrum of the RNA. The data suggest a localized and assignable alteration in RNA structure upon formation of the complex. In addition we have grown large crystals of RNAs related to 5S RNA and their complexes with a cognate protein. The properties of these crystals and the progress made in analyzing their structure are discussed.     

Isolation of E. coli from foam and effects of fluoroquinolones on E. coli and foam production in male Japanese quail

Sexually active male Japanese quail (Coturnix coutrnix Japonica) produce a foamy substance from their cloacal gland. It was postulated that bacteria played an important role in production of foam. The primary objective of this study was to isolate and identify bacteria present in the cloacal foam. The secondary objective was to evaluate the effect of fluoroquinolone treatment on bacterial counts and foam production. Healthy adult Japanese quail were maintained in individual cages under uniform husbandry conditions and allocated arbitrarily into three groups (each group consisted of 12 male and 12 female birds). Foam was collected from the cloacal gland of male birds of each group separately into sterile petri dishes and was cultured to isolate and identify bacteria and to determine their sensitivity to various antibiotics. Escherichia coli bacteria, sensitive to various antibacterials (including the fluoroquinolones ciprofloxacin and pefloxacin), were isolated. In the second part of the study, male quails of Group I (control) received I mL vehicle (normal saline 0.9% (w/v) NaCl) daily (via the intraperitoneal route) for 12 days. Male birds from groups II and III were treated intraperitoneally with ciprofloxacin or pefloxacin at the rate of 10 mg and 12 mg per/kg body weight respectively, for 12 days. In antibiotic-treated birds, there was a gradual reduction in foam production during treatment. At the end of treatment, the cloacal gland area was smaller (P < 0.05) in pefloxacin-treated birds compared to the other groups. Furthermore, a trend towards decreasing body weight and fertilizing ability was noted in the same group. A drastic reduction in bacterial counts of foam was recorded only in fluoroquinolone-treated groups during treatment period. After cessation of treatment, all end points were increasing back to pre-treatment levels. In conclusion, E. coli were present in the foam of the cloacal gland of Japanese quail and may have a role in foam production.     

Enhancing production of bio-isoprene using hybrid MVA pathway and isoprene synthase in E. coli

The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the "upper pathway" which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation.