丝状真菌液体深层发酵过程菌丝聚集的调控机制

丝状真菌是微生物发酵产品的重要表达体系,其液体深层发酵过程的典型特征是环境因素显著影响菌丝聚集,菌丝聚集影响发酵体系流变特性,进而影响质量传递、热量传递和动量传递,最终影响目标产物生物合成和生产效率。文中首先综述了丝状真菌形态调控的方法和策略,在此基础上针对丝状真菌菌丝生长和聚集过程的两大典型特征——顶端延伸生长和分枝生长,综述和展望了钙信号传导途径和几丁质生物合成途径对调控菌体聚集这一形态的重要意义。     

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.     

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.     

Induced variability in Acremonium chrysogenum, a producer of neutral-alkaline proteases and peptidases

Lethal and mutagenic effects of N-nitrosomethyl biuret on the organism producing a complex of proteolytic enzymes i. e. Acremonium chrysogenum were studied. It was shown that methionine inhibited production of the protease complex in the induced prototrophic mutants. The selected mutants were classified according to the level of enzyme biosynthesis induction by methionine.     

Regulation of aspartokinase in Lactobacillus plantarum

As a rational approach to the genetic development of a stable lysine overproducing strain of Lactobacillus plantarum for the fermentation of 'ogi', a Nigerian fermented cereal porridge, regulation of lysine biosynthesis in this species was investigated. Spontaneous lysine overproducing mutants of Lact. plantarum were obtained and their aspartokinase activities compared with those of wild-type strains under different conditions. Results showed that aspartokinase activity of Lact. plantarum cell extracts was not inhibited by either lysine, threonine, methionine or combinations of lysine and threonine. Instead, methionine enhanced aspartokinase activity in vitro. Results indicated that lysine biosynthesis in Lact. plantarum could be regulated by lysine via the control of aspartokinase production in a way different to that described for other bacteria.     

L-鸟氨酸发酵菌种的代谢工程研究进展

L-鸟氨酸是一种非蛋白类氨基酸参与尿素代谢及生物多胺类的合成,其对人体具有治疗肝脏疾病、增强免疫力等作用,被广泛应用于医疗、保健、食品等领域。工业上生产鸟氨酸主要有化学法、酶法及工业发酵法。其中,发酵法因其生产成本及环境保护等方面的优势而逐渐成为研究的焦点。本文归纳了近年来采用基因工程技术选育鸟氨酸高产菌种最新研究进展,重点讨论了产鸟氨酸谷氨酸棒杆菌的代谢工程改造策略,并对未来的研究方向进行了预测。     

曲酸的发酵生成与检测(综述)

本文概述曲霉发酵合成曲酸所需的各种营养因素及其对产酸力的影响,发酵工艺与菌种诱变,合成机理,曲酸的鉴定和检测方法,并对发酵工艺的发展趋势提出一些看法。     

L-苯丙氨酸生产的代谢工程研究

L-苯丙氨酸是一种重要的食品和医药中间体。工业上一般采用酶法和发酵法来生产L-苯丙氨酸。代谢工程的兴起,使得更加理性的改造菌株成为可能,这更加促进了发酵法的广泛应用。主要介绍了代谢工程在L-苯丙氨酸生产菌的改造中的应用情况,其中涉及苯丙氨酸生物合成途径中相关基因及其酶的调控、中央代谢途径的改造和芳香族氨基酸生物合成支路的修饰。并探讨了将来的发展前景。     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of l-threonine

l-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize l-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of l-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including l-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined l-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered.     

Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production.

Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class with commercial applicability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. Poly(hydroxyalkanoate) production in green plants promises much lower costs, but producing copolymer with the appropriate monomer composition is problematic. In this study, we have engineered Arabidopsis and Brassica to produce PHBV in leaves and seeds, respectively, by redirecting the metabolic flow of intermediates from fatty acid and amino acid biosynthesis. We present a pathway for the biosynthesis of PHBV in plant plastids, and also report copolymer production, metabolic intermediate analyses, and pathway dynamics.     

RNAi沉默lovF基因实现土曲霉一步法发酵生产辛伐他汀

辛伐他汀是重要的降胆固醇处方药物。莫那可林J是辛伐他汀合成过程的关键中间体,也是洛伐他汀生物合成的中间产物。为获得莫那可林J并通过一步法发酵生成辛伐他汀,构建了洛伐他汀生物合成关键基因lov F基因的RNAi载体p MHJ137,通过农杆菌介导的方法转化土曲霉F001,筛选整合到染色体的阳性菌,并在阳性菌株MJ1-24的发酵过程中加入了DMB-S-MMP验证其直接合成辛伐他汀的效率。结果显示MJ1-24不再产生洛伐他汀,产物中仅有莫那可林J累积。如果在发酵过程中加入前体物质,可得到产物辛伐他汀。综上,RNAi技术能够有效实现土曲霉基因沉默。此项技术推进了一步法发酵生产辛伐他汀的工艺开发。     

几种氨基酸在庆大霉素生产中作用的研究

庆大霉素是氨基糖苷类广谱抗生素,其不仅用于临床治病,而且广泛应用于畜牧业。由于其发酵周期长,产素率低,生产成本高,因此改进生产方法势在必行。本文报道了在绛红色小单孢菌产生庆大霉素的培养过程中,添加一定量的甘氨酸、蛋氨酸、赖氨酸、酪氨酸能够有效地提高微生物细胞的代谢能力,缩短发酵培养周期,提高产素率。本方法是在小试（摇瓶）成功基础上,用5L玻璃发酵罐运转一个多月,取一个月的平均罐批数据表明：新方法较原工艺发酵周期缩短30% ~45%,罐批产量增加14% 左右,产素率提高30 % ~95%（因菌种生产能力不同而异）,产品质量符合中国药典2000版（CP2000）、英国药典2000版（BP2000）、美国药典26版（USP26）,生产成本大幅度降低,具有很强的市场竞争力。     

Methionine metabolism in BHK cells: Preliminary characterization of the physiological effects of cycloleucine,an inhibitor of S-adenosylmethionine biosynthesis

Cycloleucine is in vitro a competitive inhibitor of methionine adenosyltransferase, an enzyme involved in S-adenosylmethionine biosynthesis. The physiological effects of this drug on baby hamster kidney cells have been studied. When cells are grown in a medium containing 10 μM methionine, cycloleucine is an inhibitor of cell proliferation; high concentrations of methionine are able to withdraw this inhibition suggesting that cycloleucine toxicity is related to methionine metabolism. The drug does not primarily affect methionine uptake and its subsequent use for protein biosynthesis. Cycloleucine toxicity is correlated with a block of SAM biosynthesis and nucleic acids methylations. The actions of cycloleucine on progression in the cell cycle and DNA, RNA and protein biosynthesis are studied. The implications of these results are discussed.     

Comparison of two chemical cleavage methods for preparation of a truncated form of recombinant human insulin-like growth factor I from a secreted fusion protein

We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage.     

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:204–211, 2015     

Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l(-1) of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l(-1)h(-1)) every 2 h to the production medium increased the production of methionine to 3.39 g l(-1). A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers.     

Defining an optimal carbon source/methionine feed strategy for growth and cephalosporin C formation by Cephalosporium acremonium.

The effect of the method of methionine addition, growth-limiting carbon source (glucose vs sucrose), and culture growth rate on cephalosporin C production was investigated in a Cephalosporium acremonium defined medium fed batch fermentation. Batch addition of methionine, at a concentration of 3 g/L, prior to the start of a fed sucrose fermentation was found to interfere with the ability of the culture to utilize this sugar, thus limiting growth and decreasing cephalosporin C production. Batch methionine addition had no effect on glucose-limited cultures. Concurrent exponential feeding of methionine with sucrose improved both culture growth and productivity. Under the control of identical carbon source limiting feed profiles, sucrose was observed to support greater cephalosporin C production than glucose. Optimal cephalosporin C production in a C. acremonium defined medium fed batch fermentation was obtained through controlling culture growth during the rapid growth phase at a relatively low level with respect to mumax (mu approximately 0.036 h-1) until achieving a desired cell mass with a concurrent sucrose and methionine feed, followed by maintaining relatively vigorous growth (mu approximately 0.01 h-1) with sucrose for the duration of the fermentation.     

Intracellular metabolism analysis of Clostridium cellulovorans via modeling integrating proteomics,metabolomics and fermentation

A consolidated bioprocess for cellulosic n-butanol production has been developed by engineering Clostridium cellulovorans to overexpress a bifunctional aldehyde/alcohol dehydrogenase. Rational metabolic engineering is important to further improve butanol production. This study aimed to investigate intracellular metabolism and identify the key regulators of cellulosic butanol formation in C. cellulovorans via integrated Omics and fermentation kinetics data analysis. First, comparative proteomics and metabolomics analyses of wild type and n-butanol producing mutant strain were conducted, which quantified 624 host cell proteins and 474 primary and secondary metabolites. Compared to wild type, most cellulases in cellulolysis were up-regulated, but three glycolysis enzymes and three enzymes in central pathway were down-regulated in the n-butanol producing strain. Second, a dynamic model integrating Omics and fermentation data was developed to identify key regulators in butanol biosynthesis, which were ranked by further metabolic control analysis. Finally, rational metabolic engineering was performed in C. cellulovorans by overexpressing two genes (thl and hbd) identified as important factors limiting butanol biosynthesis, which improved butanol yield and C4/C2 ratio. This study demonstrated a research approach to integrate multi-Omics and fermentation data of C. cellulovorans and guide its rational metabolic engineering, which can also be applied to other microorganisms.     

Glycine origin of the methyl substituent on C7′-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicat-ing that glycine and/or serine might be involved in the biosynthesis of apramycin. The ¹³C-NMR analysis of [2-¹³C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH₃ substituent of apramycin on the C7′ of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The ¹⁵N NMR spectra of [2-¹³C,¹⁵N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the ―NH₂ substituents of apramycin.