生物技术法生产丙酮酸的研究进展

丙酮酸是一种重要的有机酸,广泛应用于制药、日化、农用化学品和食品等工业中。相对于化工法生产的丙酮酸而言,生物技术法生产的丙酮酸具有低成本、高质量等优势。生物技术法生产丙酮酸主要包括发酵法和酶法,前者又包括直接发酵法和休止细胞法。在对比各种生产方法的基础上,考虑到球拟酵母属的多重维生素营养缺陷型菌株是目前最具竞争力的丙酮酸生产菌,因此重点介绍了发酵法生产丙酮酸在菌种、发酵条件优化等方面的研究进展,并给出了生物技术法将来可能的发展方向。      

Overproduction and secretion of α-ketoglutaric acid by microorganisms

This mini-review presents a summary of research results of biotechnological production of alpha-ketoglutaric acid (KGA) by bacteria and yeasts. KGA is of particular industrial interest due to its broad application scope, e.g., as building block chemical for the chemical synthesis of heterocycles, dietary supplement, component of infusion solutions and wound healing compounds, or as main component of new elastomers with a wide range of interesting mechanical and chemical properties. Currently KGA is produced via different chemical pathways, which have a lot of disadvantages. As an alternative several bacteria and yeasts have already been studied for their ability to produce KGA as well as for conditions of overproduction and secretion of this intermediate of the tricarboxylic acid cycle. The aim of this mini-review was to summarize the known data and to discuss the potentials of biotechnological processes of KGA production.     

Metabolic engineering strategies for improving xylitol production from hemicellulosic sugars

Xylitol is a five-carbon sugar alcohol with potential for use as a sweetener. Industrially, xylitol is currently produced by chemical hydrogenation of d-xylose using Raney nickel catalysts and this requires expensive separation and purification steps as well as high pressure and temperature that lead to environmental pollution. Highly efficient biotechnological production of xylitol using microorganisms is gaining more attention and has been proposed as an alternative process. Although the biotechnological method has not yet surpassed the advantages of chemical reduction in terms of yield and cost, various strategies offer promise for the biotechnological production of xylitol. In this review, the focus is on the most recent developments of the main metabolic engineering strategies for improving the production of xylitol.     

Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes.     

酿酒酵母产苹果酸的还原TCA路径构建及发酵调控

苹果酸广泛应用于食品、化工行业。文中通过在酿酒酵母内敲除丙酮酸脱羧酶PDC1,并通过构建胞质内还原TCA的路径,即超表达丙酮酸羧化酶和苹果酸脱氢酶,成功地实现了苹果酸的生产。在野生型菌株中基本检测不到苹果酸的生成,而在工程菌株,苹果酸发酵浓度达到了45 mmol /L,同时副产物乙醇的产量也降低了18%。进一步通过发酵调控提高第二信使Ca2+的浓度使苹果酸的产量提高了7 %,在此基础上提高丙酮酸羧化酶的辅酶生物素浓度,使苹果酸的产量达到52.5 mmol /L,较原始菌株提高了16%。     

Biotechnological routes based on lactic acid production from biomass

Lactic acid, the most important hydroxycarboxylic acid, is now commercially produced by the fermentation of sugars present in biomass. In addition to its use in the synthesis of biodegradable polymers, lactic acid can be regarded as a feedstock for the green chemistry of the future. Different potentially useful chemicals such as pyruvic acid, acrylic acid, 1,2-propanediol, and lactate ester can be produced from lactic acid via chemical and biotechnological routes. Here, we reviewed the current status of the production of potentially valuable chemicals from lactic acid via biotechnological routes. Although some of the reactions described in this review article are still not applicable at current stage, due to their “greener” properties, biotechnological processes for the production of lactic acid derivatives might replace the chemical routes in the future.     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids

Industrial microorganisms have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. Rational strain development by metabolic engineering is crucial to successful processes, and is based on efficient genetic tools and detailed knowledge of metabolic pathways and their regulation. This review summarizes recent advances in metabolic engineering of the industrial model bacteria Escherichia coli and Corynebacterium glutamicum that led to efficient recombinant biocatalysts for the production of acetate, pyruvate, ethanol, d- and l-lactate, succinate, l-lysine and l-serine.     

The use of microorganisms in L-ascorbic acid production

L-Ascorbic acid has been industrially produced for around 70 years. Over the past two decades, several innovative bioconversion systems have been proposed in order to simplify the long time market-dominating Reichstein method, a largely chemical synthesis by which still a considerable part of L-ascorbic acid is produced. Here, we describe the current state of biotechnological alternatives using bacteria, yeasts, and microalgae. We also discuss the potential for direct production of l-ascorbic acid exploiting novel bacterial pathways. The advantages of these novel approaches competing with current chemical and biotechnological processes are outlined.     

Efficient pyruvate production by a multi-vitamin auxotroph of Torulopsis glabrata: key role and optimization of vitamin levels

A multi-vitamin auxotroph, Torulopsis glabrata strain WSH-IP303, which can use ammonium chloride as a sole nitrogen source for pyruvate production, was selected. To optimize pyruvate yield and productivity, a simple but useful, orthogonal design method, was used to investigate the relationship between thiamine, nicotinic acid, pyridoxine, biotin, and riboflavin. Thiamine was confirmed to be the most important factor affecting pyruvate production. When the concentration of thiamine was 0.01 mg/l or 0.015 mg/l, glucose consumption was improved by increasing the nicotinic acid concentration. When the concentrations of nicotinic acid, thiamine, pyridoxine, biotin, and riboflavin were 8.0, 0.015, 0.4, 0.04, and 0.1 mg/l, respectively, pyruvate concentration and yield reached 52 g/l and 0.52 g/g, respectively, in a 48-h flask culture. By employing a combination of the optimum vitamin concentrations, a batch culture was conducted in a 2.5-l fermentor with an initial glucose concentration of 112 g/l; and the pyruvate concentration reached 69 g/l after 56 h (yielding 0.62 g/g).     

Enhancement of pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata

Pyruvate production by Torulopsis glabrata was used as a model to study the mechanism of product inhibition and the strategy for enhancing pyruvate production. It was found that the concentration of cell growth and pyruvate deceased with the increase of NaCl and sorbitol concentrations. To enhance the osmotic stress resistance of the strain, an NaCl-tolerant mutant RS23 was screened and selected through a pH-controlled continuous culture with 70 g/L NaCl as the selective criterion. Compared with the parent strain, mutant RS23 could grow well on the medium containing 70 g/L NaCl or 0.6 mol/L sorbitol. Pyruvate concentration by the mutant strain RS23 reached 94.3 g/L at 82 h (yield on glucose 0.635 g/g) in a 7-l fermentor with 150 g/L glucose as carbon source. Pyruvate concentration and yield of mutant RS23 were 41.1% and 11.1% higher than those of the parent strain, respectively. The strategy for enhancing pyruvate production by increasing osmotic stress resistance may provide an alternative approach to enhance organic acids production with yeast.     

Biotechnological production of bio-based long-chain dicarboxylic acids with oleogenious yeasts

Long-chain α,ω-dicarboxylic acids (DCAs) are versatile chemical intermediates of industrial importance used as building blocks for the production of polymers, lubricants, or adhesives. The majority of industrial long-chain DCAs is produced from petro-chemical resources. An alternative is their biotechnological production from renewable materials like plant oil fatty acids by microbial fermentation using oleogenious yeasts. Oleogenious yeasts are natural long-chain DCA producers, which have to be genetically engineered for high-yield DCA production. Although, some commercialized fermentation processes using engineered yeasts are reported, bio-based long-chain DCAs are still far from being a mass product. Further progress in bioprocess engineering and rational strain design is necessary to advance their further commercialization. The present article reviews the basic strategies, as well as novel approaches in the strain design of oleogenious yeasts, such as the combination of traditional metabolic engineering with system biology strategies for high-yield long-chain DCA production. Therefore a detailed overview of the involved metabolic processes for the biochemical long-chain DCA synthesis is given.     

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.     

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).     

Oxidative fermentations and exopolysaccharides production by acetic acid bacteria: a mini review

Acetic acid bacteria are versatile organisms converting a number of carbon sources into biomolecules of industrial interest. Such properties, together with the need to limit chemical syntheses in favor of more sustainable biological processes, make acetic acid bacteria appropriate organisms for food, chemical, medical, pharmaceutical and engineering applications. At current, well-established bioprocesses by acetic acid bacteria are those derived from the oxidative pathways that lead to organic acids, ketones and sugar derivates. Whereas emerging applications include biopolymers, such as bacterial cellulose and fructans, which are getting an increasing interest for the biotechnological industry. However, considering the industrial demand of high performing bioprocesses, the production yield of metabolites obtained by acetic acid bacteria, is still not satisfying. In this paper we review the major acetic acid bacteria industrial applications, considering the current status of bioprocesses. We will also describe new biotechnological advances in order to optimize the industrial production, offering also an overview on future directions.     

Biotechnological production of sphingoid bases and their applications

Sphingolipids are not only essential components of biological membranes but also play numerous other vital functions in living cells. Moreover, they are major constituents of the outermost layer of human epidermis which acts as permeability barrier of the skin. Therefore, they have a high potential to be used in a wide variety of application fields such as antibacterial and antifungal agents, active pharmaceutical ingredients of therapeutics as well as active ingredients in cosmeceutical or nutraceutical formulations. However, their chemical synthesis is a complex and cost-intensive process. As the yeast Wickerhamomyces ciferrii has been found to be a natural producer of acetylated sphingoid bases, it provides a promising alternative for their biotechnological synthesis. In the last years, this yeast has been established by classical strain improvements as well as modern genetic engineering for the industrial production of phytosphingosine. Moreover, routes for the synthesis of sphinganine and sphingosine have been implemented. This mini-review summarizes the current knowledge about biosynthesis of sphingoid bases, genetic engineering of W. ciferrii for their biotechnological production, as well as their applications in cosmetic formulations.     

New stabilized FastPrep-CLEAs for sialic acid synthesis

N-acetyl-d-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-d-neuraminic acid (sialic acid) from N-acetyl-d-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10 mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K_M for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.     

A rare sugar xylitol. Part II: biotechnological production and future applications of xylitol

Xylitol is the first rare sugar that has global markets. It has beneficial health properties and represents an alternative to current conventional sweeteners. Industrially, xylitol is produced by chemical hydrogenation of d-xylose into xylitol. The biotechnological method of producing xylitol by metabolically engineered yeasts, Saccharomyces cerevisiae or Candida, has been studied as an alternative to the chemical method. Due to the industrial scale of production, xylitol serves as an inexpensive starting material for the production of other rare sugars. The second part of this mini-review on xylitol will look more closely at the biotechnological production and future applications of the rare sugar, xylitol.     

Current advances in biological production of propionic acid

Propionic acid and its derivatives are considered “Generally Recognized As Safe” food additives and are generally used as an anti-microbial and anti-inflammatory agent, herbicide, and artificial flavor in diverse industrial applications. It is produced via biological pathways using Propionibacterium and some anaerobic bacteria. However, its commercial chemical synthesis from the petroleum-based feedstock is the conventional production process bit results in some environmental issues. Novel biological approaches using microorganisms and renewable biomass have attracted considerable recent attention due to economic advantages as well as great adaptation with the green technology. This review provides a comprehensive overview of important biotechnological aspects of propionic acid production using recent technologies such as employment of co-culture, genetic and metabolic engineering, immobilization technique and efficient bioreactor systems.