Guocheng Du

Microbial production of hyaluronic acid: current state, challenges, and perspectives

Hyaluronic acid (HA) is a natural and linear polymer composed of repeating disaccharide units of β-1, 3-N-acetyl glucosamine and β-1, 4-glucuronic acid with a molecular weight up to 6 million Daltons. With excellent viscoelasticity, high moisture retention capacity, and high biocompatibility, HA finds a wide-range of applications in medicine, cosmetics, and nutraceuticals.Traditionally HA was extracted from rooster combs, and now it is mainly produced via streptococcal fermentation. Recently the production of HA via recombinant systems has received increasing interest due to the avoidance of potential toxins. This work summarizes the research history and current commercial market of HA, and then deeply analyzes the current state of microbial production of HA by Streptococcus zooepidemicus and recombinant systems, and finally discusses the challenges facing microbial HA production and proposes several research outlines to meet the challenges.

γ-Cyclodextrin: a review on enzymatic production and applications

Cyclodextrins are cyclic α-1,4-glucans that are produced from starch or starch derivates using cyclodextrin glycosyltransferase (CGTase). The most common forms are α-, β-, and γ-cyclodextrins. This mini-review focuses on the enzymatic production, unique properties, and applications of γ-cyclodextrin as well as its difference with α- and β-cyclodextrins. As all known wild-type CGTases produce a mixture of α-, β-, and γ-cyclodextrins, the obtaining of a CGTase predominantly producing γ-cyclodextrin is discussed. Recently, more economic production processes for γ-cyclodextrin have been developed using improved γ-CGTases and appropriate complexing agents. Compared with α- and β-cyclodextrins, γ-cyclodextrin has a larger internal cavity, higher water solubility, and more bioavailability, so it has wider applications in many industries, especially in the food and pharmaceutical industries.

Identification and Characterization of Bacterial Cutinase

Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu_0883 and Tfu_0882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu_0883 and Tfu_0882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an α/β-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser170-His248-Asp216 (Tfu_0883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources.

Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy

Flavonoids are valuable natural products widely used in human health and nutrition. Recent advances in synthetic biology and metabolic engineering have yielded improved strain titers and yields. However, current fermentation strategies often require supplementation of expensive phenylpropanoic precursors in the media and separate evaluation of each strategy in turn as part of the flavonoid pathway, implicitly assuming the modifications are additive. In this study, an Escherichia coli fermentation system was developed to bypass both of these problems. An eight-step pathway, consisting of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydratase (CM/PDT), phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was assembled on four vectors in order to produce the flavonoid precursor (2S)-pinocembrin directly from glucose. Furthermore, a modular metabolic strategy was employed to identify conditions that optimally balance the four pathway modules. Once this metabolic balance was achieved, such strains were capable of producing 40.02mg/L (2S)-pinocembrin directly from glucose. These results were attained by culturing engineered cells in minimal medium without additional precursor supplementation. The fermentation platform described here paves the way for the development of an economical process for microbial production of flavonoids directly from glucose.

Optimization and scale-up of propionic acid production by propionic acid-tolerant Propionibacterium acidipropionici with glycerol as the carbon source.

Propionic acid production by Propionibacterium acidipropionici with glycerol as sole carbon source was studied in 7-L batch and 10m(3) bioreactor cultures. In batch cultures, propionic acid production increased and specific cell growth rate decreased with increasing glycerol concentrations. Maximum propionic acid production and productivity reached 44.62+/-1.12g/L and 0.20+/-0.0075gL(-1)h(-1)at 220h, respectively, when glycerol was fed at a constant rate of 0.01L/h from 72 to 120h with an initial glycerol concentration of 30g/L. In the 10m(3) bioreactor, maximum propionic acid production reached 47.28+/-0.12g/L at 240h. This glycerol feeding approach may be useful for propionic acid production on an industrial scale.

Multivariate modular metabolic engineering of Escherichia coli to produce resveratrol from l-tyrosine

Microbial fermentations and bioconversion promise to revolutionize the conventional extraction of resveratrol from natural plant sources. However, the development of efficient and feasible microbial processes remains challenging. Current fermentation strategies often require supplementation of expensive phenylpropanoic precursors and two separate fermentation protocols, which are significantly more difficult and expensive to undertake when migrating to large-scale fermentation processes. In this study, an Escherichia coli fermentation system, consisting of tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), stilbene synthase (STS), malonate synthetase, and malonate carrier protein, was developed to produce resveratrol from L-tyrosine. Multivariate modular metabolic engineering, which redefined the overall pathway as a collection of distinct modules, was employed to assess and alleviate pathway bottlenecks. Using this strategy, the optimum strain was capable of producing 35.02 mg/L of resveratrol from L-tyrosine in a single medium. The strategy described here paves the way to the development of a simple and economical process for microbial production of resveratrol and other similar stilbene chemicals.

Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation.

Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL(-1), which was enhanced 1.85-fold compared to the control (863 U mL(-1)) cultured with sorbitol added at a constant rate of 3.6 g h(-1)L(-1) after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.

Lactobacillus casei combats acid stress by maintaining cell membrane functionality

Lactobacillus casei strains have traditionally been recognized as probiotics and frequently used as adjunct culture in fermented dairy products where lactic acid stress is a frequently encountered environmental condition. We have investigated the effect of lactic acid stress on the cell membrane of L. casei Zhang [wild type (WT)] and its acid-resistant mutant Lbz-2. Both strains were grown under glucose-limiting conditions in chemostats; following challenge by low pH, the cell membrane stress responses were investigated. In response to acid stress, cell membrane fluidity decreased and its fatty acid composition changed to reduce the damage caused by lactic acid. Compared with the WT, the acid-resistant mutant exhibited numerous survival advantages, such as higher membrane fluidity, higher proportions of unsaturated fatty acids, and higher mean chain length. In addition, cell integrity analysis showed that the mutant maintained a more intact cellular structure and lower membrane permeability after environmental acidification. These results indicate that alteration in membrane fluidity, fatty acid distribution, and cell integrity are common mechanisms utilized by L. casei to withstand severe acidification and to reduce the deleterious effect of lactic acid on the cell membrane. This detailed comparison of cell membrane responses between the WT and mutant add to our knowledge of the acid stress adaptation and thus enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress.

Screening of a thiamine-auxotrophic yeast for α-ketoglutaric acid overproduction.

Aims:  To obtain a thiamine‐auxotrophic yeast strain that overproduces α‐ketoglutaric acid (α‐KG) from glycerol and to investigate nutrient effects on α‐KG production.