Strategies for the efficient biosynthesis of ß-carotene through microbial fermentation.

ß-Carotene is an orange fat-soluble compound, which has been widely used in fields such as food, medicine and cosmetics owing to its anticancer, antioxidant and cardiovascular disease prevention properties. Currently, natural ß-carotene is mainly extracted from plants and algae, which cannot meet the growing market demand, while chemical synthesis of ß-carotene cannot satisfy the pursuit for natural products of consumers. The ß-carotene production through microbial fermentation has become a promising alternative owing to its high efficiency and environmental friendliness. With the rapid development of synthetic biology and in-depth study on the synthesis pathway of ß-carotene, microbial fermentation has shown promising applications in the ß-carotene synthesis. Accordingly, this review aims to summarize the research progress and strategies of natural carotenoid producing strain and metabolic engineering strategies in the heterologous synthesis of ß-carotene by engineered microorganisms. Moreover, it also summarizes the adoption of inexpensive carbon sources to synthesize ß-carotene as well as proposes new strategies that can further improve the ß-carotene production.

Strategies for the biological synthesis of D-glucuronic acid and its derivatives.

D-glucuronic acid is a kind of glucose derivative, which has excellent properties such as anti-oxidation, treatment of liver disease and hyperlipidemia, and has been widely used in medicine, cosmetics, food and other fields. The traditional production methods of D-glucuronic acid mainly include natural extraction and chemical synthesis, which can no longer meet the growing market demand. The production of D-glucuronic acid by biocatalysis has become a promising alternative method because of its high efficiency and environmental friendliness. This review describes different production methods of D-glucuronic acid, including single enzyme catalysis, multi-enzyme cascade, whole cell catalysis and co-culture, as well as the intervention of some special catalysts. In addition, some feasible enzyme engineering strategies are provided, including the application of enzyme immobilized scaffold, enzyme mutation and high-throughput screening, which provide good ideas for the research of D-glucuronic acid biocatalysis.

Transcriptomic Analysis Reveals the Potential Mechanisms for Improving Carotenoid Production in Rhodosporidium toruloides Z11 under Light Stress.

Carotenoids, as a type of tetraterpene compound, have been widely used in food, medical, and health areas owing to their antioxidant, immune enhancement, and disease risk reduction effects. Rhodosporidium toruloides is a promising oleaginous red yeast that can industrially synthesize carotenoids. In this study, the effects of different light exposure times and intervals on carotenoid production by R. toruloides Z11 were first investigated. Results showed that a higher carotenoid content (1.29 mg/g) can be achieved when R. toruloides Z11 was exposed to light for 12 h per day, which was increased by 1.98 times compared with that of dark cultivation. Transcriptome profiling revealed that light stress could effectively promote the gene expression levels of GGPS1 and AL1 in the carotenoid biosynthesis pathway and phr in the DNA photolysis pathway of R. toruloides. This work will provide a molecular foundation to further improve the production efficiency of carotenoids by genetic engineering.

Strategies for the biological production of ectoine by using different chassis strains.

As an amino acid derivative and a typical compatible solute, ectoine can assist microorganisms in resisting high osmotic pressure. Own to its long-term moisturizing effects, ectoine shows extensive applications in cosmetics, medicine and other fields. With the rapid development of synthetic biology and fermentation engineering, many biological strategies have been developed to improve the ectoine production and simplify the production process. Currently, the microbial fermentation has been widely used for large scaling ectoine production. Accordingly, this review will introduce the metabolic pathway for ectoine synthesis and also comprehensively evaluate both wild-type and genetically modified strains for ectoine production. Furthermore, process parameters affecting the ectoine production efficiency and adoption of low cost substrates will be evaluated. Lastly, future prospects on the improvement of ectoine production will be proposed.

Microbial synthesis of pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ) is a peptide-modified natural product. PQQ has important physiological functions such as anti-oxidation, anti-aging, and immunity enhancement. However, due to the lack of in-depth understanding of PQQ biosynthesis and regulation, inefficient PQQ production level limits its wide application. Accordingly, there is still an urgent need to develop high-yielding strains for synthesis of PQQ. This paper reviewed the research and development trends on the PQQ biosynthetic pathways, catalytic reaction mechanism of key enzymes, and the selection of high-yielding strains, which also prospects for the future construction of PQQ biosynthetic microbial cell factories.

Characterization of a novel esterase and construction of a Rhodococcus-Burkholderia consortium capable of catabolism bis (2-hydroxyethyl) terephthalate.

Bis (2-hydroxyethyl) terephthalate (BHET) is one of the main compounds produced by enzymatic hydrolysis or chemical depolymerization of polyethylene terephthalate (PET). However, the lack of understanding on BHET microbial metabolism is a main factor limiting the bio-upcycling of PET. In this study, BHET-degrading strains of Rhodococcus biphenylivorans GA1 and Burkholderia sp. EG1 were isolated and identified, which can grow with BHET as the sole carbon source. Furthermore, a novel esterase gene betH was cloned from strain GA1, which encodes a BHET hydrolyzing esterase with the highest activity at 30 °C and pH 7.0. In addition, the co-culture containing strain GA1 and strain EG1 could completely degrade high concentration of BHET, eliminating the inhibition on strain GA1 caused by the accumulation of intermediate metabolite ethylene glycol (EG). This work will provide potential strains and a feasible strategy for PET bio-upcycling.

Isolation of a newly Trichoderma asperellum LYS1 with abundant cellulase-hemicellulase enzyme cocktail for lignocellulosic biomass degradation.

As the most abundant and renewable natural resource in the world, lignocellulose is a promising alternative to fossil energy to relieve environmental concerns and resource depletion. However, due to its recalcitrant structure, strains with efficient degradation capability still need exploring. In this study, a fungus was successfully isolated from decayed wood and named as Trichoderma asperellum LYS1 by phylogenetic and draft genomic analysis. The further investigations showed that strain LYS1 had an outstanding performance on lignocellulose degradation, especially for hemicellulose-rich biomass. After the analysis of encoded CAZymes, mainly on GH family, a large amount of genes coding ß-glucosidase and xylanase may contribute to the high degradation of cellulose and hemicellulose. Collectively, the results generated in this study demonstrated that T. asperellum LYS1 is a potential cell factory for lignocellulose biorefinery.

Construction of Coculture System Containing Escherichia coli with Different Microbial Species for Biochemical Production.

Microbial synthesis of target chemicals usually involves multienzymatic reactions in vivo, especially for compounds with a long metabolic pathway. However, when various genes are introduced into one single strain, it leads to a heavy metabolic burden. In contrast, the microbial coculture system can allocate metabolic pathways into different hosts, which will relieve the metabolic burdens. Escherichia coli is the most used chassis to synthesize biofuels and chemicals owing to its well-known genetics, high transformation efficiency, and easy cultivation. Accordingly, cocultures containing the cooperative E. coli with other microbial species have received great attention. In this review, the individual applications and boundedness of different combinations will be summarized. Additionally, the strategies for the self-regulation of population composition, which can help enhance the stability of a coculture system, will also be discussed. Finally, perspectives for the cocultures will be proposed.

Customized spatial niches for synthetic microbial consortia.

The construction of synthetic microbial consortia has been considered a new frontier. However, maintaining artificial microbial communities remains challenging because the dominant strain eventually outcompetes the others. Inspired by natural ecosystems, one promising approach to assemble stable consortia is to construct spatial niches partitioning subpopulations and overlapping abiotic requirements.

Enhanced ß-carotene production in Yarrowia lipolytica through the metabolic and fermentation engineering.

ß-Carotene is a kind of high-value tetraterpene compound, which shows various applications in medical, agricultural, and industrial areas owing to its antioxidant, antitumor, and anti-inflammatory activities. In this study, Yarrowia lipolytica was successfully metabolically modified through the construction and optimization of ß-carotene biosynthetic pathway for ß-carotene production. The ß-carotene titer in the engineered strain Yli-C with the introduction of the carotenogenesis genes crtI, crtE, and crtYB can reach 34.5 mg/L. With the overexpression of key gene in the mevalonate pathway and the enhanced expression of the fatty acid synthesis pathway, the ß-carotene titer of the engineered strain Yli-CAH reached 87 mg/L, which was 152% higher than that of the strain Yli-C. Through the further expression of the rate-limiting enzyme tHMGR and the copy number of ß-carotene synthesis related genes, the ß-carotene production of Yli-C2AH2 strain reached 117.5 mg/L. The final strain Yli-C2AH2 produced 2.7 g/L ß-carotene titer by fed-batch fermentation in a 5.0-L fermenter. This research will greatly speed up the process of developing microbial cell factories for the commercial production of ß-carotene. ONE-SENTENCE SUMMARY: In this study, the ß-carotene synthesis pathway in engineered Yarrowia lipolytica was enhanced, and the fermentation conditions were optimized for high ß-carotene production.

Report of genome sequence of Rhodococcus biphenylivorans strain GA1, an isolate capable of efficiently degrading lignin and its derivates.

Rhodococcus biphenylivorans GA1 was successfully isolated, which can efficiently degrade alkali lignin and a variety of lignin-derived aromatic compounds as the sole carbon source. Whole genome sequencing of strain GA1 showed that it possessed G + C content of 68% with the size of 6.0 Mb and 4319 putative open reading frames (ORFs). Four replicons consisting of one circular chromosome (ChrA1) and three circular plasmids (pGA1, pGA2, pGA3) were found. Among these annotated proteins, lignin depolymerizing peroxidases (Dyp) and two lignin-derived aromatic compounds cleavage dioxygenases, protocatechuate 3,4-dioxygenase(P34D) and catechol-1,2-dioxygenase (C12D) play key roles in the catabolism of lignin.

Enhanced 1,3-propanediol production with high yield from glycerol through a novel Klebsiella-Shewanella co-culture.

BACKGROUND: 1,3-Propanediol (1,3-PDO) is a platform compound, which has been widely used in food, pharmaceutical and cosmetic industries. Compared with chemical methods, the biological synthesis of 1,3-PDO has shown promising applications owing to its mild conditions and environmental friendliness. However, the biological synthesis of 1,3-PDO still has the problem of low titer and yield due to the shortage of reducing powers. RESULTS: In this study, Klebsiella sp. strain YT7 was successfully isolated, which can synthesize 11.30 g/L of 1,3-PDO from glycerol in flasks. The intracellular redox regulation strategy based on the addition of electron mediators can increase the 1,3-PDO titer to 28.01 g/L. Furthermore, a co-culturing system consisting of strain YT7 and Shewanella oneidensis MR-1 was established, which can eliminate the supplementation of exogenous electron mediators and reduce the by-products accumulation. The 1,3-PDO yield reached 0.44 g/g and the final titer reached 62.90 g/L. The increased titer and yield were attributed to the increased redox levels and the consumption of by-products. CONCLUSIONS: A two-bacterium co-culture system with Klebsiella sp. strain YT7 and S. oneidensis strain MR-1 was established, which realized the substitution of exogenous electron mediators and the reduction of by-product accumulation. Results provided theoretical basis for the high titer of 1,3-PDO production with low by-product concentration.

Construction of a Microbial Consortium for the De Novo Synthesis of Butyl Butyrate from Renewable Resources.

Butyl butyrate has shown wide applications in food, cosmetic, and biofuel sectors. Currently, biosynthesis of butyl butyrate still requires exogenous addition of precursors and lipase, which increases the production cost and limits further large-scale development. In this study, a microbial consortium was first designed to realize direct butyl butyrate production from lignocellulose. The highest butyl butyrate concentration of 34.42 g/L was detected in the solvent phase from 60 g/L glucose using a microbial coculture system composed of Clostridium acetobutylicum NJ4 and Clostridium tyrobutyricum LD with the elimination of butyric acid supplementation. Meanwhile, 13.52 g/L butyl butyrate was synthesized from 60 g/L glucose using a microbial consortium composed of three strains including strain NJ4, strain LD, and Escherichia coli BL21- pET-29a(+)-LE without the addition of any exogenous precursors and lipase. In addition, 2.94 g/L butyl butyrate could be directly produced from 60 g/L microcrystalline cellulose when Trichoderma asperellum was added to the above-mentioned three-strain microbial consortium. This four-strain microbial consortium represents the first study regarding the direct butyl butyrate production from lignocellulose without the supplementation of exogenous precursors and lipase, which may be extended to the biosynthesis of other short-chain esters, such as ethyl acetate and butyl lactate.

Carotenoids production and genome analysis of a novel carotenoid producing Rhodococcus aetherivorans N1.

Carotenoids are a series of natural pigments with unique structure and physiological functions. In this study, a novel Rhodococcus aetherivorans strain N1 was discovered, which can produce 6.4 mg/g carotenoids including ß-carotene, zeaxanthin and isorenieratene from glucose. Moreover, strain N1 can directly produce 3.0 mg/g carotenoids from the undetoxified straw hydrolysate, representing the highest carotenoids production from the undetoxified lignocellulosic hydrolysate. The crude carotenoid extracts of strain N1 showed efficient free radical scavenging activity and stability. Strain N1 has complete methylerythritol 4-phosphate (MEP) pathway and related genes for carotenoid synthesis, especially the rare aromatic carotenoid of isorenieratene. Genomic comparison between strain N1 and other carotenoid producing Rhodococcus sp. strains showed the conservatism and universality of carotenoids synthesis gene. These results proved that R. aetherivorans strain N1 can serve as a promising producer for the industrialization of carotenoid production.

The De Novo Synthesis of 2-Phenylethanol from Glucose by the Synthetic Microbial Consortium Composed of Engineered Escherichia coli and Meyerozyma guilliermondii.

Synthetic microbial consortia show promising applications for fine chemical production, especially with long metabolic pathways. In this study, a synthetic microbial consortium consisting of Escherichia coli YLC20 and Meyerozyma guilliermondii MG57 was successfully constructed, which could achieve efficient de novo 2-phenylethanol (2-PE) production from glucose. A tyrosine-deficient E. coli YLC20 overexpressing genes of aroF and pheA was first constructed, which could accumulate 29.5 g/L of l-phenylalanine (l-Phe) within 96 h from glucose accompanied by the coproduction of acetate and α-ketoglutarate (α-KG). Furthermore, the engineered M. guilliermondii MG57 was constructed through the stepwise metabolic engineering strategy, which could facilitate the 2-PE synthesis from l-Phe. Moreover, the cosubstrate and material intervention strategies were applied to improve the stability of the microbial consortium and 2-PE production. Finally, the synthetic microbial consortium could de novo synthesize 3.77 g/L of 2-PE from 80 g/L of glucose, providing a reference for the de novo synthesis of fine chemicals with long metabolic pathways.

Biological production of xylitol by using nonconventional microbial strains.

Xylitol (C5H12O5), an amorphous sugar alcohol of crystalline texture has received great interest on the global market due to its numerous applications in different industries. In addition to its high anticariogenic and sweetening properties, characteristics such as high solubility, stability and low glycemic index confer xylitol its fame in the food and odontological industries. Moreover, it also serves as a building-block in the production of polymers. As a result of the harmful effects of the chemical production of xylitol, the biotechnological means of producing this polyol have evolved over the decades. In contrast to the high consumption of energy, long periods of purification, specialized equipment and high production cost encountered during its chemical synthesis, the biotechnological production of xylitol offers advantages both to the economy and the environment. Non-Saccharomyces yeast strains, also termed as nonconventional, possess the inherent capacity to utilize D-xylose as a sole carbon source, unlike Saccharomyces species.

The draft genome sequence of Rhodosporidium toruloides strain Z11, an isolate capable of co-producing lipids and carotenoids from waste molasses.

A wild-type Rhodosporidium toruloides strain Z11 which could utilize molasses to co-produce high amount of lipid and carotenoids was isolated and characterized. The genome of strain Z11 with a G + C content of 59.0% was estimated to be 22.6 Mb and contained 5290 encoded protein sequences. Among these annotated genes, the ATP citrate (pro-S)-lyase, two malic enzymes (MaeA and MaeB) and the geranylgeranyl pyrophosphate synthase play key roles for the production of lipids and carotenoids. In addition, a ß-fructofuranosidase (SacA) was identified, which may contribute to the utilization of molasses.

Comparative Genomic Analysis of Carbofuran-Degrading Sphingomonads Reveals the Carbofuran Catabolism Mechanism in Sphingobium sp. Strain CFD-1.

The worldwide use of the carbamate insecticide carbofuran has caused considerable concern about its environmental fate. Degradation of carbofuran by Sphingobium sp. strain CFD-1 is initiated via the hydrolysis of its ester bond by carbamate hydrolase CehA to form carbofuran phenol. In this study, another carbofuran-degrading strain, Sphingobium sp. CFD-2, was isolated. Subsequently, a cfd gene cluster responsible for the catabolism of carbofuran phenol was predicted by comparing the genomes of strains CFD-1, CFD-2, and Novosphingobium sp. strain KN65.2. The key genes verified to be involved in the catabolism of carbofuran phenol within the cfd cluster include the hydroxylase gene cfdC, epoxide hydrolase gene cfdF, and ring cleavage dioxygenase gene cfdE and are responsible for the successive conversion of carbofuran phenol, resulting in complete ring cleavage. These carbofuran-catabolic genes (cehA and the cfd cluster) are distributed on two plasmids in strain CFD-1 and are highly conserved among the carbofuran-degrading sphingomonad strains. The mobile genetic element IS6100 flanks cehA and the cfd gene cluster, indicating the importance of horizontal gene transfer in the formation of carbofuran degradation gene clusters. The elucidation of the molecular mechanism of carbofuran catabolism provides insights into the evolutionary scenario of the conserved carbofuran catabolic pathway. IMPORTANCE Owing to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. In this study, the cfd gene cluster, responsible for the catabolism of carbofuran phenol, was predicted by comparing sphingomonad genomes. The function of key enzymatic genes in this gene cluster was identified. Furthermore, the carbamate hydrolase gene cehA and the cfd gene cluster are highly conserved in different carbofuran-degrading strains. Additionally, the horizontal gene transfer elements flanking the cfd gene cluster were investigated. These findings help elucidate the molecular mechanism of microbial carbofuran degradation and enhance our understanding of the evolutionary mechanism of the carbofuran catabolic pathway.

Current status, challenges and prospects for lignin valorization by using Rhodococcus sp.

Lignin represents the most abundant renewable aromatics in nature, which has complicated and heterogeneous structure. The rapid development of biotransformation technology has brought new opportunities to achieve the complete lignin valorization. Especially, Rhodococcus sp. possesses excellent capabilities to metabolize aromatic hydrocarbons degraded from lignin. Furthermore, it can convert these toxic compounds into high value added bioproducts, such as microbial lipids, polyhydroxyalkanoate and carotenoid et al. Accordingly, this review will discuss the potentials of Rhodococcus sp. as a cell factory for lignin biotransformation, including phenol tolerance, lignin depolymerization and lignin-derived aromatic hydrocarbon metabolism. The detailed metabolic mechanism for lignin biotransformation and bioproducts spectrum of Rhodococcus sp. will be comprehensively discussed. The available molecular tools for the conversion of lignin by Rhodococcus sp. will be reviewed, and the possible direction for lignin biotransformation in the future will also be proposed.

The Novel Amidase PcnH Initiates the Degradation of Phenazine-1-Carboxamide in Sphingomonas histidinilytica DS-9.

Phenazines are an important class of secondary metabolites and are primarily named for their heterocyclic phenazine cores, including phenazine-1-carboxylic acid (PCA) and its derivatives, such as phenazine-1-carboxamide (PCN) and pyocyanin (PYO). Although several genes involved in the degradation of PCA and PYO have been reported so far, the genetic foundations of PCN degradation remain unknown. In this study, a PCN-degrading bacterial strain, Sphingomonas histidinilytica DS-9, was isolated. The gene pcnH, encoding a novel amidase responsible for the initial step of PCN degradation, was cloned by genome comparison and subsequent experimental validation. PcnH catalyzed the hydrolysis of the amide bond of PCN to produce PCA, which shared low identity (only 26 to 33%) with reported amidases. The Km and kcat values of PcnH for PCN were 33.22 ± 5.70 µM and 18.71 ± 0.52 s-1, respectively. PcnH has an Asp-Lys-Cys motif, which is conserved among amidases of the isochorismate hydrolase-like (IHL) superfamily. The replacement of Asp37, Lys128, and Cys163 with alanine in PcnH led to the complete loss of enzymatic activity. Furthermore, the genes pcaA1A2A3A4 and pcnD were found to encode PCA 1,2-dioxygenase and 1,2-dihydroxyphenazine (2OHPC) dioxygenase, which were responsible for the subsequent degradation steps of PCN. The PCN-degradative genes were highly conserved in some bacteria of the genus Sphingomonas, with slight variations in the sequence identities. IMPORTANCE Phenazines have been widely acknowledged as a natural antibiotic for more than 150 years, but their degradation mechanisms are still not completely elucidated. Compared with the studies on the degradation mechanism of PCA and PYO, little is known regarding PCN degradation by far. Previous studies have speculated that its initial degradation step may be catalyzed by an amidase, but no further studies have been conducted. This study identified a novel amidase, PcnH, that catalyzed the hydrolysis of PCN to PCA. In addition, the PCA 1,2-dioxygenase PcaA1A2A3A4 and 2OHPC dioxygenase PcnD were also found to be involved in the subsequent degradation steps of PCN in S. histidinilytica DS-9. And the genes responsible for PCN catabolism are highly conserved in some strains of Sphingomonas. These results deepen our understanding of the PCN degradation mechanism.