Engineering industrial yeast for improved tolerance and robustness.

As an important cell factory, industrial yeast has been widely used for the production of compounds ranging from bulk chemicals to complex natural products. However, various adverse conditions including toxic products, extreme pH, and hyperosmosis etc., severely restrict microbial growth and metabolic performance, limiting the fermentation efficiency and diminishing its competitiveness. Therefore, enhancing the tolerance and robustness of yeasts is critical to ensure reliable and sustainable production of metabolites in complex industrial production processes. In this review, we provide a comprehensive review of various strategies for improving the tolerance of yeast cells, including random mutagenesis, system metabolic engineering, and material-mediated immobilization cell technology. It is expected that this review will provide a new perspective to realize the response and intelligent regulation of yeast cells to environmental stresses.

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.

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.

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.

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.

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.

3D Printed Biocatalytic Living Materials with Dual-Network Reinforced Bioinks.

The field of living materials seeks to harness living cells as microfactories that can construct a material itself or enhance the performance of material in some manner. While recent advances in 3D printing allow microbe manipulation to create bespoke living materials, the effective coupling of these living components in reinforced bioink designs remains a major challenge due to the difficulty in building a robust and cell-friendly microenvironment. Here, a type of dual-network bioink is reported for the 3D printing of living materials with enhanced biocatalysis capabilities, where bioinks are readily printable and provide a biocompatible environment along with desirable mechanical performance. It is demonstrated that integrating microbes into these bioinks enables the direct printing of catalytically living materials with high cell viability and maintains metabolic activity, which those living materials can be preserved and reused. Further, a bacteria-algae coculture system is fabricated for the bioremediation of chemicals, giving rise to its potential field applications.

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.

N-acyl-homoserine lactones in extracellular polymeric substances from sludge for enhanced chloramphenicol-degrading anode biofilm formation in microbial fuel cells.

Exploring an efficient acclimation strategy to obtain robust bioanodes is of practical significance for antibiotic wastewater treatment by bioelectrochemical systems (BESs). This study investigated the effects of two acclimation conditions on chloramphenicol (CAP)-degrading anode biofilm formation in microbial fuel cells (MFCs). The one was continuously added the extracellular polymeric substances (EPS) extracted from anaerobic sludge and increasing concentrations of CAP after the first start-up phase, while the other was added the EPS-1 (N-acyl-homoserine lactones, namely AHLs were extracted from the EPS) at the same conditions. The results demonstrated that AHLs in the sludge EPS played a crucial role for enhanced CAP-degrading anode biofilm formation in MFCs. The AHL-regulation could not only maintain stable voltage outputs but also significantly accelerate CAP removal in the EPS MFC. The maximum voltage of 653.83 mV and CAP removal rate of 1.21 ± 0.05 mg/L·h were attained from the EPS MFC at 30 mg/L of CAP, which were 0.84 and 1.57 times higher than those from the EPS-1 MFC, respectively. These improvements were largely caused by the thick and 3D structured biofilm, strong and homogeneous cell viability throughout the biofilm, and high protein/polysaccharide ratio along with more conductive contents in the biofilm EPS. Additionally, AHLs facilitated the formation of a biofilm with rich biodiversity and balanced bacterial proportions, leading to more beneficial mutualism among different functional bacteria. More bi-functional bacteria (for electricity generation and antibiotic resistance/degradation) were specifically enriched by AHLs as well. These findings provide quorum sensing theoretical knowledge and practical instruction for rapid antibiotic-degrading electrode biofilm acclimation in BESs.

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.

Metabolic engineering of Pichia pastoris for malic acid production from methanol.

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by-product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

Quantitative proteomic analysis to reveal expression differences for butanol production from glycerol and glucose by Clostridium sp. strain CT7.

Clostridium sp. strain CT7 is a new emerging microbial cell factory with high butanol production ratio owing to its non-traditional butanol fermentation mode with uncoupled acetone and 1,3-propanediol formation. Significant changes of metabolic products profile were shown in glycerol- and glucose-fed strain CT7, especially higher butanol and lower volatile fatty acids (VFAs) production occurred from glycerol-fed one. However, the mechanism of this interesting phenomenon was still unclear. To better elaborate the bacterial response towards glycerol and glucose, the quantitative proteomic analysis through iTRAQ strategy was performed to reveal the regulated proteomic expression levels under different substrates. Proteomics data showed that proteomic expression levels related with carbon metabolism and solvent generation under glycerol media were highly increased. In addition, the up-regulation of hydrogenases, ferredoxins and electron-transferring proteins may attribute to the internal redox balance, while the earlier triggered sporulation response in glycerol-fed media may be associated with the higher butanol production. This study will pave the way for metabolic engineering of other industrial microorganisms to obtain efficient butanol production from glycerol.

Microbial application of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery.

Recently, thermophilic Thermoanaerobacterium species have attracted increasing attentions in consolidated bioprocessing (CBP), which can directly utilize lignocellulosic materials for biofuels production. Compared to the mesophilic process, thermophilic process shows greater prospects in CBP due to its relatively highly efficiency of lignocellulose degradation. In addition, thermophilic conditions can avoid microbial contamination, reduce the cooling costs, and further facilitate the downstream product recovery. However, only few reviews specifically focused on the microbial applications of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery. Accordingly, this review will comprehensively summarize the recent advances of Thermoanaerobacterium species in lignocellulosic biorefinery, including their secreted xylanases and bioenergy production. Furthermore, the co-culture can significantly reduce the metabolic burden and achieve the more complex work, which will be discussed as the further perspectives. KEY POINTS: • Thermoanaerobacterium species, promising chassis for lignocellulosic biorefinery. • Potential capability of hemicellulose degradation for Thermoanaerobacterium species. • Efficient bioenergy production by Thermoanaerobacterium species through metabolic engineering.

Luteimonas wenzhouensis Sp. Nov., A Chitinolytic Bacterium Isolated from a Landfill Soil.

A Gram-staining-negative, aerobic, non-motile, rod-shaped bacterium with degradation ability of chitin, designated strain YD-1 T, was isolated from landfill soil sample collected in Wenzhou, Zhejiang province, China. The growth of strain YD-1 T occurred optimally in the tryptone soy broth (TSB) with 1.0% NaCl at pH 7.0-8.0, 30 °C. Ubiquinone-8 (Q-8) was the predominant quinone. The polar lipids of strain YD-1 T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, five glycolipids and four lipids. The major fatty acids were iso-C15:0 (30.7%), iso-C17:1ω9c (23.2%), iso-C11:0 (18.9%), iso-C11:0 3-OH (6.8%) and iso-C17:0 (5.9%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain YD-1 T was affiliated to the genus Luteimonas with the highest similarity to Luteimonas marina KCTC 12327 T (97.3%), followed by Luteimonas aquatica DSM 22088 T (96.5%) and Luteimonas composti CCUG 53595 T (96.4%). The genomic DNA G + C content of strain YD-1 T was 71.8 mol%. Average nucleotide identity (ANI) and the digital DNA-DNA hybridizations (dDDH) for draft genomes between strain YD-1 T and Luteimonas marina KCTC 12327 T were 82.7% and 26.1%, respectively. On the basis of genotypic, phenotypic and chemotaxonomic data, strain YD-1 T is considered to represent a novel species to degrade chitin in the genus Luteimonas, for which the name Luteimonas wenzhouensis sp. nov. is proposed, with YD-1 T (= KCTC 72425 T = CCTCC AB 2019153 T) as the type strain.

Challenges and Future Perspectives of Promising Biotechnologies for Lignocellulosic Biorefinery.

Lignocellulose is a kind of renewable bioresource containing abundant polysaccharides, which can be used for biochemicals and biofuels production. However, the complex structure hinders the final efficiency of lignocellulosic biorefinery. This review comprehensively summarizes the hydrolases and typical microorganisms for lignocellulosic degradation. Moreover, the commonly used bioprocesses for lignocellulosic biorefinery are also discussed, including separated hydrolysis and fermentation, simultaneous saccharification and fermentation and consolidated bioprocessing. Among these methods, construction of microbial co-culturing systems via consolidated bioprocessing is regarded as a potential strategy to efficiently produce biochemicals and biofuels, providing theoretical direction for constructing efficient and stable biorefinery process system in the future.

Recent progress on bio-succinic acid production from lignocellulosic biomass.

Succinic acid is a valuable bulk chemical, which has been extensively applied in food, medicine, surfactants and biodegradable plastics industries. As a substitute for chemical raw material, bio-based succinic acid production has received increasing attention due to the depletion of fossil fuels and environmental issues. Meanwhile, the effective bioconversion of lignocellulosic biomass has always been a hot spot of interest owning to the advantages of low expense, abundance and renewability. Consolidated bioprocessing (CBP) is considered to be an alternative approach with outstanding potential, as CBP can not only improve the product yield and productivity, but also reduce the equipment and operating costs. In addition, the current emerging microbial co-cultivation systems provide strong competitiveness for lignocellulose utilization through CBP. This article comprehensively discusses different strategies for the bioconversion of lignocellulose to succinic acid. Based on the principles and technical concepts of CBP, this review focuses on the progress of succinic acid production under different CBP strategies (metabolic engineering based and microbial co-cultivation based). Moreover, the main challenges faced by CBP-based succinic acid fermentation are analyzed, and the future direction of CBP production is prospected.

Consolidated bioprocessing performance of a two-species microbial consortium for butanol production from lignocellulosic biomass.

Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.

Metabolic engineering of Escherichia coli for L-malate production anaerobically.

BACKGROUND: L-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce L-malate from renewable resources has attracted significant attention. RESULTS: A potential L-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in L-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final L-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied. CONCLUSIONS: The availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of L-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of L-malate.

Perspectives for the microbial production of ethyl acetate.

Ethyl acetate is one of the short-chain esters and widely used in the food, beverage, and solvent areas. The ethyl acetate production currently proceeds through unsustainable and energy intensive processes, which are based on natural gas and crude oil. Microbial conversion of biomass-derived sugars into ethyl acetate may provide a sustainable alternative. In this review, the perspectives of bio-catalyzing ethanol and acetic acid to ethyl acetate using lipases in vitro was introduced. Besides, the crucial elements for high yield of ethyl acetate in fermentation was expounded. Also, metabolic engineering in yeasts to product ethyl acetate in vivo using alcohol acyl transferases (AAT) was discussed. KEY POINTS: •The accumulation of acetyl-CoA is crucial for synthesizing ethyl acetate in vivo; AAT-mediated metabolic engineering could efficiently improve ethyl acetate production.

Techniques for enhancing the tolerance of industrial microbes to abiotic stresses: A review.

The diversity of stress responses and survival strategies evolved by microorganism enables them to survive and reproduce in a multitude of harsh environments, whereas the discovery of the underlying resistance genes or mechanisms laid the foundation for the directional enhancement of microbial tolerance to abiotic stresses encountered in industrial applications. Many biological techniques have been developed for improving the stress resistance of industrial microorganisms, which greatly benefited the bacteria on which industrial production is based. This review introduces the main techniques for enhancing the resistance of microorganisms to abiotic stresses, including evolutionary engineering, metabolic engineering, and process engineering, developed in recent years. In addition, we also discuss problems that are still present in this area and offer directions for future research.